Peptides with antitumour activity

Abstract

The invention relates to organic chemistry, pharmacology and medicine and concerns novel peptides characterized in that they exhibit antitumour activity, which can be used, in particular, for the prophylaxis and treatment of neoplastic diseases in a subject. In addition, the present invention also includes the use of the claimed peptides for the treatment of neoplastic diseases, as well as pharmaceutical compositions for the treatment of neoplastic diseases which contain the claimed peptides.

Description

[0001] Peptides with antitumor activity

[0002] Field of technology

[0003] The invention relates to the chemistry of organic compounds, pharmacology and medicine and concerns new peptides characterized by antitumor activity, which, in particular, can be used for the prevention and treatment of tumor diseases in a subject.

[0004] State of the art

[0005] Cancer is one of the most common diseases worldwide, and currently widely used treatment methods include chemotherapy, surgery, radiation therapy, hematopoietic stem cell transplantation, and immunotherapy. Tumors pose a significant threat to human health and remain one of the most dangerous diseases with a high mortality rate. Therefore, the discovery of new, highly effective, and high-quality anticancer drugs is an important topic of research worldwide. Research into the molecular mechanisms of cancer is actively underway. Despite the progress made, many of the currently developed treatments rely on surgical intervention.

[0006] Peptides are increasingly being developed for use as therapeutic agents for the treatment of many diseases, including cancer. Therapeutic peptides offer the advantages of target specificity and low toxicity. Peptides can induce cell death through numerous mechanisms, including membrane disruption and subsequent necrosis, apoptosis, inhibition of tumor angiogenesis, immune regulation, disruption of cellular signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways [Caroline M. Li et al / Novel Peptide Therapeutic Approaches for Cancer Treatment / Cells. 2021 Nov; 10(11): 2908].

[0007] The prior art discloses de novo developed peptides and modified peptides with antitumor activity (CN109071622, WO2023277514,

[0008] WO2021112662). A number of antitumor peptides are currently in clinical trials [Wararat Chiangjong et al / Anticancer peptide: Physicochemical property, functional aspect and trend in clinical application (Review) / Int J Oncol. 2020 Sep; 57(3): 678–696].

[0009] Thus, attempts to create new peptide molecules with pharmacological activity seem appropriate and relevant.

[0010] Disclosure of the invention The objective of the present invention is to develop and create new effective antitumor agents that are promising for use in clinical practice for the treatment and / or prevention of antitumor diseases.

[0011] The technical result of the invention is the development and production of new peptide compounds that have an antitumor (cytotoxic and antiproliferative) effect and are promising for use in the treatment of tumor diseases in a subject, in particular in the treatment of melanoma, metastatic adenocarcinoma of the pancreas, and lung carcinoma.

[0012] The specified technical result is achieved by developing and creating a peptide with antitumor activity, having the sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 13, 14 or 15, or its pharmaceutically acceptable salt.

[0013] The present invention also includes the use of the peptide of the invention as an antitumor agent.

[0014] The subject of the present invention is also the use of a peptide according to the invention for the production of a pharmaceutical composition with antitumor activity for the treatment and / or prevention of a tumor disease in a subject.

[0015] In particular embodiments of the invention, the disease is melanoma, metastatic pancreatic adenocarcinoma, lung carcinoma, breast cancer, colorectal cancer, prostate cancer, liver cancer, esophageal cancer.

[0016] In particular embodiments of the invention, the subject is a human being.

[0017] The subject of the present invention is also a pharmaceutical composition with antitumor activity for the treatment and / or prevention of a tumor disease in a subject, containing an effective amount of a peptide according to the invention and at least one pharmaceutically acceptable excipient.

[0018] In particular embodiments of the invention, the pharmaceutically acceptable excipient is a carrier, filler and / or solvent.

[0019] In particular embodiments of the invention, the disease is melanoma, metastatic pancreatic adenocarcinoma, lung carcinoma, breast cancer, colorectal cancer, prostate cancer, liver cancer, esophageal cancer.

[0020] In particular embodiments of the invention, the subject is a human being.

[0021] The present invention also includes the production of compounds (peptides) according to the invention. The present invention also includes a method for the treatment and / or prevention of a tumor disease in a subject by administering to the subject a peptide according to the invention or a pharmaceutical composition according to the invention.

[0022] The present invention also includes a method for blocking and / or inhibiting and / or suppressing tumor growth using a peptide of the invention or pharmaceutical compositions of the invention.

[0023] Detailed disclosure of the invention

[0024] Definitions and terms

[0025] For a better understanding of the present invention, certain terms used in this description of the invention are provided below. The following definitions apply throughout this document unless otherwise specified.

[0026] In the description of this invention, the terms "includes" and "comprising" are interpreted to mean "includes, among other things." These terms are not intended to be construed as meaning "consists solely of."

[0027] The term "and / or" means one, more than one, or all of the listed elements.

[0028] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0029] The term "optional" or "optional" or "optionally" as used herein means that the subsequently described event or circumstance may, but does not necessarily, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0030] The term "amino acid" also refers to naturally occurring amino acids (including both L-amino acids and D-amino acids). Amino acids are designated by standard abbreviations, for example: arginine (Arg; R), leucine (Leu; L), lysine (Lys; K), phenylalanine (Phe; F), tryptophan (Trp; W), isoleucine (He; I), valine (Vai; V), proline (Pro; P), tyrosine (Tyr; Y), methionine (Met; M), serine (Ser; S), alanine (Ala; A), aspartic acid (Asp; D), glycine (Gly; G).

[0031] The terms "isolated" and "purified," when applied to a substance (e.g., a peptide, etc.), indicate that the substance is substantially free of at least one substance that may also be present in the natural source. Thus, an isolated or purified peptide refers to a peptide that is substantially free of other cellular material, such as carbohydrate, lipid, and other contaminating proteins, from the cell or tissue source from which the peptide is obtained. If a peptide is chemically synthesized, an isolated or purified peptide refers to a peptide that is substantially free of the precursor substance or other chemical. The phrase "substantially free of cellular material" includes peptide production in which the peptide is separated from cellular components of the cells from which the peptide was isolated or where it was recombinantly produced.Thus, a peptide that is substantially free of cellular material includes peptide preparations that contain less than about 30%, 20%, 10%, or 5%, 3%, 2%, or 1% (based on dry weight) of other cellular materials.

[0032] If the peptide is produced recombinantly, an isolated or purified peptide that is substantially free of culture medium, and a peptide that is substantially free of culture medium, includes peptide preparations that contain culture medium less than about 20%, 10%, or 5%, 3%, 2%, or 1% (based on dry weight) of the volume of the peptide preparation.

[0033] Alternatively, if the peptide is synthesized chemically, the isolated or purified peptide is substantially free of a precursor substance or other chemical, and a peptide that is substantially free of a precursor substance or other chemical includes peptide preparations that contain a precursor substance or other chemicals in amounts less than about 30%, 20%, 10%, 5%, 3%, 2%, or 1% (based on dry weight) of the volume of the peptide preparation. In a preferred embodiment, the peptides of the present invention are isolated or purified.

[0034] The compounds that form the essence of this invention may exist in radioisotope-labeled form, i.e., said compounds may contain one or more atoms whose atomic mass or mass number differs from the atomic mass or mass number of the most common natural isotopes. Radioisotopes of hydrogen, carbon, phosphorus, and chlorine include 3H, 14 WITH, 32 R, 35 S, and 36 C1, respectively. Compounds of the present invention that contain such radioisotopes and / or other radioisotopes of other atoms are within the scope of the present invention. Tritiated, i.e. 3 H and carbon, i.e. 14 Radioisotopes are particularly preferred due to their ease of preparation and detection.

[0035] The radiolabeled compounds of the present invention can be prepared using methods well known to those skilled in the art. Labeled compounds can be prepared using the procedures described herein by simply replacing unlabeled reagents with the appropriate labeled reagents.

[0036] The compounds of the present invention may exist in free form or, if desired, as a pharmaceutically acceptable salt or other derivative. The term "pharmaceutically acceptable salt" as used herein refers to those salts that, within the scope of medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, etc., and that meet a reasonable benefit-risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates, and other types of compounds are well known in the medical field. Salts can be prepared in situ during the isolation or purification of the compounds of the invention, and can also be prepared separately by reacting the free acid or free base of the compound of the invention with a suitable base or acid, respectively.Examples of pharmaceutically acceptable, non-toxic acid salts include salts of the amino group formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or organic acids such as acetic, oxalic, maleic, tartaric, succinic or malonic acids, or obtained by other methods used in the art, such as ion exchange.Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, Pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valeriate, and the like. Typical alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and others.In addition, pharmaceutically acceptable salts may contain, if desired, non-toxic ammonium, quaternary ammonium and amine cations prepared using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0037] The peptides of the present invention may contain modifications such as glycosylation, side chain oxidation, and phosphorylation, provided that such modifications do not destroy the biological activity of the original peptide. Other illustrative modifications include the introduction of D-amino acids or other amino acid mimetics, which can be used, for example, to increase the serum half-life of the peptides.

[0038] The claimed peptides can be incorporated into other large molecules (proteins, peptides, nucleic acids, carbohydrates, lipids) without altering their pharmacological activity or to impart new properties. Furthermore, derivatives of the claimed peptide can be obtained by chemical modification of the terminal amino acid regions.

[0039] The peptides of the invention can be synthesized with additional chemical groups at their amino and / or carboxyl termini to increase the stability, bioavailability, and / or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or tert-butyloxycarbonyl groups can be added to the amino termini of the peptides. Similarly, an acetyl group or a 9-fluorenylmethoxycarbonyl group can be placed at the amino termini of the peptides. In addition, a hydrophobic group, tert-butyloxycarbonyl, or amide group can be added to the carboxyl termini of the peptides.

[0040] A "therapeutically effective amount" is defined as the amount of a compound administered or delivered to a patient that is most likely to produce the desired response to treatment. The exact amount required may vary from subject to subject depending on the patient's age, body weight, and general condition, the severity of the disease, the method of administration, combination therapy with other drugs, etc.

[0041] A "prophylactically effective amount" is defined as the amount of a compound administered or delivered to a patient that is most likely to produce the desired tumor prophylaxis response. The exact amount required may vary from subject to subject depending on the patient's age, body weight, and general condition, the method of administration, the combination with other drugs, etc. For prophylactic treatment, a therapeutically or prophylactically effective amount is the amount that will effectively prevent tumor disease.

[0042] The term "patient" ("subject") encompasses all mammalian species, preferably humans, that utilize the compounds of the invention, either by self-administration and / or administration to the patient by another person, for the treatment and / or prevention of a disease or medical condition.

[0043] The terms "treatment" and "therapy" cover the treatment of pathological conditions in mammals, preferably in humans, and include: a) blocking (stopping) the course of the disease, b) alleviating the severity of the disease, i.e. inducing regression of the disease.

[0044] The terms "prophylaxis," "avoidance," and "preventive therapy" encompass the elimination of risk factors, as well as prophylactic treatment of subclinical stages of disease in humans, aimed at reducing the likelihood of developing clinical stages of the disease. Patients for prophylactic therapy are selected based on factors that, based on known data, increase the risk of developing clinical stages of the disease compared to the general population. Preventive therapy includes (a) primary prevention and (b) secondary prevention. Primary prevention is defined as prophylactic treatment in patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of recurrence of the same or a similar clinical state of the disease.

[0045] The term "risk reduction" encompasses therapies that reduce the incidence of clinical disease. Examples of risk reduction include primary and secondary disease prevention.

[0046] Provided that the methods and compositions of the present invention are suitable for treating a malignant tumor, the treatment is considered effective if it achieves clinical benefits, such as reducing the size, spread, or metastatic properties of a malignant tumor, slowing the progression of a malignant tumor, alleviating the clinical symptoms of a malignant tumor, prolonging the survival period, or suppressing postoperative recurrences in a subject. If the treatment is administered prophylactically, effectiveness means that the treatment slows or prevents the formation of a malignant tumor, or prevents or alleviates the clinical symptoms of a malignant tumor. Efficacy is determined relative to any generally known methods for diagnosing or treating a specific tumor type.

[0047] Provided that the methods and compositions of the present invention are suitable for the prevention of malignant tumors, the term "prevention" as used herein includes any activity that alleviates the burden of mortality or morbidity associated with malignant tumors. Prevention can be carried out at the primary, secondary, and tertiary levels. While primary prevention helps avoid the development of a disease, secondary and tertiary prevention includes the prevention of disease progression and the onset of symptoms, as well as activities designed to reduce the adverse effects of an existing disease by restoring function and reducing complications associated with the disease.On the other hand, prevention (prophylaxis) may involve mitigating the severity of a specific disorder, such as extensive prophylactic therapy designed to reduce tumor growth and metastasis.

[0048] In the context of the present invention, treatment and / or prevention of a malignant tumor and / or prevention of its postoperative recurrence include any of the following events: inhibition of malignant cell proliferation, tumor regression, initiation of remission and suppression of malignant tumor development, tumor regression, as well as reduction or inhibition of metastasis, suppression of postoperative malignant tumor recurrences, and prolongation of survival. Effective treatment and / or prevention of a malignant tumor reduces mortality, improves the prognosis of an individual with a malignant tumor, reduces the level of tumor markers in the blood, and alleviates detectable symptoms associated with a malignant tumor.For example, relief or improvement of symptoms constitutes effective treatment and / or prevention (prophylaxis), and includes a state in which symptoms are stable or improved, in particular by 10%, 20%, 30% or more.

[0049] One skilled in the art will appreciate that the peptide described herein can exist and is often used in the form of its pharmaceutically acceptable derivatives, such as salts, prodrugs, metabolites, esters, ethers, hydrates, polymorphs, solvates, complexes, enantiomers, or other pharmaceutically acceptable derivatives. Therefore, reference to a peptide described herein is intended to include such pharmaceutically acceptable salts, prodrugs, metabolites, esters, ethers, hydrates, polymorphs, solvates, complexes, enantiomers, or any other pharmaceutically acceptable derivatives thereof.

[0050] Pharmaceutical compositions

[0051] The invention also relates to pharmaceutical compositions that comprise a peptide of the invention (or a prodrug, a pharmaceutically acceptable salt or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, solvents and / or excipients, such that can be administered to a patient together with the compound that is the essence of this invention and that do not destroy the pharmacological activity of this compound and are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0052] The pharmaceutical compositions referred to in this invention comprise the peptides of the present invention together with pharmaceutically acceptable carriers, which may include any solvents (in particular water), diluents, dispersions or suspensions, surfactants, isotonic agents, thickeners and emulsifiers, preservatives, binders, lubricants, etc., suitable for a particular dosage form.Materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, mono- and oligosaccharides and derivatives thereof; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut, cottonseed, sesame, olive, corn and soybean oils and others; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; depyrogenated water; isotonic solution, Ringer's solution; alcohol and phosphate buffer solutions. The composition may also contain other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, film agents, sweeteners, flavorings and aromatizers, preservatives and antioxidants.

[0053] Pharmaceutical compositions of the present invention may include a combination of two or more peptides of the present invention. The combination of peptides may be in the form of a cocktail of mixed peptides, or they may be conjugated to each other using standard methods. For example, the peptides may be chemically linked or expressed as single fusion polypeptide sequences.

[0054] The subject of the present invention is also dosage forms - a class of pharmaceutical compositions, the structure of which is optimized for a specific method of administration into the body in a therapeutically effective dose, for example, for administration into the body intravenously, orally, intramuscularly, subcutaneously, intraocularly, by inhalation, intranasally and sublingually, in recommended dosages.

[0055] The dosage forms of this invention may contain structures obtained by methods of using liposomes, microencapsulation methods, methods of producing nanoforms of the drug, or other methods known in pharmaceuticals.

[0056] For parenteral administration, aqueous suspensions, isotonic saline solutions or sterile injection solutions are used, the compatible agents of which contain pharmacological agents, for example, propylene glycol or butylene glycol.

[0057] In particular embodiments of the invention, the compositions of the invention may include a peptide of the present invention conjugated to polyethylene glycol, namely, conjugation of the N-terminus of the peptide to polyethylene glycol (hereinafter referred to as PEG) [ACS Appl. Mater. Interfaces 2020, 12, 41, 46991-47001; https: / / doi.org / 10.1021 / acsami.0c13492]. There are various variations of PEG, depending on its molecular weight.

[0058] This approach allows to reduce immunogenicity, increase solubility and half-life of protein drugs [Pasut G. Pegylation of biological molecules and potential benefits: pharmacological properties of certolizumab pegol. BioDrugs. 2014;28 Suppl 1:S15-S23. doi:10.1007 / s40259-013-0064-z], PEG is highly soluble in water and many organic solvents, non-toxic and non-immunogenic. PEGylation of biologically active compounds allows to increase metabolic stability by creating steric hindrances that protect the molecule from proteases, and, thus, to increase the circulation time in vivo. Methods of therapeutic application

[0059] The compounds of the present invention are antitumor agents and are therefore useful for the treatment and / or prevention of tumor disease.

[0060] The present invention relates to methods for the treatment and / or prevention of a malignant tumor and / or prevention, in particular its postoperative recurrence, which comprise administering to a subject a therapeutically or prophylactically effective amount of a peptide of the present invention or a pharmaceutical composition of the present invention.

[0061] In another aspect, the invention also relates to methods for treating or preventing a tumor in a subject, comprising administering to the subject an effective amount of a peptide of the invention. In one embodiment, the subject is a human, and the peptide of the invention can be delivered intravenously, intramuscularly, orally, epidermally, subcutaneously, intraosseously, intraperitoneally, intraocularly, by inhalation, intranasally and / or sublingually, as well as by systemic administration or local administration in close proximity to target sites, but is not limited to the above. In some embodiments, the route of administration includes subcutaneous or intravenous injection. Administration can be carried out by a single administration or divided into multiple administrations.

[0062] Malignant tumors for the treatment of which the peptides of the invention can be used include bladder cancer, breast cancer, cervical cancer, ovarian cancer, acute myeloid leukemia, chronic myeloid leukemia (CML), lymphoma, colon cancer, gastric cancer, esophageal cancer, liver cancer, melanoma, lung cancer, lymphoma, osteosarcoma, prostate cancer, pancreatic cancer, renal cell carcinoma, kidney cancer, hepatocellular carcinoma, non-small cell lung cancer and gastrointestinal stromal tumors, soft tissue tumors, etc., but are not limited to the above.

[0063] The compound or compositions described herein can also be used for prophylactic purposes. Accordingly, the compound or composition can be administered to a subject potentially at risk of developing a tumor.

[0064] For therapeutic use, the compounds of the invention can be administered using a pharmaceutical composition in any pharmaceutical dosage form by any route of administration. Dosage forms typically include a pharmaceutically acceptable carrier suitable for the particular dosage form selected. In particular, a compound of the invention can be administered daily for a period of time necessary to treat and / or prevent diseases relevant to the patient, including a course of therapy lasting days, months, years, or the patient's entire life. Routes of administration include, but are not limited to, intravenous, intramuscular, oral, epidermal, subcutaneous, intraosseous, intraperitoneal, intraocular, inhalation, intranasal, and / or sublingual. The preferred route of administration is intravenous.

[0065] The invention also relates to a pharmaceutical composition comprising a daily dose of said compound in the form of a fixed dosage unit, and to a combination comprising said pharmaceutical composition or said compound. In a preferred embodiment, said composition for use in accordance with the invention is administered once daily at a dosage of 1 mg or more of the selected compound according to the invention. The preferred dosage is 1-500 mg. The most preferred dosage is 10-200 mg.

[0066] One or more additional pharmacologically active agents may be administered in combination with the peptide of the invention. Generally, any additional single or multiple active agents other than the compounds of the invention, including, but not limited to, other antitumor drugs, antibodies, hormonal therapy agents, and others, may be used in any combination with the compound of the invention in a single or separate dosage form, allowing for the simultaneous or sequential therapeutic action of the active agents.

[0067] Implementation of the invention

[0068] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

[0069] Obtaining a peptide according to the invention

[0070] General methods for obtaining the peptide according to the invention

[0071] Well-known methods can be used to produce the peptides of the present invention. For example, recombinant DNA technology or chemical synthesis can be used to produce the peptides of the present invention. A person skilled in the art can readily synthesize the peptides of the invention. Standard methods for producing synthetic peptides are well known in the art. The peptides of the invention can be synthesized using commonly used methods such as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise synthesis, with one amino acid added at each step, starting from the carboxyl terminus of the peptide. The peptides of the invention can also be synthesized using solid-phase peptide synthesis methods well known in the art.

[0072] Furthermore, the peptides of the invention can be obtained not only by chemical synthesis but also by biotechnological methods: nucleotide sequences encoding the amino acid sequences of the claimed peptides can be synthesized. These nucleotide sequences can be introduced into cells (using vectors or native nucleic acids), and the cells thus transformed can be used to express the claimed peptides. These vectors and nucleotide sequences can be introduced into the human body (and other living organisms) for direct in vivo expression of the claimed peptides. These methods are also well known to those skilled in the art.

[0073] The peptides of the present invention can be isolated from host cells or the products of a synthetic reaction, after they have been produced in host cells using recombinant DNA technology or after they have been chemically synthesized. That is, the peptides of the present invention can be purified or isolated in such a way that they are substantially free of other proteins and fragments thereof from the host cell, or any other chemicals.

[0074] Synthesis of peptides according to the invention

[0075] The peptides of the invention were synthesized by solid-phase Fmoc synthesis using a JBMS-96-A automated synthesizer (Jianbang Pharmacy Technology Co., Ltd.). The peptides were purified (purity >95%) using reversed-phase high-performance liquid chromatography (Table 2).

[0076] Some particular embodiments of the invention are disclosed in Table 1 below.

[0077] Table 1. Examples of peptides according to the invention.

[0078] HPLC of the peptides according to the invention was performed on a YMC-Triart C18 column (4.6*250mm*5um), eluting with 0.1% trifluoroacetic acid in 100% water (solvent A) and 0.1% trifluoroacetic acid in 100% acetonitrile (solvent B), at a flow rate of 1 ml / min.

[0079] Table 2. HPLC and mass spectroscopy data for the peptide of the invention.

[0080] Characteristics of biological activity

[0081] To study the antitumor activity of the claimed compounds, the following cell lines were used: SK-MEL-2 (ATCC NTB-68; malignant melanoma; BioloT LLC, Russia), metastatic pancreatic adenocarcinoma (AsPC-1; Collective Use Center “Collection of Vertebrate Cell Cultures”, Institute of Cytology of the Russian Academy of Sciences, Russia) and A549 (lung carcinoma; Collective Use Center “Collection of Vertebrate Cell Cultures”, Institute of Cytology of the Russian Academy of Sciences, Russia).

[0082] The cells were cultured with the addition of 10% FBS at 37°C and 5% CO2 in the following media: Igla MEM - for the SK-MEL-2 line; RPMI 1640 - for AsPC-1; DMEM - for A549. The media were changed 2-3 times a week. The cells were passaged 1:3 upon reaching 80-90% confluency of the monolayer using 0.25% trypsin solution and 0.02% Versene solution in a ratio of 1:1.

[0083] Cell counting was performed using a Luna-FM automated cell counter using trypan blue staining. Cells were plated in 24-well plates at 5x10 4 living cells per well in 450 µl of medium 24 hours before adding the test substances to form a monolayer.

[0084] The studied peptide solutions were then added in 50 µl aliquots, resulting in a final concentration in the medium of 7 µg / ml (n = 6 replicates) and 70 µg / ml (n = 6 replicates). To obtain a positive control, 50 µl of nutrient medium supplemented with 10% FBS was added to the wells instead of the active substance. The sample plates were incubated overnight (16 hours) at 37°C and 5% CO2.

[0085] The following day, the EZ4U reagent kit (a modified MTT assay) was used to determine cell viability in the experimental and control groups. The medium was replaced with 450 µl of fresh medium (Igla MEM, RPMI 1640, or DMEM, depending on the cell line) supplemented with 10% FBS. 50 µl of activated EZ4U solution was added per well, and the plates were incubated for 2 hours at 37°C.

[0086] The optical density of the solution in the wells of the plate was measured at a wavelength of 450 nm, subtracting the optical density at 620 nm as background, using a Cytation 1 multifunctional photometer-imager and expressed as a percentage relative to the positive control. The results are presented in Tables 3-5.

[0087] Table 3. Results of the study of the antitumor activity of peptides against malignant melanoma cells (SK-MEL-2) (* - significant differences from the control).

[0088] Table 4. Results of the study of the antitumor activity of peptides against metastatic pancreatic adenocarcinoma cells (AsPC-1) (* - significant differences from the control).

[0089] Table 5. Results of the study of the antitumor activity of peptides NKM9 and ACP301 (70 μg / ml) in relation to lung carcinoma cells (A549) (* - significant differences from the control). Thus, the study results presented in Tables 3-5 demonstrate that the compounds of the invention exhibit cytotoxic and antiproliferative activity against the cell lines studied. Furthermore, a dose-dependent enhancement of the antitumor activity was observed for the ABP40 and NKM9 peptides.

[0090] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Claims

Invention formula 1. A peptide with antitumor activity, or a pharmaceutically acceptable salt thereof, having a sequence selected from the sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

2. Use of the peptide according to paragraph 1 as an antitumor agent.

3. Use of the peptide according to claim 1, for obtaining a pharmaceutical composition with antitumor activity for the treatment and / or prevention of a tumor disease in a subject.

4. Use according to paragraph 3, characterized by the fact that the disease is melanoma, metastatic adenocarcinoma of the pancreas, lung carcinoma, breast cancer, colorectal cancer, prostate cancer, liver cancer, esophageal cancer.

5. Application under paragraph 3, where the subject is a human being.

6. A pharmaceutical composition with antitumor activity for the treatment and / or prevention of a tumor disease in a subject, containing an effective amount of a peptide according to claim 1 and at least one pharmaceutically acceptable excipient.

7. A pharmaceutical composition according to claim 6, characterized in that the pharmaceutically acceptable excipient is a carrier, filler and / or solvent.

8. The pharmaceutical composition according to claim 6, characterized in that the disease is melanoma, metastatic adenocarcinoma of the pancreas, lung carcinoma, breast cancer, colorectal cancer, prostate cancer, liver cancer, esophageal cancer.

9. The pharmaceutical composition according to claim 6, wherein the subject is a human.

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