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 pentapeptides, and more particularly to improved pentapeptides and their uses. [Background technology]

[0002] Research into techniques for treating diseases such as cancer with peptides is ongoing (see, for example, International Publication No. WO2017 / 014604A1). In the course of conducting 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 problem to be solved by the present invention is to provide a new use for 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 here will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0007] The present 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 unforeseen uses.

[0008] The present invention provides a pentapeptide in which the N-terminus of a peptide consisting of the amino acid sequence (QLHLD) of SEQ ID NO: 1 is acetylated, or a pharmaceutically 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 variants, which are naturally or artificially mutated without changing the main activity and in which a part of the peptide structure is mutated.

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

[0013] The present invention also provides pharmaceutical uses of the pentapeptide of the present invention or a pharmaceutically 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 anticancer uses. Anticancer uses include uses for treating or preventing cancer, where treatment encompasses the improvement or alleviation of symptoms, and prevention encompasses the inhibition of progression from a pre-disease stage to 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 group consisting of suppressing resistance to other anti-cancer drugs, assisting the anti-cancer effects of other anti-cancer drugs, suppressing autophagy in tumor cells, enhancing immunity, activating immune cells, promoting anti-tumor immune responses, promoting tumor cell death, and suppressing tumor cell proliferation.

[0017] The present invention provides a pharmaceutical composition for suppressing anticancer drug resistance, which comprises the pentapeptide of the present invention or a pharmaceutically 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 any other anticancer drug other than the active ingredient.

[0020] The anti-cancer agent can 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 inhibiting autophagy in tumor cells, which comprises the pentapeptide of the present invention or a pharmaceutically 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 the inhibition of protective autophagy.

[0026] The inhibition of autophagy can be achieved by suppressing the 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 within the cancer cells. As a result, organelles and breakdown products damaged by the anticancer drug are reused as metabolic products by the activated autophagy, thereby preventing cell death and promoting cancer cell proliferation, thereby conferring resistance to the anticancer drug. Therefore, it is also possible to suppress anticancer drug resistance 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 pharmaceutically 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 pharmaceutically acceptable salt thereof as an active ingredient.

[0032] The immune cells can be natural killer cells or cytotoxic T cells.

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

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

[0035] Activation of these immune cells can promote the 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 cause anti-tumor effects.

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

[0039] The active ingredient is intended for administration in combination with other anticancer drugs. It is administered in combination with other anticancer drugs. obtain.

[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 the 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 can 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 can 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 to suppress resistance or side effects caused by the other anti-cancer drug or to enhance the anti-cancer effect of the anti-cancer drug.

[0053] The pharmaceutical composition may be a pharmaceutical composition for inhibiting 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 pharmaceutically acceptable additive, and consists of the pentapeptide of the present invention or a pharmaceutically acceptable salt thereof and the additive.

[0056] The pentapeptide of the present invention can be prepared by acetylating the N-terminus of the peptide of SEQ ID NO: 1. First, the peptide of SEQ ID NO: 1 can be prepared by a method for forming a peptide bond. For example, it can be prepared by solution phase synthesis or solid phase synthesis. Examples of methods for forming a peptide bond include the acyl azide method, acyl halide method, acylimidazole method, carbodiimide method, phosphonium method, anhydride method, mixed anhydride method, oxidation-reduction method, and a method using Woodward's reagent K. Before the condensation reaction, carboxyl groups, amino groups, etc. that do not participate in the reaction can be protected, and carboxyl groups, etc. that participate in the condensation reaction can be activated by methods known in the art. Examples of groups that protect carboxyl groups include ester-forming groups such as methyl, t-butyl, aryl, pentafluorophenyl, benzyl, para-methoxybenzyl, or methoxyethoxymethyl. Examples of groups 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, and 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 include benzene, toluene, hexane, acetone, nitromethane, cyclohexane, ether, chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol, and ethanol, or a mixture thereof. The reaction temperature can be in the range of about -70°C to 100°C, which is commonly used for reactions, and more preferably in the range of -30°C to 30°C.The reaction for removing a peptide protecting group varies depending on the type of protecting group, but can be removed using an acid compound, a base compound, or a transition metal, which can cleave the protecting group without affecting the peptide bond. Protecting groups can be removed by acid treatment, for example, with hydrogen chloride, hydrogen bromide, hydrogen fluoride, acetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, trimethylchlorosilane, or a mixture thereof. When performing the reaction for removing a protecting group by acid treatment, an auxiliary agent such as anisole, phenol, or thioanisole can be added to promote the reaction. Protecting groups can also be removed by base treatment, for example, with ammonia, diethylamine, hydrazine, morpholine, N-methylpyrrolidine, piperidine, sodium carbonate, or a mixture of these bases. Protecting groups can also be removed by transition metal treatment, for example, with zinc, mercury, or palladium / hydrogen.

[0057] The N-terminal acyl substituent of a peptide can be formed by a known method using an activated acyl derivative. Specifically, an acetylation reagent can be used to acetylate the N-terminus of the peptide of SEQ ID NO: 1. Examples of the acetylation reagent include acetic anhydride and acyl halides (e.g., acetyl chloride, acetyl bromide, acetyl iodide). It is also possible to introduce an acetyl group into the N-terminus of a peptide by direct condensation with acetic acid.

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

[0059] The dosage of the pentapeptide of the present invention or a pharmaceutically acceptable salt thereof, when administered parenterally, varies depending on the subject and condition, but may be, for example, 0.8 to 2000 mg / kg. Within this range, 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, more specifically, 2 to 8 mg / kg for humans (body weight 60 kg). Such dosages can be more safely effective. These dosages may be administered on a single basis, one to seven times per week depending on the subject. For oral administration, the dosage is 2 to 5 times the parenteral dosage. The pentapeptides of the present invention are primarily administered 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 pharmaceutically acceptable additives into the form of injections, suppositories, powders, nasal drops, granules, tablets, and the like.

[0061] Pharmaceutically acceptable additives can be selected based on several factors well known to those skilled in the art, including, but not limited to, the specific physiologically active substance used, its concentration, stability, and intended bioavailability; the disease or condition to be treated; the individual receiving treatment, their age, size, and general condition; and the route used to administer the composition, such as nasal, oral, ocular, topical, transdermal, and intramuscular. Pharmaceutically acceptable additives typically used for administering physiologically active substances via routes other than oral administration include D5W (5% glucose in water), an aqueous solution containing dextrose and physiological salts at up to 5% by volume, and various injectable hydrogels can be used for local intralesional injection to enhance and prolong the therapeutic effect. Pharmaceutically acceptable additives can also contain additional ingredients, such as preservatives and antioxidants, that can enhance the stability of the active ingredient. The pentapeptides or compositions of the present invention can be prepared by any suitable method used in the relevant field, or can be formulated according to the disease or ingredient, referring to methods disclosed in well-known pharmaceutical textbooks.

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

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

[0064] The present invention also provides use of the pentapeptide of the present invention or a pharmaceutically acceptable salt thereof for the production 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, all references to the pentapeptide or pharmaceutically acceptable salt thereof, use, composition, and method of the present invention are applicable to each other to the same extent unless they contradict each other. [Effects 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 explanation 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. [Figure 2] 1 is a graph showing the results of stability experiments for Examples and Comparative Examples at a storage temperature of 25° C. [Figure 3]1 is a graph showing the results of a stability experiment for Examples and Comparative Examples at a storage temperature of 40° C. [Figure 4] 1 is a graph showing the results of a stability experiment for Examples and Comparative Examples at a storage temperature of 40° C. [Figure 5] 1 is a graph showing the results of tumor volume measurement according to the number of days elapsed since 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 the survival rate of mice over time after sample administration 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 anticancer effect. [Figure 12] 1 is a graph showing the expression intensity of Granzyme B for each experimental group in an experiment to confirm anticancer effect. [Figure 13] 1 is a graph showing the ratio of the expression level of cleaved PARP to PARP for each experimental group in an experiment to confirm 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 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 anticancer effect. [Figure 16] 1 is a graph showing the survival rate of A549 cells according to the culture time for each experimental group in an experiment to confirm anticancer effect. [Figure 17] 1 is a graph showing the survival rate of MDA-MB-231 cells according to the culture time for each experimental group in an experiment to confirm 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 anticancer effect. DETAILED DESCRIPTION OF THE INVENTION

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

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

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

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

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

[0073] In another reactor (hereafter referred to as the second reactor for convenience), Fmoc-Leu-OH (1.5 equivalents per starting material equivalent) and hydroxybenzotriazole (HOBt, 1.5 equivalents per starting material equivalent) were added, and N,N-dimethylformamide (DMF, 4.5 mL per gram of starting material) was added and dissolved. Diisopropylethylamine (DIPEA, 1.5 equivalents per starting material equivalent) was then added. The reaction solution was cooled to 0-5°C, and O-(benzotriazol-1yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1.5 equivalents per 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. While stirring, the activated amino acid derivative solution prepared in the second reactor was slowly added, and the mixture was stirred for 2 hours at a reaction temperature of 10-30°C. The reaction was confirmed using the ninhydrin (Kaiser) test. After the reaction was complete, the peptide resin was filtered and washed once each with the solvents N,N-dimethylformamide (DMF) and methyl t-butyl ether (MTBE), and then washed twice with N,N-dimethylformamide (DMF) (75 mL, 6 mL / g resin).

[0075] The next step, fluorenylmethyloxycarbonyl (Fmoc) deprotection, was carried out by treating the peptide-reacted resin with a 5% piperidine mixture (v / v / w / v: 5% piperidine / 1.25% DBU / 1% HOBt / DMF) twice (75 mL, 6 mL / g resin, stirred for 10 minutes, then filtered). The filtered peptide-reacted resin was washed twice with N,N-dimethylformamide (DMF), twice with methyl t-butyl ether (MTBE), and twice with N,N-dimethylformamide (DMF) (6 mL / g resin). The fluorenylmethyloxycarbonyl (Fmoc) deprotection was confirmed by a 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 Fmoc deprotection cycles for the amino acid derivatives #3, #4, and #5, i.e., Fmoc-His(Trt)-OH (#3), Fmoc-Leu-OH (#4), and Fmoc-Gln(Trt)-OH (#5). However, for the Fmoc-His(Trt)-OH (#3) reaction, the coupling reaction was carried out in dichloromethane / N,N-dimethylformamide (DCM / DMF) followed by 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 removed by adding a 5% piperidine mixed solvent (v / v / w / v: 5% piperidine / 1.25% DBU / 1% HOBt / DMF), stirring, filtering, and washing to synthesize H-Gln(Trt)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide resin.

[0078] The synthesized peptide resin was then subjected to N-terminal acetylation. Specifically, the synthesized peptide resin was added to a solution of dichloromethane / N,N-dimethylformamide (DCM / DMF) solvent (1 / 3, v / v, 4-6 mL per gram of resin), 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. 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) once, methyl t-butyl ether (MTBE) once, N,N-dimethylformamide (DMF) three times, and methyl t-butyl ether (MTBE) three times (6 ml per gram of resin) and dried under vacuum at 26 °C for approximately 44.8 hours to give 21.42 g of peptide resin. The yield was 109.5% based on the weight gain relative to the starting H-Asp(OtBu)-2-ClTrt resin.

[0079] Another reactor (hereafter referred to as the third reactor for convenience) was charged with a deprotection mixture solution of trifluoroacetic acid / triisopropylsilane / dichloromethane (TFA / TIS / DCM: 55 / 5 / 40, v / v / v, 45 mL, 10 mL per gram of peptide resin) and cooled to 0-5°C. The resulting peptide resin was slowly added to the cooled solution while maintaining the reaction temperature below 17°C (maximum temperature, 13.8-14.6°C). The reaction mixture was then heated to 23-27°C and stirred at this temperature for 90 min. The reaction mixture was filtered using a polytetrafluoroethylene (PTFE) filter, washed twice with trifluoroacetic acid (TFA) (1.0 mL x 2 per gram of peptide resin), and the filtrate was completely recovered. The recovered filtrate was then concentrated under reduced pressure at 30°C or below to remove 70-80% of the solvent. Pre-chilled methyl t-butyl ether (MTBE) solvent (approximately 10 times the volume of the evaporation residue) was added to the concentrated residue to produce the crude peptide as a solid precipitate. The resulting solid precipitate was stirred at room temperature for 1 hour, filtered, and washed five times with chilled methyl t-butyl ether (MTBE) solvent (50% MTBE for precipitation). The filtered crude peptide was dried in vacuo at 24 °C for approximately 30 hours to synthesize 5.74 g of crude peptide with a 98.4% deprotected peptide yield 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. The solution was then filtered to remove insoluble material. The crude peptide filtrate was loaded onto a Prep RP-HPLC column packed with C18 reverse-phase resin, and purification was performed using a water-acetonitrile mixture.

[0081] The purified fractions were concentrated under reduced pressure at 35-40°C to remove most of the acetonitrile (AN) solvent. The concentrated solution was stirred with a mechanical stirrer, and the pH was adjusted to 4.00 ± 0.10 by slowly adding acetic acid (AcOH) dropwise at room temperature. 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 / HO, pH 4.0) solution. The washed solid was added to 50 mL of purified water to produce a homogeneous slurry, which was then loaded into a freeze-dryer tray for lyophilization. Finally, 2.8 g of acetyl-QLHLD pentapeptide with an HPLC purity of 99.6% was obtained as a white or cream-white lyophilized compound. The molecular weight of the synthesized pentapeptide was 666.71.

[0082] <Comparative Example 1> Preparation of peptide

[0083] The peptide disclosed in International Publication No. WO 2017 / 014604 A1 was prepared as Comparative Example 1. That is, the peptide (QLHLD) of Comparative Example 1 was produced 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 attached to a solid-phase resin (Wang resin; Sigma-Aldrich, substitution rate 0.55 mmol / g).

[0084] The amino acid derivative coupling reaction was performed 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 resin cleavage were carried out using a 95:5 (v / v) mixture of trifluoroacetic acid and water at room temperature for 3 hours. The cleaved solution was filtered through a glass filter, and diethyl ether 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 under vacuum. The dried crude peptide was dissolved in a small amount of purified water and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (20 x 250 mm). The purified and separated peptide solution was checked for purity by HPLC, 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 testing for each peptide was performed by liquid chromatography purity analysis at different temperatures and pHs depending on the analysis time point (initial, 24 hours, and 130 hours). The HPLC analysis conditions are shown in Table 2, with the mobile phase consisting of 0.1 M sodium dihydrogen phosphate (0.1 M NaH2PO4) buffer and acetonitrile. Samples were analyzed by injecting 20 μl of each sample using an automatic sample injection system. The analytical column was a Dikma Inspire C18 (4.6 × 250 mm, 5 μm, 12 nm), the analytical temperature was 40°C, and the UV wavelength was 215 nm. [Table 2]

[0089] From the HPLC analysis results, the relative purity of Comparative Example 1 and Example 1 was calculated for each storage condition. The relative purity was calculated as a percentage, with the initial purity of each sample set at 100%, and the relative purity was calculated over time. 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 Examples and Comparative Examples at a storage temperature of 25°C {graph of the experimental results after 24 hours of storage (Figure 1) and the experimental results after 130 hours of storage (Figure 2)}. Figures 3 and 4 are graphs showing the results of stability experiments for Examples and Comparative Examples at a storage temperature of 40°C {graph of the experimental results after 24 hours of storage (Figure 3) and the experimental results after 130 hours of storage (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, the stability of Comparative Example 1 varied depending on the storage conditions, and in some cases, it was difficult to ensure stability. However, Example 1 was shown to be very stable regardless of the storage conditions. In particular, when stored at high temperatures for a long period of time, Example 1 maintained high purity regardless of the pH, unlike Comparative Example 1. Therefore, it can be said that the pentapeptide of the present invention exhibits significant effects that cannot be predicted from at least Comparative Example 1.

[0091] <Experimental Example 2> Confirmation of suppression of anti-cancer chemotherapy drug resistance 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 anticancer drug resistance and whether the peptide of Example 1 exhibits an anticancer effect.

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

[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 into the fat pad of Balb / c mice (6-week-old, female) to examine changes in cell death and autophagy markers using Western blot analysis. 4T1 cells (1 × 10) were transplanted into the fat pad of the mice. 6 After cell / head transplantation, the tumor volume was 70mm 3 Drug administration began when the tumor volume reached 100 mg / kg. In the first week, paclitaxel was administered at 20 mg / kg on days 1 and 2, and the peptide of Example 1 (10 and 100 mg / kg) was co-administered on days 3, 5, and 7. The same administration was performed for the second and third weeks following the first week (n=3). Because tumor growth was very rapid, tumor volume was measured daily, and the mice were necropsied the day after the third week of administration. Changes in the tumor cell death marker cleaved PARP and the autophagy marker LC3-II were analyzed by 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 animal experiments. 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% reduction in tumor volume compared to the paclitaxel alone group, and the group administered 100 mg / kg in combination showed a 40% reduction in tumor volume 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 animal experiments, and Figure 7 is a graph showing changes in the expression level of LC3II, an autophagy marker, by treatment group in animal experiments.

[0098] As shown in Figure 6, the expression level of cleaved PARP was increased by 2-fold and 3-fold in the groups administered with 100 mg / kg of peptides from Example 1 compared to the paclitaxel group, with statistical significance shown in the group administered with 100 mg / kg of peptides from 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%, respectively, in the groups administered with 100 mg / kg of peptides from Example 1 compared to the paclitaxel alone group, with statistical significance shown in the group administered with 100 mg / kg of peptides from Example 1 (p<0.05).

[0099] These results indicate that autophagy is reduced and resistance to anticancer drugs is suppressed when anticancer drugs and the peptide of Example 1 are co-administered to tumor cells resistant to anticancer drugs. Furthermore, it was confirmed that co-administration of the peptide of Example 1 improves the cell death efficacy of anticancer drugs and inhibits tumor growth.

[0100] 2-2. Confirmation of anti-cancer 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 effectively than the group administered paclitaxel alone. Human triple-negative breast cancer cell line MDA-MB-231 (ATCC, American Type Culture Collection) 5 x 10 6 The cells / head were mixed with Matrigel and PBS at a ratio of 1:1, and 100 μl of the mixture 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 began from 0:00. Paclitaxel was administered at 20 mg / kg on days 0 and 1, for a total of two doses, and the peptide of Example 1 was administered at 100 mg / kg on days 2, 4, and 6, for a total of three doses. Tumor volume was measured three times a week for three weeks after the first dose, and tumor weight was measured at autopsy after the experiment. Ten mice were used for each group.

[0102] As a result of measuring tumor volume for each individual, in the group administered paclitaxel alone, tumor volume steadily increased from one week after administration in all but two animals, and the tumor disappeared in one animal on day 16 (CR=10%). In the group administered paclitaxel in combination with the peptide of Example 1, tumor volume decreased or was maintained until two weeks after the first administration, and the tumor disappeared in two animals on day 14 (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 initial administration for each group is shown in Figure 9. Figure 8 is a graph showing the change in tumor volume over time after sample administration in an animal experiment. The x-axis of Figure 8 represents the time (days) after administration, and the y-axis represents tumor volume (mm3 9 is a graph showing tumor weights by experimental group in animal experiments. The x-axis of FIG. 9 represents experimental group, and the y-axis represents tumor weight (mg).

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

[0105] In conclusion, the anti-cancer effect of 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 for up to about 2 weeks.

[0106] 2-3. Confirmation of anti-cancer effects II

[0107] The following experiment was carried out to examine the survival rate of a group administered paclitaxel in combination with the peptide of Example 1, compared to a group administered paclitaxel alone. 6 The cells / head were mixed with Matrigel and PBS at a ratio of 1:1, and 100 μl of the mixture was injected into the right third fat pad of 7-week-old female BalB / c nude mice. The average tumor size was 70 mm 3Drug administration began from 10:00 AM. Paclitaxel was administered at 20 mg / kg once a week, and 48 hours after paclitaxel administration, 100 mg / kg of the peptide of Example 1 was administered subcutaneously 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 daily. The results are shown in Figure 10. Figure 10 is a graph showing the survival rate over time after sample administration in the animal experiment. In Figure 10, the x-axis represents the time (days) elapsed since administration, and the y-axis represents the survival rate (%). As shown in Figure 10, the first death occurred in the paclitaxel alone group on day 21 of administration, and the first death occurred in the group administered in combination with the peptide of Example 1 on day 60 of administration. After the experiment was completed, the paclitaxel alone group showed a survival rate of 30%, while the group administered in combination with the peptide of Example 1 showed a survival rate of 50%.

[0108] These results demonstrate that the combined administration of paclitaxel and the peptide of Example 1 results in a higher survival rate for cancer-affected individuals than the group administered paclitaxel alone, demonstrating that the peptide of the present invention exhibits anti-cancer effects.

[0109] It is also clear that an effective anti-cancer effect can be achieved by administering the peptide of Example 1 sequentially after administering an anti-cancer drug such as paclitaxel.

[0110] 2-4. Confirmation of anti-cancer effects III

[0111] The following experiment was carried out to examine the cell viability in a group administered with the anti-cancer chemotherapeutic agent doxorubicin in combination with the peptide of Example 1, compared to a group administered with doxorubicin alone. 3 Cells were seeded into 96-well plates and treated with 20 nM doxorubicin and 300 μM of the peptide from Example 1 under each condition. After 24 hours of culture, the viability of EL4 cells was measured by adding CCK-8. The results are shown in Figure 11. Figure 11 is a graph showing the EL4 cell viability as a function of culture time for each experimental group in an experiment to confirm anticancer effects. In Figure 11, the x-axis represents the culture time, and the y-axis represents cell viability (%).

[0112] As shown in Figure 11, the survival rate of cancer cells was significantly reduced 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 demonstrate that the pentapeptide of Example 1 promotes tumor cell death and exhibits anti-cancer effects.

[0113] It is also evident that simultaneous administration of the peptide of Example 1 together with an anticancer agent such as doxorubicin effectively exhibits anticancer effects.

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

[0115] Using an immune checkpoint inhibitor that exhibits immune anticancer activity, it was confirmed whether the peptide of Example 1 suppresses resistance to anticancer immunotherapy agents and whether the peptide of Example 1 exhibits anticancer effects.

[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 the immune checkpoint inhibitor anti-PD-L1 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. 7.8 × 10 PC3 cells were cultured at 47 °C. 4The cells were seeded into a 4-well chamber and cultured overnight to stabilize. An equal number of NK92 cells were then seeded and treated with 10 μg / ml of anti-PD-L1 and 300 μM of the peptide from Example 1 under each condition. After 12 hours, the NK92 cells were removed by washing, and the tumor cells were fixed and examined for Granzyme B expression levels using immunofluorescence staining. Granzyme B expression levels were calculated as a relative percentage to the control group. The results are shown in Figure 12. Figure 12 is a graph showing the Granzyme B expression levels by experimental group in an experiment to confirm anti-cancer effect. The x-axis represents the experimental group, and the y-axis represents the Granzyme B expression level (%) relative to the control group.

[0118] As shown in Figure 12, Granzyme B expression in tumor cells was significantly increased in the anti-PD-L1 combination treatment group compared to the anti-PD-L1 alone treatment group. These results suggest that the peptide of Example 1 activates immune cells such as NK92 cells to increase Granzyme B expression, and induces a decrease in autophagy in tumor cells, inhibiting the degradation of Granzyme B that has penetrated into tumor cells, thereby increasing intratumoral cell death.

[0119] Western blotting was also used to confirm the expression of the autophagy marker LC3II and the cell death marker cleaved PARP. 6The cells were seeded into 6 wells and cultured overnight to stabilize. An equal number of NK92 cells were then seeded and treated with 10 μg / ml of anti-PD-L1 and 300 μM of the peptide from Example 1 under each condition. After a set period of treatment, the NK92 cells were removed by washing, and proteins were isolated from the tumor cells to examine changes in LC3II (12 h) and PARP (24 h) expression. The expression levels were calculated relative to the control group. The results are shown in Figures 13 and 14. Figure 13 is a graph showing the expression levels of cleaved PARP relative to PARP by experimental group in the anti-cancer effect confirmation experiment. 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 levels of LC3II relative to actin by experimental group in the anti-cancer effect confirmation experiment. 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, LC3II expression was significantly decreased (P<0.001) and cleaved PARP expression was significantly increased (P<0.05) in the pentapeptide combination treatment group of Example 1 compared to the anti-PD-L1 monotherapy 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 autophagy in tumor cells, thereby suppressing resistance to anti-cancer immunotherapy drugs and promoting tumor cell death.

[0121] 3-2. Confirmation of anti-cancer 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 lines (PC3) were treated with PD-L1. 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. 3 cells (in PC3 and MDA-MB-231, respectively) and 2.5 × 10 3 NK92 cells (in A549) were seeded with 10 μg / ml of anti-PD-L1 and 300 μM of the peptide from Example 1 under each condition. After co-culture (24 / 48 / 72 hours for PC3 and MDA-MB-231, or 24 / 48 hours for A549), the 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 as a function of culture time for each experimental group in an experiment to confirm anticancer effect. In Figure 15, the x-axis represents culture time, and the y-axis represents cell viability (%). Figure 16 is a graph showing the A549 cell viability as a function of culture time for each experimental group in an experiment to confirm anticancer effect. In Figure 16, the x-axis represents culture time, and the y-axis represents cell viability (%). 17 is a graph showing the MDA-MB-231 cell viability as a function of culture time for each experimental group in an experiment to confirm anticancer effect, in which the x-axis represents culture time and the y-axis represents cell viability (%).

[0123] As shown in Figures 15 to 17, as the co-culture time increased, the survival rate of cancer cells in the anti-PD-L1 co-treatment group was significantly reduced compared to the anti-PD-L1 mono-treatment group in the peptide co-treatment group of Example 1. These experimental results demonstrate that the pentapeptide of Example 1 exhibits anti-cancer effects by promoting tumor cell death.

[0124] 3-3. Confirmation of anti-cancer 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. 3 NK92 cells were seeded on the cells, and the cells were treated with 10 μg / ml of anti-PD-1 and 100 or 300 μM of the peptide of Example 1 under each condition. After 48 or 72 hours of co-culture, the NK92 cells were removed by washing, and the viability of the 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 as a function of culture time for each experimental group in an experiment to confirm anticancer effects. In Figure 18, the x-axis represents culture time, and the y-axis represents cell viability (%).

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

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

[0128] The above experimental results demonstrate that the pentapeptide of Example 1 exhibits anticancer effects by suppressing tumor resistance to other anticancer agents (e.g., anticancer chemotherapeutic agents, anticancer immunotherapeutic agents) through the suppression of autophagy in tumor cells, enhancement of immunity, activation of immune cells, and promotion of antitumor immune responses.

[0129] <Experimental Example 4> Toxicity test

[0130] Toxicity and safety evaluation experiments were conducted by subcutaneously administering the pentapeptide of Example 1 to 10 male and female rats at doses of 500, 1000, and 2000 mg / kg daily for four weeks. Specifically, general observations, body weight measurements, food intake measurements, ophthalmologic examinations, and urinalysis were performed. After the observation period, hematological and blood biochemistry tests, organ weights, and macroscopic and histopathological examinations were performed at necropsy. As a result, no animals died in any test group, and no abnormal findings related to the pentapeptide of Example 1 were observed. Based on these results, the no observed adverse effect level (NOEAL) of the pentapeptide of Example 1 administered subcutaneously to rats repeatedly for four weeks was determined to be 2000 mg / kg / day or higher for both males and females.

[0131] In addition, three male and female beagle dogs per group were subcutaneously administered the pentapeptide of Example 1 at doses of 40, 120, and 400 mg / kg daily for four weeks. During the test period, general symptoms were observed, body weight and food intake were measured, ophthalmological examinations were performed, electrocardiograms were performed, urinalysis was performed, hematological and blood biochemistry tests were performed, and toxicokinetic tests were conducted. Organ weights were measured at necropsy, and macroscopic examinations and histopathological tests were also conducted on the necropsy animals. No deaths were observed in any of the animals, male or female, including the control group, during the test period. No abnormal changes due to the 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 conditions of this test is determined to be 400 mg / kg / day.

[0132] From the above results, the human equivalent dose (HED) was calculated based on 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 calculated to be 0.8 to 8.1 mg / kg for mice, 81 to 324.3 mg / kg for rats, and 21.6 to 216 mg / kg for dogs (beagles). Therefore, the pentapeptide of Example 1 can be administered to humans at 0.8 to 324.3 mg / kg. [Table 4]

[0133] These experimental results demonstrate that the pentapeptide of Example 1 is safe and effective when administered parenterally (e.g., by injection) at a single dose of 0.8 to 2000 mg / kg depending on the subject. Specifically, it can be administered by single injection 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 one to seven times per week depending on the subject. It also demonstrates that other anticancer agents can be administered once per week at an effective amount (e.g., 2 to 20 mg / kg). These experimental results also demonstrate that the pentapeptide of Example 1 is effective when administered simultaneously or sequentially with other anticancer agents. Specifically, the pentapeptide of Example 1 can be administered parenterally (e.g., by injection) to a human subject 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 subject, paclitaxel can be administered at 2 to 20 mg / kg, followed by parenteral administration (e.g., by injection) of the peptide of Example 1 at a content of 0.8 to 324.3 mg / kg 24 hours later (preferably within 48±12 hours). In this case, paclitaxel and the peptide of Example 1 can each be administered once a week. It can be said that administering the pentapeptide of Example 1 in this manner more effectively suppresses anticancer drug resistance caused by other anticancer drugs and exerts 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 (600 mg / vial). [Industrial Applicability]

[0136] The pentapeptide according to the present invention is stable and effective in suppressing anticancer drug resistance, inhibiting 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 in which the N-terminus of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is acetylated, or a pharmaceutically acceptable salt thereof.

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

3. A pharmaceutical composition for inhibiting autophagy in tumor cells, comprising the pentapeptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

4. A pharmaceutical composition for promoting anti-tumor immune responses, comprising the pentapeptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

5. 10. An anti-cancer pharmaceutical composition comprising the pentapeptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

6. The anti-cancer pharmaceutical composition according to claim 5, wherein the active ingredient is for administration in combination with another anti-cancer drug.

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

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