Pentapeptides and their uses

Abstract

The present invention provides a pentapeptide obtained by acetylating the N-terminus of a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a pharma- ceutically acceptable salt thereof, and uses thereof. The pentapeptide according to the present invention is stable, and is effective in suppressing anticancer drug resistance, suppressing autophagy in tumor cells, enhancing immunity, activating immune cells, promoting antitumor immune responses, and / or anticancer.

Description

[Technical field]

[0001] The present invention relates to a pentapeptide, and more particularly to an improved pentapeptide and its uses. [Background technology]

[0002] Research into techniques for treating diseases such as cancer with peptides is ongoing (see International Publication No. WO2017 / 014604A1, etc.). In the course of research into such peptides, the present inventors have come to recognize the need for unexpected improvements. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. WO2017 / 014604A1, January 26, 2017, Specification Summary of the Invention [Problem to be solved by the invention]

[0004] One problem to be solved by the present invention is to provide an improved pentapeptide.

[0005] Another object of the present invention is to provide a novel use of the improved pentapeptide.

[0006] The problems to be solved by the present invention are not limited to those mentioned above, and other problems not mentioned herein will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0007] The inventors recognized that the peptide consisting of the amino acid sequence (QLHLD) of SEQ ID NO:1 needed to be improved in terms of stability, and as a result of their research, they have completed the present invention which is not only excellent in terms of stability, but also, surprisingly, has previously unanticipated uses.

[0008] The present invention provides a pentapeptide in which the N-terminus of a peptide consisting of the amino acid sequence of SEQ ID NO:1 (QLHLD) is acetylated, or a pharma- ceutical acceptable salt thereof.

[0009] In the amino acid sequence, Q represents glutamine (Gln), L represents leucine (Leu), H represents histidine (His), and D represents aspartate (Asp).

[0010] The amino acids constituting the peptides include L-, D- and DL-forms, and the amino acids constituting the peptides of the present invention include all of these.

[0011] The peptides include mutants, and include those in which the main activity is not changed by natural or artificial mutations and a part of the peptide structure is mutated.

[0012] Examples of the pharma- ceutically acceptable salt include hydrochloride, sulfate, phosphate, acetate, trifluoroacetate, citrate, tartrate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, paratoluenesulfonate, sodium salt, potassium salt, magnesium salt, or calcium salt.

[0013] The present invention also provides a medical use of the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof, preferably for suppressing anticancer drug resistance, suppressing autophagy in tumor cells, enhancing immunity, activating immune cells, promoting antitumor immune responses, and / or for anticancer use. Anticancer use includes use in treating or preventing cancer, where treatment includes the improvement and alleviation of symptoms, and prevention includes the inhibition of progression from a pre-disease stage to the disease.

[0014] The cancer may be a metastatic cancer.

[0015] The cancer may be one or more selected from breast cancer, prostate cancer, lymphoma, and lung cancer.

[0016] The anti-cancer effect may be achieved by one or more selected from the following: suppression of resistance to other anti-cancer drugs, supporting the anti-cancer effect of other anti-cancer drugs, suppression of autophagy in tumor cells, enhancement of immunity, activation of immune cells, promotion of anti-tumor immune responses, promotion of tumor cell death, and suppression of tumor cell proliferation.

[0017] The present invention provides a pharmaceutical composition for suppressing resistance to anticancer drugs, which comprises the pentapeptide of the present invention or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0018] The anticancer drug resistance includes a decrease in sensitivity to an anticancer drug.

[0019] The anticancer drug may be an anticancer drug other than the active ingredient.

[0020] The anti-cancer agent may be an anti-cancer chemotherapeutic agent or an anti-cancer immunotherapeutic agent.

[0021] The suppression of anticancer drug resistance includes suppression of anticancer drug resistance against cancer.

[0022] The suppression of anticancer drug resistance can be due to the suppression of autophagy activity in tumor cells or the activation of immune cells.

[0023] The present invention provides a pharmaceutical composition for suppressing autophagy in tumor cells, comprising the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0024] The pharmaceutical composition may be an autophagy inhibiting agent that inhibits autophagy activity in cancer cells.

[0025] The inhibition of autophagy includes inhibition of protective autophagy.

[0026] The inhibition of autophagy can be achieved by suppressing expression of LC3II protein.

[0027] The active ingredient can suppress activated autophagy in cancer cells and inhibit the progression of cancer.

[0028] When an anticancer drug is administered, autophagy, an intracellular self-digestion process, is activated in the cancer cells, and the organelles and decomposition products damaged by the anticancer drug are reused as metabolic products of the cancer cells by the activated autophagy, thereby preventing cell death and promoting the proliferation of cancer cells, thereby conferring resistance to the anticancer drug. Therefore, it is also possible to suppress resistance to anticancer drugs by inhibiting autophagy with the active ingredient.

[0029] The present invention provides a pharmaceutical composition for enhancing immunity, which comprises the pentapeptide of the present invention or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0030] The immune enhancement can be due to activation of immune cells within the tumor.

[0031] The present invention provides a pharmaceutical composition for activating immune cells, which comprises the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0032] The immune cell may be a natural killer cell or a cytotoxic T cell.

[0033] The pharmaceutical composition may be an immune cell activator.

[0034] The pharmaceutical composition may be a natural killer cell activator.

[0035] Activation of the immune cells can promote expression of cell-killing factors in tumor cells, promote immune cell (e.g., natural killer cell)-mediated lysis in tumor cells, kill tumor cells, and / or induce anti-tumor effects.

[0036] The present invention provides a pharmaceutical composition for promoting anti-tumor immune response, comprising the pentapeptide of the present invention or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0037] The present invention provides an anti-cancer pharmaceutical composition comprising the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0038] The present invention also provides a pharmaceutical composition for treating or preventing cancer, which comprises the pentapeptide of the present invention or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0039] The active ingredient may be for administration in combination with other anti-cancer agents.

[0040] The other anti-cancer agent may be an anti-cancer chemotherapeutic agent or an anti-cancer immunotherapeutic agent.

[0041] The anti-cancer chemotherapeutics may be one or more selected from paclitaxel and doxorubicin.

[0042] The anti-cancer immunotherapy can be an immune checkpoint inhibitor.

[0043] The immune checkpoint inhibitor may be one or more selected from anti-PD-L1 and anti-PD-1.

[0044] The active ingredient may be administered simultaneously or sequentially with the other anti-cancer agent.

[0045] The active ingredient may be administered 24 hours after administration of the other anti-cancer agent.

[0046] The active ingredient may be administered within 48±12 hours after administration of the other anti-cancer agent.

[0047] The active ingredient may be administered at a content of 0.8 to 2000 mg / kg.

[0048] The active ingredient may be administered 1 to 7 times per week.

[0049] The active ingredient may be administered by injection.

[0050] The other anticancer agent may be administered at a content of 2 to 20 mg / kg.

[0051] The pharmaceutical composition may be an anti-cancer pharmaceutical composition for supporting the anti-cancer effect of another anti-cancer agent.

[0052] The support may be one that suppresses resistance or side effects caused by the other anti-cancer agent or promotes the anti-cancer effect of the anti-cancer agent.

[0053] The pharmaceutical composition may be a pharmaceutical composition for suppressing autophagy induced by other anticancer drugs or enhancing the immune activity of immune cells.

[0054] The enhancement of the immune activity of the immune cells may be enhancement of granzyme expression in the immune cells by the active ingredient.

[0055] The pharmaceutical composition further comprises a pharma- ceutically acceptable excipient, and consists of the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof and the excipient.

[0056] The pentapeptide of the present invention can be produced by acetylating the N-terminus of the peptide of SEQ ID NO: 1. First, the peptide of SEQ ID NO: 1 can be produced by a method for forming a peptide bond. For example, it can be produced by a method such as solution phase synthesis or solid phase synthesis. Examples of the method for forming a peptide bond include the acyl azide method, the acyl halide method, the acylimidazole method, the carbodiimide method, the phosphonium method, the anhydride method, the mixed anhydride method, the oxidation-reduction method, and the method using Woodward's reagent K. Before carrying out the condensation reaction, carboxyl groups, amino groups, etc. that are not involved in the reaction can be protected, and carboxyl groups, etc. that are involved in the condensation reaction can be activated by a method known in the art. Examples of groups for protecting carboxyl groups include ester-forming groups such as methyl, t-butyl, aryl, pentafluorophenyl, benzyl, para-methoxybenzyl, or methoxyethoxymethyl. Examples of groups for protecting amino groups include tritylcarbonyl, aryloxycarbonyl, cyclohexyloxycarbonyl, trichloroethyloxycarbonyl, benzyloxycarbonyl, t-butoxycarbonyl, and / or 9-fluorenylmethyloxycarbonyl, etc. Examples of activated forms of carboxyl groups include mixed anhydrides, azides, acyl chlorides, or activated esters [esters with alcohols (e.g., pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxysuccinimide, N-hydroxyoxyphthalimide, or 1-hydroxybenzotriazole)], etc. Solvents that can be used in the condensation reaction to form peptide bonds can be a single solvent or a mixture of solvents such as benzene, toluene, hexane, acetone, nitromethane, cyclohexane, ether, chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethylsulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol, or ethanol. The reaction temperature can be in the range of about -70°C to 100°C, which is generally used in the reaction, and more preferably in the range of -30°C to 30°C.The reaction for removing the peptide protecting group varies depending on the type of protecting group, but it can be removed using an acid compound, a base compound, or a transition metal that can detach the protecting group without affecting the peptide bond. The protecting group can be removed by acid treatment, for example, treatment with hydrogen chloride, hydrogen bromide, hydrogen fluoride, acetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, trimethylchlorosilane, or a mixture thereof. When the reaction for removing the protecting group is performed by the acid treatment, anisole, phenol, thioanisole, or the like can be added as an auxiliary agent to promote the reaction. The protecting group can be removed by base treatment, for example, using ammonia, diethylamine, hydrazine, morpholine, N-methylpyrrolidine, piperidine, sodium carbonate, or a mixture of these bases. The protecting group can also be removed by transition metal treatment, for example, using zinc, mercury, palladium / hydrogen, or the like.

[0057] The N-terminal acyl substituent of the peptide can be formed by a known method using an active acyl derivative. Specifically, an acetylation reagent can be used to acetylate the N-terminus of the peptide of SEQ ID NO: 1. The acetylation reagent can be acetic anhydride, acyl halides (e.g., acetyl chloride, acetyl bromide, acetyl iodide), etc. Of course, an acetyl group can be introduced into the N-terminus of the peptide by direct condensation with acetic acid.

[0058] The peptides can also be purified by conventional peptide purification methods, such as extraction, phase separation, solid precipitation, recrystallization, or column chromatography.

[0059] The dosage of the pentapeptide of the present invention or a pharma- ceutical acceptable salt thereof, when administered parenterally, may vary depending on the subject and condition, and may be, for example, 0.8 to 2000 mg / kg. Within such ranges, the dosage may vary depending on the species. For example, the dosage may be 10 to 100 mg / kg for mice (body weight 20 g), 500 to 2000 mg / kg for rats (body weight 250 g), 40 to 400 mg / kg for dogs (body weight 7 kg), and 0.8 to 324.3 mg / kg for humans (body weight 60 kg), more specifically, 2 to 8 mg / kg. With such dosages, it is possible to exert an effect more safely. Such dosages may be on a one-time basis, and may be administered 1 to 7 times a week depending on the subject. In the case of oral administration, the dosage is 2 to 5 times the parenteral dosage. The pentapeptide of the present invention is administered mainly parenterally, for example, by local injection, intravenous or subcutaneous injection, intracerebroventricular or intrathecal administration, or nasal or rectal administration. It can also be administered orally if necessary.

[0060] The pentapeptide or composition of the present invention can be formulated together with pharma- ceutically acceptable additives to form injections, suppositories, powders, nasal drops, granules, tablets, and the like.

[0061] Pharmaceutically acceptable additives may be applied according to several factors well known to those skilled in the art, including, but not limited to, the specific bioactive agent used, its concentration, stability and intended bioavailability; the disease and illness or condition to be treated; the individual to be treated, age, size and general condition; the route used to administer the composition, such as nasal, oral, ocular, topical, transdermal and muscular. Pharmaceutically acceptable additives generally used for administration of bioactive agents other than oral routes include D5W (5% glucose in water), aqueous solutions containing dextrose and physiological salts within 5% by volume, and in the case of local injection into the lesion, various injectable hydrogels can be used to enhance the therapeutic effect and prolong its duration. Pharmaceutically acceptable additives may also include additional ingredients that can reinforce the stability of the active ingredient, such as preservatives and antioxidants. The pentapeptide or composition of the present invention may be prepared by any suitable method used in the relevant field, and may be formulated according to each disease or ingredient with reference to methods disclosed in well-known books on pharmaceutical science.

[0062] The pentapeptide of the present invention can be stored as a saline solution or, after the addition of mannitol or sorbitol, lyophilized into ampoules, which can be dissolved in saline, water for injection, etc., when used for administration.

[0063] The present invention also provides a method for suppressing anti-cancer drug resistance, a method for suppressing autophagy in tumor cells, a method for enhancing immunity, a method for activating immune cells, a method for promoting anti-tumor immune responses, and / or a method for treating and / or preventing cancer, which comprises administering the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof to an individual. The individual may require the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof. The individual may include or exclude humans. The individual may be a mammal. The pentapeptide or a pharma- ceutically acceptable salt thereof administered to the individual may be an effective amount of the pentapeptide or a pharma- ceutically acceptable salt thereof. The administration may be performed simultaneously or sequentially with other anti-cancer drugs. The individual may be an individual in which anti-cancer drug resistance has developed.

[0064] The present invention also provides use of the pentapeptide of the present invention or a pharma- ceutically acceptable salt thereof for the manufacture of a formulation of an autophagy inhibitor, an immune enhancer, an immune cell activator, an anti-tumor immune response promoter, and / or an anti-cancer agent.

[0065] Unless otherwise specified, the matters mentioned in the pentapeptide or pharma- ceutically acceptable salt, use, composition, and method of the present invention are applicable to each other to the same extent unless they are inconsistent with each other. Effect of the Invention

[0066] The pentapeptides according to the present invention are stable and effective in suppressing anti-cancer drug resistance, suppressing autophagy in tumor cells, enhancing immunity, activating immune cells, promoting anti-tumor immune responses, and / or anti-cancer. [Brief description of the drawings]

[0067] [Figure 1] 1 is a graph showing the results of stability experiments for Examples and Comparative Examples at a storage temperature of 25° C. [Diagram 2] 1 is a graph showing the results of stability experiments for Examples and Comparative Examples at a storage temperature of 25° C. [Diagram 3]1 is a graph showing the results of stability experiments for Examples and Comparative Examples at a storage temperature of 40° C. [Figure 4] 1 is a graph showing the results of stability experiments for Examples and Comparative Examples at a storage temperature of 40° C. [Diagram 5] 1 is a graph showing the results of tumor volume measurement according to the number of days after treatment in an animal experiment. [Figure 6] 1 is a graph showing the change in the expression level of cleaved PARP, a cell death marker, by treatment group in an animal experiment. [Figure 7] 1 is a graph showing the change in the expression level of LC3II, an autophagy marker, by treatment group in an animal experiment. [Figure 8] 1 is a graph showing the change in tumor volume over time after sample administration in an animal experiment. [Figure 9] 1 is a graph showing tumor weights by experimental group in an animal experiment. [Figure 10] 1 is a graph showing mouse survival rates over time after administration of a sample in an animal experiment. [Figure 11] 1 is a graph showing the survival rate of cancer cells by experimental group in an experiment to confirm an anticancer effect. [Figure 12] 1 is a graph showing the expression intensity of Granzyme B for each experimental group in an experiment to confirm an anticancer effect. [Figure 13] 1 is a graph showing the expression ratio of cleaved PARP to PARP in each experimental group in an experiment to confirm an anticancer effect. [Figure 14] 1 is a graph showing the expression level of LC3II relative to actin for each experimental group in an experiment to confirm an anticancer effect. [Figure 15] 1 is a graph showing the survival rate of PC3 cells according to the culture time for each experimental group in an experiment to confirm an anticancer effect. [Figure 16] 1 is a graph showing the survival rate of A549 cells according to culture time for each experimental group in an experiment to confirm an anticancer effect. [Figure 17] 1 is a graph showing the survival rate of MDA-MB-231 cells according to culture time for each experimental group in an experiment to confirm an anticancer effect. [Figure 18] 1 is a graph showing the survival rate of PC3 cells according to the culture time for each experimental group in an experiment to confirm an anticancer effect. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The present invention will be described in more detail below with reference to examples and manufacturing examples. However, the following examples and manufacturing examples are intended to illustrate the present invention, and the contents of the present invention are not limited to the following examples and manufacturing examples.

[0069] The reagents and materials used in the following examples were commercially available and of the highest quality. Unless otherwise specified, the materials used were purchased from Sigma-Aldrich.

[0070] Example 1: Preparation of N-terminal acetylated pentapeptide

[0071] A pentapeptide consisting of an acetylated N-terminus of a peptide consisting of the amino acid sequence (QLHLD) of SEQ ID NO: 1 was manufactured by Shanghai AmbioPharm, Inc. (China). Specifically, the starting material was H-Asp(OtBu)-2-ClTrt solid resin (substitution ratio: 0.63 to 0.67 mmol / g) and the standard fluorenylmethyloxycarbonyl solid phase peptide synthesis (Fmoc-SPPS) method was used to manufacture the peptide.

[0072] First, 12.50 g of the starting material, H-Asp(OtBu)-2-ClTrt peptide resin, was placed in a thoroughly dried reactor (hereinafter referred to as the first reactor for convenience), and N,N-dimethylformamide (DMF, 125 ml, 10 mL / g resin) was added and stirred for 20 minutes to swell the resin. The resin was then filtered, the filtrate was discarded, and the resin was washed twice with N,N-dimethylformamide (DMF, 75 ml, 6 mL / g resin).

[0073] In addition, Fmoc-Leu-OH (1.5 equivalents relative to the starting material equivalent) and hydroxybenzotriazole (HOBt, 1.5 equivalents relative to the starting material equivalent) were added to another reactor (hereinafter, for convenience, referred to as the second reactor), and N,N-dimethylformamide (DMF, 4.5 mL per gram of starting material) was added and dissolved, and then diisopropylethylamine (DIPEA, 1.5 equivalents relative to the starting material equivalent) was added. After cooling the reaction solution to 0-5°C, O-(benzotriazol-1yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1.5 equivalents relative to the starting material equivalent) was added to activate the amino acid derivative.

[0074] Dichloromethane (1.5 mL per 1 g of starting material) was added to the first reactor containing the starting peptide resin, and the activated amino acid derivative solution produced in the second reactor was slowly added while stirring, and the reaction solution was stirred for 2 hours at a temperature of 10 to 30°C. The reaction was confirmed using the ninhydrin (Kaiser) test method. After the reaction was completed, the peptide resin was filtered and washed once each with the solvents N,N-dimethylformamide (DMF) and methyl t-butyl ether (MTBE), and then the resin reaction product was washed twice with N,N-dimethylformamide (DMF) (75 mL, 6 mL / g resin).

[0075] The next fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction was carried out by treating the peptide reaction resin with a 5% piperidine mixed solution (v / v / w / v: 5% piperidine / 1.25% DBU / 1% HOBt / DMF) twice (75 ml, 6 mL / g resin, filtered after stirring for 10 minutes), and then filtering. The filtered peptide reaction resin was washed with the solvent N,N-dimethylformamide (DMF) x 2 times, methyl t-butyl ether (MTBE) x 2 times, and N,N-dimethylformamide (DMF) x 2 times (6 mL / g resin). The fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction was confirmed by the ninhydrin (Kaiser) test.

[0076] The Fmoc-Gln(Trt)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide resin was synthesized by repeating the coupling and fluorenylmethyloxycarbonyl (Fmoc) deprotection cycles for the #3, #4, and #5 amino acid derivatives, i.e., #3 reaction Fmoc-His(Trt)-OH, #4 reaction Fmoc-Leu-OH, and #5 reaction Fmoc-Gln(Trt)-OH, respectively, to sequentially attach amino acids to the peptide resin. However, in the case of the #3 Fmoc-His(Trt)-OH reaction, the coupling reaction was carried out from dichloromethane / N,N-dimethylformamide (DCM / DMF) to N-hydroxybenzotriazole / N,N'-diisopropylcarbodiimide.

[0077] The N-terminal fluorenylmethyloxycarbonyl (Fmoc)-group of Fmoc-Gln(Trt)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide resin was synthesized by adding 5% piperidine mixed solvent (v / v / w / v: 5% piperidine / 1.25% DBU / 1% HOBt / DMF), stirring, filtering and washing to remove the fluorenylmethyloxycarbonyl (Fmoc) group, and obtaining H-Gln(Trt)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide resin.

[0078] Then, the synthesized peptide resin was subjected to N-terminal acetylation reaction. Specifically, the synthesized peptide resin was added to a solution of dichloromethane / N,N-dimethylformamide (DCM / DMF) solvent (1 / 3 per gram of resin, v / v, 4-6 mL), acetic anhydride (1 equivalent relative to the starting material equivalent) and pyridine (10 equivalents relative to the starting material equivalent), and the mixture was stirred at room temperature for 2 hours to carry out the acetylation reaction. The reaction was confirmed to be complete using the ninhydrin (Kaiser) test method. The completed Ac-H-Gln(Trt)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide resin was washed with the solvents N,N-dimethylformamide (DMF) x 1, methyl t-butyl ether (MTBE) x 1, N,N-dimethylformamide (DMF) x 3, and methyl t-butyl ether (MTBE) x 3 (6 ml per gram of resin) and dried in vacuum at 26°C for about 44.8 hours to obtain 21.42 grams of peptide resin. The yield was 109.5% based on the weight increase relative to the starting material H-Asp(OtBu)-2-ClTrt resin.

[0079] In addition, a deprotection mixed solution of trifluoroacetic acid / triisopropylsilane / dichloromethane (TFA / TIS / DCM: 55 / 5 / 40, v / v / v, 45 ml, 10 mL per 1 g of peptide resin) was added to another reactor (hereinafter referred to as the third reactor for convenience), and the temperature was cooled to 0-5°C. The obtained peptide resin was slowly added to the cooled solution while maintaining the reaction solution temperature below 17°C (maximum temperature, 13.8-14.6°C). Next, the reaction solution was heated to 23-27°C and stirred at the same temperature for 90 minutes. The reaction solution was filtered to remove solid resin using a polytetrafluoroethylene (PTFE) filter, washed twice with trifluoroacetic acid (TFA) (1.0 mL x 2 times per gram of peptide resin) to recover all the filtrate, and the recovered filtrate was vacuum concentrated under reduced pressure at 30°C or less to remove 70-80% of the solvent. Pre-cooled methyl t-butyl ether (MTBE) solvent (approximately 10 times the volume of the evaporated residue) was added to the concentrated residue to produce the crude peptide as a solid precipitate. The solid precipitate was stirred at room temperature for 1 hour, filtered and washed five times with cooled methyl t-butyl ether (MTBE) solvent (50% of MTBE for precipitation). The filtered crude peptide was dried in vacuum at 24°C for approximately 30 hours to synthesize 5.74 g of crude peptide with a deprotected peptide yield of 98.4% and an HPLC purity of 91.3 to 97.5%.

[0080] For purification, 5.74 g of crude peptide was added to 20 ml of purified water, and aqueous ammonia (NH3·H2O, 50 mL) was slowly added dropwise to adjust the pH to 9.00 ± 0.10 to dissolve the crude peptide, and the solution was filtered to remove insoluble material. The crude peptide filtrate was loaded onto a Prep RP-HPLC column packed with C18 reversed-phase resin, and purification was performed using a water-acetonitrile mixture.

[0081] In order to remove the solvent from the purified fraction, the fraction was concentrated under reduced pressure at a temperature of 35-40°C to remove most of the acetonitrile (AN) solvent. The concentrated solution was stirred with a stirrer while slowly adding acetic acid (AcOH) dropwise at room temperature to adjust the pH to 4.00 ± 0.10, and the solution was stirred at the same temperature for 16 hours to crystallize the target peptide as a solid precipitate. The crystallized precipitate was filtered and washed with an acetic acid / water (AcOH / H2O, pH 4.0) solution. The washed solid was added to 50 ml of purified water to produce a homogenous slurry, which was then loaded into a freeze-drier tray for freeze-drying, and finally 2.8 g of Acetyl-QLHLD pentapeptide with an HPLC purity of 99.6% was obtained as a white or cream-white freeze-dried compound. The molecular weight of the synthesized pentapeptide was 666.71.

[0082] Comparative Example 1: Preparation of peptides

[0083] The peptide disclosed in International Publication No. WO2017 / 014604A1 was prepared as Comparative Example 1. That is, the peptide (QLHLD) of Comparative Example 1 was manufactured by AnyGen Co., Ltd. (Korea). Specifically, it was synthesized by fluorenylmethyloxycarbonyl solid phase peptide synthesis (Fmoc Solid Phase Peptide Synthesis). More specifically, the peptide was synthesized by a method in which the C-terminus of an amino acid was bound to a solid phase resin (Wang resin; Sigma-Aldrich, substitution rate 0.55 mmol / g) using a solid phase resin.

[0084] The amino acid derivative coupling reaction was carried out using O-(benzotriazole-1yl)-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU) and amino acid derivatives protected with tert-butyl and tert-butyloxycarbonyl groups. After the reaction was completed, deprotection and cleavage with resin were carried out at room temperature for 3 hours using a solution of trifluoroacetic acid and water in a ratio of 95:5 (v / v). The cleaved solution was filtered using a glass filter, and diethyl ether solvent was added to the filtrate to crystallize the crude peptide precipitate. The crude peptide precipitate was filtered, washed repeatedly with diethyl ether, and then dried in vacuum. The dried crude peptide was dissolved in a small amount of purified water and then purified by reverse-phase high performance liquid chromatography (RP-HPLC) using a C18 column (20 x 250 mm). The purified and separated peptide solution was checked for HPLC purity, and the filtrate that reached the target purity was used to synthesize peptide QLHLD using a freeze dryer. The HPLC purity of the synthesized peptide (Comparative Example 1) was 97.6%, and the molecular weight was 624.67.

[0085] <Experimental Example 1> Stability test

[0086] For Example 1 and Comparative Example 1, stability tests were carried out at different temperatures and pH levels.

[0087] The storage conditions for each sample are as shown in Table 1. The liquid state in Table 1 indicates that the peptide is completely dissolved in the buffer (1 mg / ml). [Table 1]

[0088] Stability tests for each peptide were performed by liquid chromatography purity analysis at different temperatures and pHs depending on the analysis time points (initial, 24 hours, 130 hours). The HPLC analysis conditions were as shown in Table 2, and the mobile phase used was 0.1M sodium dihydrogen phosphate (0.1M NaH2PO4) buffer and acetonitrile. The samples were analyzed by injecting 20 ul using an automatic sample injection device, the analysis column was Dikma Inspire C18 (4.6 x 250 mm, 5 μm, 12 nm), the analysis temperature was 40°C, and the UV wavelength was 215 nm. [Table 2]

[0089] From the HPLC analysis results, the relative purity was calculated for each storage condition for Comparative Example 1 and Example 1. The relative purity was calculated as a percentage by setting the initial purity of each sample to 100% and then calculating the relative purity according to the storage period. The results are shown in Table 3 and Figures 1 to 4. Figures 1 and 2 are graphs showing the results of stability experiments for the Examples and Comparative Examples at a storage temperature of 25°C {graph of the experimental results after storage for 24 hours (Figure 1) and graph of the experimental results after storage for 130 hours (Figure 2)}, and Figures 3 and 4 are graphs showing the results of stability experiments for the Examples and Comparative Examples at a storage temperature of 40°C {graph of the experimental results after storage for 24 hours (Figure 3) and graph of the experimental results after storage for 130 hours (Figure 4)}. In each graph, the x-axis represents the experimental group by pH, and the y-axis represents the relative purity (%). [Table 3]

[0090] As shown in Table 3 and Figures 1 to 4, Comparative Example 1 showed that its stability varied depending on the storage conditions, and in some cases, it was not easy to ensure stability. However, Example 1 showed that it was very stable regardless of the storage conditions. In particular, when stored at high temperature for a long period of time, Example 1 maintained high purity regardless of pH, unlike Comparative Example 1. Therefore, it can be said that the pentapeptide of the present invention shows remarkable effects that cannot be predicted from at least Comparative Example 1.

[0091] <Experimental Example 2> Confirmation of inhibition of resistance to anti-cancer chemotherapy drugs and anti-cancer effect

[0092] Using 4T1 cells that are resistant to paclitaxel, an anticancer chemotherapeutic agent, it was confirmed whether the peptide of Example 1 suppresses the resistance to anticancer drugs or not. It was also confirmed whether the peptide of Example 1 exhibits an anticancer effect or not.

[0093] 2-1. Confirmation of the effect of suppressing anticancer drug resistance

[0094] We used a syngeneic model in which 4T1 (ATCC, American Type Culture Collection), a mouse triple-negative breast cancer tumor cell line that exhibits paclitaxel resistance, was transplanted to examine changes in cell death and autophagy markers using Western blot analysis. 1 × 10 4T1 cells were transplanted into the fat pad of a Balb / c mouse (6 weeks old, female). 6 After cell / head transplantation, the tumor volume was 70 mm 3 Drug administration was started when the tumor volume reached 100 mg / kg. In the first week, paclitaxel was administered at 20 mg / kg on the first and second days, and the peptide of Example 1 (10, 100 mg / kg) was co-administered on the third, fifth, and seventh days. In addition, the same procedure was performed in the second and third weeks following the first week (n=3). Since the tumors grew very fast, the tumor volume was measured every day, and the tumors were autopsied the day after the third week of administration, and the changes in the cell death marker cleaved PARP and the autophagy marker LC3-II in the tumor were compared and analyzed using Western blotting.

[0095] The results of tumor volume measurement are shown in Figure 5. Figure 5 is a graph showing the results of tumor volume measurement according to the number of days elapsed since treatment in an animal experiment. In Figure 5, the x-axis represents the number of days elapsed since treatment, and the y-axis represents the change in tumor volume (mm 3 )

[0096] As shown in Figure 5, the tumor volume was slightly reduced in the paclitaxel (PTX) alone group compared to the control group. The group administered 10 mg / kg of the peptide of Example 1 in combination showed a 20% tumor volume reduction compared to the paclitaxel alone group, and the group administered 100 mg / kg in combination showed a 40% tumor volume reduction compared to the paclitaxel alone group.

[0097] Changes in cleaved PARP, a cell death marker, are shown in Figure 6, and changes in LC3-II, an autophagy marker, are shown in Figure 7. Figure 6 is a graph showing changes in the expression level of cleaved PARP, a cell death marker, by treatment group in an animal experiment, and Figure 7 is a graph showing changes in the expression level of LC3II, an autophagy marker, by treatment group in an animal experiment.

[0098] As shown in Figure 6, the expression level of cleaved PARP was increased by 2-fold and 3-fold in the group administered with peptide 10 and 100 mg / kg of Example 1 compared to the group administered with paclitaxel, and the expression level was statistically significant in the group administered with peptide 100 mg / kg of Example 1 (p<0.05). As shown in Figure 7, the expression level of LC3-II was increased by 2.4-fold in the group administered with paclitaxel alone compared to the control group, and decreased by 30% and 86% in the group administered with peptide 10 and 100 mg / kg of Example 1 compared to the group administered with paclitaxel alone, and the expression level was statistically significant in the group administered with peptide 100 mg / kg of Example 1 (p<0.05).

[0099] These results indicate that autophagy is reduced and resistance to the anticancer drug is suppressed when anticancer drugs and the peptide of Example 1 are administered in combination to tumor cells that exhibit resistance to anticancer drugs. It was also confirmed that the combined administration of the peptide of Example 1 improves the cell death efficacy of the anticancer drug and inhibits tumor growth.

[0100] 2-2. Confirmation of anticancer effects I

[0101] The following experiment was carried out to confirm whether the administration of the peptide of Example 1 after paclitaxel administration could inhibit tumor growth more than the group administered paclitaxel alone. 6 The cells / head were mixed with Matrigel and PBS at a ratio of 1:1, and 100 ul was injected into the right third fat pad of 7-week-old female athymic nude mice, resulting in an average tumor size of 80 mm 3 Drug administration begins from 0:00. Paclitaxel is administered at 20 mg / kg on days 0 and 1, for a total of two doses, and the peptide of Example 1 is administered at 100 mg / kg on days 2, 4, and 6, for a total of three doses. Tumor volume is measured three times a week for three weeks after the first dose, and tumor weight is measured after autopsy at the end of the experiment. Ten mice are used for each group.

[0102] As a result of measuring the tumor volume for each individual, in the group administered paclitaxel alone, the tumor volume steadily increased from one week after administration except for two individuals, and one individual's tumor disappeared on the 16th day (CR=10%). In the group administered paclitaxel in combination with the peptide of Example 1, the tumor volume decreased or was maintained until two weeks after the first administration, and two individuals' tumors disappeared on the 14th day (CR=20%).

[0103] The change in tumor volume over time for each group is shown in Figure 8, and the tumor weight on day 18 after the first administration for each group is shown in Figure 9. Figure 8 is a graph showing the change in tumor volume over time after administration of a sample in an animal experiment. The x-axis of Figure 8 represents the time (days) after administration, and the y-axis represents the tumor volume (mm3 9 is a graph showing tumor weights by experimental group in an animal experiment. The x-axis of FIG. 9 represents the experimental group, and the y-axis represents the tumor weight (mg).

[0104] As shown in Figure 8, the results of measuring the tumor volume 18 days after the initial administration showed a 33% decrease in the group administered paclitaxel alone, and a 48% decrease in the group administered paclitaxel in combination with the peptide of Example 1, compared to the control group. Also, as shown in Figure 9, the results of measuring the tumor weight 18 days after the initial administration showed a 34.5% decrease in the group administered paclitaxel alone, and a 51% decrease in the group administered paclitaxel in combination with the peptide of Example 1, compared to the control group.

[0105] In conclusion, the anticancer effect of the combined administration of paclitaxel and the peptide of Example 1 was confirmed, and the sustained effect of one cycle of drug treatment can be seen to last up to about 2 weeks.

[0106] 2-3. Confirmation of anticancer effects II

[0107] The following experiment was carried out to examine the survival rate of the group administered paclitaxel in combination with the peptide of Example 1, compared to the group administered paclitaxel alone. 6 The cells / head were mixed with Matrigel and PBS at a ratio of 1:1, and 100ul was injected into the right third fat pad of 7-week-old female BalB / c nude mice. The average tumor size was 70mm 3Drug administration begins from 20 mg / kg once a week, and when 48 hours have passed since paclitaxel administration, 100 mg / kg of the peptide of Example 1 is subcutaneously administered three times a week at 48-hour intervals. Each experimental group was administered for 12 weeks, and the survival of the experimental animals was observed every day. The results are shown in FIG. 10. FIG. 10 is a graph showing the survival rate over time after sample administration in an animal experiment. In FIG. 10, the x-axis represents the time (days) elapsed after administration, and the y-axis represents the survival rate (%). As shown in FIG. 10, the first dead individual occurred in the paclitaxel alone administration group on the 21st day of administration, and the first dead individual occurred in the peptide co-administration group of Example 1 on the 60th day of administration. After the experiment was completed, the paclitaxel alone administration group showed a survival rate of 30%, and the peptide co-administration group of Example 1 showed a survival rate of 50%.

[0108] These results show that the survival rate of cancer-affected individuals is higher when paclitaxel is administered in combination with the peptide of Example 1 compared to the group administered paclitaxel alone, and therefore the peptide of the present invention shows an anticancer effect.

[0109] It is also evident that the administration of an anticancer drug such as paclitaxel followed by the administration of the peptide of Example 1 also effectively exhibits an anticancer effect.

[0110] 2-4. Confirmation of anticancer effects III

[0111] The following experiment was carried out to examine the cell viability when doxorubicin, an anticancer chemotherapy drug, was administered in combination with the peptide of Example 1, in comparison with a group administered only doxorubicin. 3 The cells were seeded in 96 wells and treated with 20 nM doxorubicin and 300 uM of the peptide of Example 1 under each condition. After 24 hours of culture, the survival rate of EL4 cells was measured by adding CCK-8. The results are shown in Figure 11. Figure 11 is a graph showing the survival rate of EL4 cells according to the culture time for each experimental group in the experiment to confirm the anticancer effect. In Figure 11, the x-axis represents the culture time, and the y-axis represents the cell survival rate (%).

[0112] As shown in Figure 11, the survival rate of cancer cells was significantly decreased in the group treated with the peptide of Example 1 compared to the group treated with doxorubicin (Dox) alone (24 hours, p<0.05). These experimental results show that the pentapeptide of Example 1 promotes the death of tumor cells and exhibits an anticancer effect.

[0113] It was also found that the simultaneous administration of an anticancer drug such as doxorubicin and the peptide of Example 1 showed an effective anticancer effect.

[0114] <Experimental Example 3> Suppression of resistance to anti-cancer immunotherapy drugs and confirmation of anti-cancer effects

[0115] Using an immune checkpoint inhibitor that exhibits immune anticancer activity, it was confirmed whether the peptide of Example 1 exhibits suppression of resistance to anticancer immunotherapy agents. It was also confirmed whether the peptide of Example 1 exhibits an anticancer effect.

[0116] 3-1. Confirmation of inhibitory effect against resistance to anti-cancer immunotherapy drugs

[0117] The following experiment was conducted to confirm the mechanism of tumor suppression by combined treatment of anti-PD-L1, an immune checkpoint inhibitor, and the peptide of Example 1. First, prostate cancer cell line PC3 (ATCC) and NK92 cells (Immunotherapeutic Agent Research Center, Korea Institute of Bioscience and Biotechnology) were co-cultured. PC3 cells 7.8 × 10 4The cells were seeded into a 4-well chamber and cultured overnight to stabilize, and the same number of NK92 cells were additionally seeded and treated with 10ug / ml of anti-PD-L1 and 300uM of the peptide of Example 1 under each condition. After 12 hours, the NK92 cells were removed by washing, and only the tumor cells were fixed and the expression level of Granzyme B was confirmed using immunofluorescence staining. The expression level of Granzyme B was calculated as a relative ratio to the control group. The results are shown in Figure 12. Figure 12 is a graph showing the expression intensity of Granzyme B by experimental group in the experiment to confirm the anti-cancer effect, where the x-axis represents the experimental group and the y-axis represents the expression intensity (%) of Granzyme B relative to the control group.

[0118] As shown in Figure 12, the expression of Granzyme B in tumor cells was significantly increased in the group treated with the peptide of Example 1 compared to the group treated with anti-PD-L1 alone. These results suggest that the peptide of Example 1 can increase the expression of Granzyme B by activating immune cells such as NK92 cells, and can increase cell death in tumors by inducing a decrease in autophagy in tumor cells and inhibiting the degradation of Granzyme B that has penetrated into tumor cells.

[0119] In addition, the expression of the autophagy marker LC3II and the cell death marker cleaved PARP was confirmed by Western blotting. 6The cells were seeded in 6 wells, cultured overnight to stabilize, and the same number of NK92 cells were seeded and treated with 10ug / ml anti-PD-L1 and 300uM of the peptide of Example 1 under each condition. After a certain period of treatment, the NK92 cells were removed by washing, and proteins were isolated from the tumor cells to confirm the expression changes of LC3II (12h) and PARP (24h). The expression levels of each were calculated as a relative ratio to the control group. The results are shown in Figures 13 and 14. Figure 13 is a graph showing the expression level of cleaved PARP relative to PARP by experimental group in the experiment to confirm the anticancer effect, where the x-axis represents the experimental group and the y-axis represents the expression level of cleaved PARP relative to PARP (%). Figure 14 is a graph showing the expression level of LC3II relative to actin by experimental group in the experiment to confirm the anticancer effect, where the x-axis represents the experimental group and the y-axis represents the expression level of LC3II relative to actin (%).

[0120] As shown in Figures 13 and 14, the expression of LC3II was significantly decreased (P<0.001) and the expression of cleaved PARP was significantly increased (P<0.05) in the pentapeptide combination treatment group of Example 1 compared to the anti-PD-L1 single treatment group. This suggests that the pentapeptide combination treatment of Example 1 promotes immune cell-mediated lysis (e.g., natural killer cell-mediated lysis) and inhibits the autophagy of tumor cells, thereby suppressing resistance to anti-cancer immunotherapy drugs and promoting the death of tumor cells.

[0121] 3-2. Confirmation of anticancer effects I

[0122] The following experiment was carried out to confirm the tumor cell proliferation inhibitory effect of combined treatment of the immune checkpoint inhibitor anti-PD-L1 and the peptide of Example 1. First, 5 × 10 human prostate cancer cell line (PC3) 3 Cell, human lung cancer cell line {A549 (Korean Cell Line Bank)} 5 × 10 3 cell, human triple negative breast cancer cell line {MDA-MB-231(ATCC)}1×10 4The cells were seeded into 96-well plates and cultured overnight to stabilize. Then, 5 × 10 3 cells (in PC3 and MDA-MB-231, respectively) and 2.5 × 10 3 NK92 cells (in A549) were seeded with 10ug / ml of anti-PD-L1 and 300uM of the peptide of Example 1 under each condition. After co-culture {24 / 48 / 72 hours (for PC3, MDA-MB-231), 24 / 48 hours (for A549)}, NK92 cells were removed by washing, and cancer cell viability was measured by adding CCK-8. The results are shown in Figures 15 to 17. Figure 15 is a graph showing the PC3 cell viability by culture time in the experiment to confirm the anticancer effect, divided into experimental groups. In Figure 15, the x-axis represents the culture time, and the y-axis represents the cell viability (%). Figure 16 is a graph showing the A549 cell viability by culture time in the experiment to confirm the anticancer effect, divided into experimental groups. In Figure 16, the x-axis represents the culture time, and the y-axis represents the cell viability (%). 17 is a graph showing the MDA-MB-231 cell viability according to the culture time for each experimental group in the anticancer effect confirmation experiment, in which the x-axis represents the culture time and the y-axis represents the cell viability (%).

[0123] As shown in Figures 15 to 17, as the co-culture time increased, the survival rate of cancer cells was significantly decreased in the anti-PD-L1 combined treatment group with the peptide of Example 1 compared to the anti-PD-L1 alone treatment group. These experimental results indicate that the pentapeptide of Example 1 exhibits an anti-cancer effect by promoting the death of tumor cells.

[0124] 3-3. Confirmation of anticancer effects II

[0125] In addition, the following experiment was carried out to confirm the tumor cell proliferation inhibitory effect of combined treatment of anti-PD-1, another immune checkpoint inhibitor, and the peptide of Example 1. First, human prostate cancer cell line PC3 5×10 3Cells were seeded into 96-well plates and cultured overnight to stabilize. Then, 5 × 10 3 NK92 cells were additionally seeded and treated with 10ug / ml anti-PD-1 and 100 or 300uM peptide of Example 1 under each condition. After 48 / 72 hours of co-culture, NK92 cells were removed by washing and the viability of PC3 cells was measured by adding CCK-8. The results are shown in Figure 18. Figure 18 is a graph showing the PC3 cell viability according to the culture time in the experiment to confirm the anticancer effect, divided by experimental group. In Figure 18, the x-axis represents the culture time, and the y-axis represents the cell viability (%).

[0126] As shown in Figure 18, as the co-culture time increased, the survival rate of cancer cells was significantly decreased in the group treated with 300uM of peptide of Example 1 compared to the group treated with anti-PD-1 alone (P<0.05). These experimental results indicate that the pentapeptide of Example 1 promotes the death of tumor cells and exerts an anti-cancer effect.

[0127] It is also evident that simultaneous administration of an anti-cancer immunotherapeutic agent and the peptide of Example 1 effectively exerts an anti-cancer effect.

[0128] From the above experimental results, it is evident that the pentapeptide of Example 1 exhibits anti-cancer effects by suppressing the resistance of tumors to other anti-cancer agents (e.g., anti-cancer chemotherapeutic agents, anti-cancer immunotherapy agents) through the inhibition of autophagy in tumor cells, enhancement of immunity, activation of immune cells, and promotion of anti-tumor immune responses.

[0129] <Experimental Example 4> Toxicity test

[0130] The pentapeptide of Example 1 was subcutaneously administered to 10 male and female rats at doses of 500, 1000, and 2000 mg / kg every day for 4 weeks to investigate toxicity and conduct safety evaluation experiments. Specifically, general symptom observation, body weight measurement, food intake measurement, ophthalmologic examination, and urinalysis were performed, and after the observation period, hematological and blood biochemical examinations, organ weights, and macroscopic and histopathological examinations at autopsy were performed. As a result, no animals died in any of the test groups, and no abnormal findings related to the pentapeptide of Example 1 were observed. Taking the above results into consideration, it was determined that the No Observed Adverse Effect Level (NOEAL) was 2,000 mg / kg / day or more for both males and females when the pentapeptide of Example 1 was repeatedly administered subcutaneously to rats for 4 weeks.

[0131] In addition, the pentapeptide of Example 1 was repeatedly administered subcutaneously daily for four weeks at doses of 40, 120, and 400 mg / kg using three male and female beagle dogs per group. During the test period, general symptoms were observed, body weight and food intake were measured, ophthalmological examinations, electrocardiograms, urinalysis, hematological and blood biochemistry tests, and toxicokinetic tests were performed. Organ weights were measured at necropsy, and macroscopic examinations and histopathological examinations were performed on the necropsy animals. During the test period, no deaths were observed in any of the animals, including the control group. No abnormal changes due to administration of the pentapeptide of Example 1 were observed in body weight, food intake, ophthalmological examinations, electrocardiograms, urinalysis, hematological and blood biochemistry tests, macroscopic examinations at necropsy, or organ weights. As a result of the toxicokinetic test, the systemic exposure (AUC last ,C max ) increased almost in proportion to the administered dose, and no accumulation of the pentapeptide of Example 1 was observed. Therefore, the no observed adverse effect level (NOAEL) of the pentapeptide of Example 1 for male and female beagle dogs under the test conditions is determined to be 400 mg / kg / day.

[0132] From the above results, the human equivalent dose (HED) was converted based on the body surface area (see J Basic Clin Pharm. 2016 Mar; 7(2): 27-31), and the results are shown in Table 4. As shown in Table 4, the human equivalent dose of the pentapeptide of Example 1 was converted to 0.8 to 8.1 mg / kg on a mouse basis, 81 to 324.3 mg / kg on a rat basis, and 21.6 to 216 mg / kg on a dog (beagle) basis. Therefore, the pentapeptide of Example 1 can be administered to humans at 0.8 to 324.3 mg / kg. [Table 4]

[0133] From the above experimental results, it is found that the pentapeptide of Example 1 is safe and effective when administered parenterally (e.g., by injection) at a content of 0.8 to 2000 mg / kg per administration depending on the subject. Specifically, it can be administered by injection once at a dose of 10 to 100 mg / kg to mice (body weight 20 g), 500 to 2000 mg / kg to rats (body weight 250 g), 40 to 400 mg / kg to dogs (body weight 7 kg), and 0.8 to 324.3 mg / kg to humans (body weight 60 kg). In this case, it can be administered once to seven times a week depending on the subject. It is also found that the other anticancer drugs can be administered once a week at an effective amount (e.g., 2 to 20 mg / kg). It is also found that the above experimental results show that the pentapeptide of Example 1 is effective when administered simultaneously or sequentially with the administration of the other anticancer drugs. Specifically, the pentapeptide of Example 1 can be administered parenterally (e.g., by injection) to a human after administration of paclitaxel, or can be administered simultaneously with an anticancer immunotherapeutic agent or an anticancer chemotherapeutic agent other than paclitaxel. For example, when the peptide of Example 1 is administered in combination with paclitaxel to a human, the peptide of Example 1 can be administered parenterally (e.g., by injection) at a content of 0.8 to 324.3 mg / kg 24 hours later (preferably within 48±12 hours) after administration of 2 to 20 mg / kg of paclitaxel. In this case, each of paclitaxel and the peptide of Example 1 can be administered once a week. It can be said that by administering the pentapeptide of Example 1 in this way, it is possible to more effectively suppress the anticancer drug resistance caused by other anticancer drugs and exert an anticancer effect.

[0134] <Production Example 1> Production of injections

[0135] The peptide (raw drug) prepared in Example 1 is diluted 5 times (w / w based on the weight of the raw drug) with water for injection, and then 2N NaOH (2 equivalents based on the weight of the raw drug) is added to adjust the pH to 7.0 and dissolved. The solution is then sterile filtered through a filter (0.22 um) and freeze-dried to prepare the final drug product (600 mg / vial). [Industrial Applicability]

[0136] The pentapeptide according to the present invention is stable and is effective in suppressing anticancer drug resistance, suppressing autophagy in tumor cells, enhancing immunity, activating immune cells, promoting antitumor immune responses, and / or anticancer. Therefore, the present invention has industrial applicability.

Claims

1. A pentapeptide having an acetylated N-terminus of a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a pharma- ceutically acceptable salt thereof.

2. A pharmaceutical composition for suppressing resistance to anticancer drugs, comprising the pentapeptide according to claim 1 or a pharma- ceutical acceptable salt thereof as an active ingredient.

3. A pharmaceutical composition for inhibiting autophagy in tumor cells, comprising the pentapeptide according to claim 1 or a pharma- ceutically acceptable salt thereof as an active ingredient.

4. A pharmaceutical composition for promoting anti-tumor immune response, comprising the pentapeptide according to claim 1 or a pharma- ceutical acceptable salt thereof as an active ingredient.

5. 10. An anti-cancer pharmaceutical composition comprising the pentapeptide according to claim 1 or a pharma- ceutically acceptable salt thereof as an active ingredient.

6. The anticancer pharmaceutical composition according to claim 5, wherein the active ingredient is for administration in combination with another anticancer agent.

7. 10. A method for suppressing anticancer drug resistance, comprising administering the pentapeptide of claim 1 or a pharma- ceutically acceptable salt thereof to an individual other than a human.

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