Ras-gtpase inhibitor and use thereof to treat oncological diseases

Abstract

The invention relates to the field of biotechnology and medicine, and more particularly to a polypeptide having the amino acid sequence SEQ ID NO: 1 and being capable of inhibiting RAS-GTPase activity, to the use of said polypeptide to produce a drug for treating stomach cancer or rectal cancer in a dose of 0.81-3.00 mg / kg, as well as to a drug based on said polypeptide for the treatment of stomach cancer or rectal cancer, and to a method for treating stomach cancer or rectal cancer by intraperitoneally administering said drug to a subject in an amount of 150 ml twice at an interval of 7 days.

Description

[0001] RAS-GTPase inhibitor and its use in the treatment of oncological diseases

[0002] The invention relates to the field of biotechnology and medicine, in particular, to the amino acid sequence of the polypeptide SEQ ID NO: 1, capable of inhibiting the activity of RAS-GTPase, to the use of the polypeptide for obtaining a medicinal product for treating gastric cancer or rectal cancer, for treating gastric cancer or rectal cancer at a dose of 0.81-3.00 mg / kg, as well as to a medicinal product based on the polypeptide intended for treating gastric cancer or rectal cancer, to a method for treating gastric cancer or rectal cancer, including administering the medicinal product to a subject intraperitoneally in a volume of 150 ml twice with an interval of 7 days.

[0003] State of the art

[0004] Ras is the most common oncogene in human tumors that is not affected by any of the registered drugs. Mutations in the Ras-GTP genes, leading to hyperactivation of the MAPK / ERK signaling pathway, are found in 25% of all human tumors. Mutations result in Ras kinase being constantly in a complex with GTP. The dual Ras-GTP complex binds Raf kinase with high affinity, resulting in the formation of an active ternary Ras-Raf-GTP complex, which phosphorylates MEK kinase and, thereby, transmits the signal further along the kinase cascade, activating proliferative activity. Signals transmitted along the RAS / MAPK / ERK signaling pathway determine the activity of the tumor cell, the ability to grow, metastasize, and also determine the lifespan of cancer cells.The isolated polypeptide binds to the active Ras-GTP complex at the Raf kinase binding site and prevents the formation of the active Ras-Raf-GTP ternary complex, thereby inhibiting the MAPK / ERK signaling pathway, causing proliferation arrest and / or cell elimination by apoptosis.

[0005] The key reaction of Ras activation and subsequent signal transmission is GTP binding. Therefore, approaches to influence specifically the G-domain of Ras have been actively studied. These approaches were based on attempts to increase the rate of hydrolysis of the mutant form of Ras (1,2) and inhibition of GDP-GTP exchange (3, 4).

[0006] The high mutation rate of Ras in malignant diseases makes it an attractive target for antitumor therapy. Methods for achieving this goal can be based on inhibition of the enzymes of posttranslational modification of the Ras protein. The most common targets for achieving this goal are farnesyl transferase and geranyl geranyl transferase I, which are involved in the addition of a prenyl group to the amino acid residues of the ras protein. (Prenylation is the process of transferring a prenyl group (15-carbon farnesylor 20-carbon geranyl-geranyl) to the cysteine ​​residue of Ras at its C-terminus.) An inhibitor, FTI, has been described, which has shown itself to be a potential candidate for an antitumor agent in Ras-activated tumor variants (5, 6, 7). Although FTI showed activity against cancer variants with H-RAS and N-RAS mutations, it was not active against tumors with mutant variants of K-RAS.Therefore, although a number of farnesyltransferase inhibitors have shown encouraging antitumor activity, there is a need to find ways to inhibit mutant forms of K-RAS.

[0007] An additional motive for the search for technologies for the use of natural protein proliferation inhibitors was the discovery of short amino acid sequences (n=15–30) capable of performing vector (transport) functions in relation to peptide sequences and compounds of a different chemical nature (RNA, DNA) (9).

[0008] Until now, attempts have been made to solve the problem of restoring the impaired function of intracellular proteins using gene delivery methods (gene therapy) (10). However, this technology has not yet been widely adopted into clinical practice due to a number of fundamental problems.

[0009] An alternative method for solving this problem, based on the technology of peptide vectors that have the ability to penetrate cells without damaging the plasma membrane, is very promising due to the weak immunogenicity of such compounds and the ability to transport fairly large molecules.

[0010] The combination of the possibility of targeted delivery of peptides into the cell and the discovery of short functional domains in proteins regulating various cellular functions created the prerequisites for constructing molecules with a pathogenetic focus (11). The relative simplicity of the synthesis of such molecules allows us to talk about the fundamental possibility of creating individual chemotherapeutic agents based on them, i.e. affecting the pathological changes inherent in a given specific tumor (Perea SE et al., 2004). The discovery of peptides capable of penetrating into the cell without the participation of membrane proteins and capable of carrying out intracellular transport of protein fragments and oligonucleotides associated with them opens a new stage in the development of biology and medicine. One of the effective carriers of large molecules into cells is the pAntp peptide. Its properties are known, in particular, from the publications of Derossi D. et al.. The third helix of the Antennapedia homeodamain translocates through membranes. / / J. Biol. Chem.269 ​​(1994) 10444-10450 and Morris MC. et al. A peptides carrier for the delivery of biologically active proteins in mammalian cells. / / Nat. Biotechnology. 19 (2001) 1173-1176.

[0011] Disclosure of invention

[0012] The objective of the invention is to create a chimeric peptide with an increased therapeutic effect and not requiring a complex and labor-intensive scheme for its production, as well as to obtain a medicinal product based on it that has a cytotoxic and cytostatic effect on tumor cells of various localizations. Efficiency depends on the cell type and may be associated with the presence or absence of mutations in the KRAS gene.

[0013] The technical result of the present invention is a medical and biological effect, objectively manifested in the pronounced cytotoxic effect of the said polypeptide on tumor cells of such diseases as colorectal cancer and stomach cancer, in addition, the creation of a medicinal product based on it allows its use in the treatment of oncological diseases. An additional technical result is the use of the medicinal product for the treatment of colorectal cancer and stomach cancer.

[0014] The technical result is achieved by obtaining a polypeptide with antiproliferative activity, namely, capable of inhibiting the activity of RAS-GTPase, represented by the amino acid sequence SEQ ID NO: 1, containing a functional fragment and a transport sequence, as well as obtaining a drug based on it for treating tumors. The drug has specific pharmacological activity in vivo, high antitumor activity, low nonspecific toxicity. An effective dose of the drug was selected and possible routes of administration of the drug were determined. These properties of the drug can also be regarded as additional technical results.

[0015] In one embodiment, the present invention relates to a polypeptide with antiproliferative activity, namely, capable of inhibiting RAS-GTPase activity, represented by the amino acid sequence SEQ ID NO: 1.

[0016] In another embodiment, the polypeptide is used to produce a medicament for the treatment of gastric cancer or rectal cancer.

[0017] In a preferred embodiment, the polypeptide is used to treat gastric cancer or rectal cancer at a dose of 0.81-3.00 mg / kg.

[0018] In one embodiment, the present invention relates to a polypeptide-based medicament for the treatment of gastric cancer or rectal cancer.

[0019] In a preferred embodiment, the medicinal product is presented in the form of a lyophilisate.

[0020] In one embodiment, the invention relates to a method for treating gastric cancer or rectal cancer using a polypeptide.

[0021] In a preferred embodiment, the method for treating gastric cancer or rectal cancer using a medicinal product comprises administering the medicinal product to the subject intraperitoneally in a volume of 150 ml twice with an interval of 7 days.

[0022] The term "polypeptide" or "Ras-GTPase / Ras-GTP inhibitor" or "chimeric peptide" or "fusion protein" or K26K as used herein includes a peptide having a defined binding sequence of a functional and transport fragment in a chimeric peptide molecule represented by the amino acid sequence SEQ ID NO: 1: KQRTVVNVRSRQIKIWFQNRR-Nle-KWKK.

[0023] The amino acid sequence of the peptide contains a Ras-GTPase inhibitor peptide as a functional group, and a transport sequence - an internalizable peptide. Additionally, any sequence of any length can be used for binding, including by means of additional amino acid residues (from 1 to 50) linking the two above-mentioned fragments, while the transport amino acid sequence, as an option, can be attached to the C-terminus of the said functional sequence by means of a group X, where X is an amino acid sequence containing from 1 to 50 amino acid residues, and inserts of various chemical natures can also be used. The approach used to create new antitumor drugs is based on the technology of intracellular delivery of high-molecular compounds based on CPP (cell penetrating peptides) or internalizable peptides.

[0024] Internalizable peptides (IPs) are short peptide sequences (less than 30 amino acids) that are able to penetrate cell membranes and transfer various compounds into cells. It has now been established that INPs penetrate all eukaryotic cells, have low immunogenicity, high penetration rate (5-20 minutes), and do not use the receptor pathway.

[0025] Methods for obtaining a chimeric peptide include both a solid-phase method, presented in documents RU 2297241, 22.11.2004 and RU 2435783, 29.09.2010, and a genetic engineering method, presented in documents RU 2297241, 22.11.2004 and RU 2435783, 29.09.2010. A study of the properties of these chimeric peptides is presented in document RU 2435783.

[0026] As used herein, the term "antiproliferative activity" includes the ability of cyclin kinase inhibitors or compositions comprising cyclin kinase inhibitors to exert a cytostatic and cytotoxic effect on malignant and benign tumor cells.

[0027] The term "transport sequence" as used herein includes any transport sequence. Any transport sequence may be used, provided that the claimed functional sequence included in any other polypeptide sequence of any size or any other structure or molecule performs the functions specified herein. In particular, the sequences of the VP22 peptide (pAntp) of the herpes simplex virus may be used as a transport agent for transferring a cyclin kinase inhibitor into target cells, for example, KWKK. In a particular case, the protein transduction region also binds or is otherwise linked to the Ras kinase / anti-cancer component conjugate.

[0028] The skilled artisan knows that a variant can be readily screened or tested to determine whether the variant retains antitumor properties. In other words, according to the method provided herein, a variant of the amino acid sequence of SEQ ID NO:1 or another variant of the internalizing peptide can be coupled to a detectable marker introduced into a cell and analyzed for internalization by the cell. A preferred assay method is fluorescence microscopy, although the skilled artisan knows that the assay method should be selected depending on the marker used. In addition to or instead of this in vitro method, an in vivo assay can be used. For example, a variant coupled to a detectable marker is introduced into an animal, such as a nude mouse with a tumor or cancerous tissue, and then the tumor tissue of the animal is analyzed to detect the marker.A skilled artisan may use other methods or variations of such methods to detect an internalizing peptide, such as SEQ ID NO:1, in a cell.

[0029] As used herein, the term "cancer" includes both malignant and benign tumors, including colorectal cancer, gastric cancer, pancreatic cancer, and other malignancies complicated by peritoneal or pleural carcinomatosis.

[0030] The term "Ras (Retrovirus Associated DNA Sequences)" as used in this document refers to a family of 3 genes:

[0031] KRAS, HRAS, NRAS, and the proteins they encode are small G proteins (small GTPases). Ras are membrane-bound proteins and participate in a cascade of kinases that lead to the activation of signaling pathways and transcription of genes that regulate cell differentiation and proliferation.

[0032] The term "Medicinal product / drug" as used herein means a Ras GTPase inhibitor that binds to the Ras-Raf-GTP complex and prevents its activation, thereby inhibiting the MAPK / ERK signaling pathway and may be in the form of a lyophilisate for the preparation of an injection solution, 10 mg.

[0033] As used herein, the term "intraperitoneal" refers to administration intraperitoneally via pressurized intraperitoneal aerosol chemotherapy (PIPAC).

[0034] Pressurized intraperitoneal aerosol chemotherapy is a simple, minimally invasive and safe method of intraperitoneal drug delivery.

[0035] The drug will be administered twice with an interval of 7 days.

[0036] The volume of the drug administered will be 150 ml. The dosage of the drug will be based on the patient's weight, measured in the morning on the day of drug administration.

[0037] In Phase I of the study, patients will receive the drug in one of the following doses:

[0038] Cohort 1 (0.45 mg / kg);

[0039] Cohort 2 (0.9 mg / kg);

[0040] Cohort 3 (1.8 mg / kg).

[0041] Dose calculations were based on the results of preclinical studies, taking into account the FDA guidelines (Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. FDA, 2005).

[0042] The toxic effect of the drug in rats is observed when the drug is administered at a dose of 30 mg / kg.

[0043] When converted (based on body surface area), the equivalent dose in humans is:

[0044] HED (human equivalent dose) = 30 / 6.2 = 4.8 mg / kg (6.2. - conversion factor for rats, according to Table 1 of the FDA guidelines) Maximum recommended starting dose:

[0045] MRSD (maximum recommended starting dose) = HED / 10 = 4.8 mg / kg / 10 = 0.48 mg / kg

[0046] The effective dose for mice when administered intraperitoneally is 10 mg / kg. When recalculated, the equivalent human dose (target dose) is:

[0047] 10 / 12.3=0.81 mg / kg (12.3 conversion factor for mice)

[0048] Thus, the dose regimens for the study cohorts were determined:

[0049] Cohort 1-0.45 mg / kg (dosage not exceeding the MRSD, * of the effective dose in animals); Cohort 2-0.9 mg / kg (~ the effective dose in animals);

[0050] Cohort 3-1.8 mg / kg (dose 2 times higher than the effective dose in animals).

[0051] DESCRIPTION OF FIGURES

[0052] FIG. 1. Effect of the K26K sequence on HT-29, OVCAR-3, OAW-42 cell cultures, incubation time 48 hours.

[0053] FIG. 2. Results of the LDH test of HT-29, OVCAR-3, OAW-42 cell cultures after 24 and 48 hours of incubation with the peptide sequence of the Ras-GTPase inhibitor K26K, in concentrations from 5 to 40 μM. The control results - cell cultures incubated with the medium for 24 hours - are taken as 1.

[0054] FIG. 2. Comparison of the cytotoxic effect of K26K relative to HT-29, OVCAR-3 and OAW-42 cultures. CFDA-SE / PI staining, dependence of the change in the number of PI-positive particles on the concentration of the drug, incubation time 24 hours.

[0055] FIG. 3. Changes in the level of apoptosis and necrosis in the culture of HT-29, OVCAR-3, OAW-42 cell lines when exposed to the K26K sequence at concentrations of 10 - 40 μM. Flow cytometry method, Annexin V-FITC (FL1) / PI (FL3) staining.

[0056] FIG. 5. Dynamics of tumor node growth in BALBc mice with subcutaneously transplanted human colon adenocarcinoma HT-29 with intraperitoneal administration of the Ras-GTPase inhibitor, K26K, at doses of 10 mg / kg to 20 mg / kg, for groups 1 and 2, respectively.

[0057] FIG. 4. Inhibition of growth of subcutaneously transplanted HT-29 tumor with intraperitoneal administration of the Ras-GTPase inhibitor, K26K.

[0058] Implementation of the invention

[0059] The possibility of implementing the invention with the implementation of the stated purpose and achieving the technical result is confirmed by the following research results, however, the presented results do not limit the options for using the declared technical solution.

[0060] Example 1. Study of the specific pharmacological activity in vivo of a drug based on the peptide inhibitor of Ras-GTPase, K26K

[0061] In vitro experiments were used to evaluate the cytotoxic and cytostatic effects of a drug based on a peptide inhibitor of Ras-GTPase against ovarian cancer and colon cancer cells at concentrations of the studied drug from 5 to 40 μM. The peptide inhibitor was diluted in phosphate-buffered saline to the desired concentration immediately before adding to the culture medium. The results were assessed 24 and 48 hours after incubation of the cell culture with the peptide inhibitor of Ras-GTPase for the LDH test, MTT test, flow cytometer and every 15 minutes of 96-hour incubation for the real-time proliferation assessment method (iCELLigence system).

[0062] In vivo studies were performed to evaluate the antitumor efficacy of a drug based on a peptide Ras-GTPase inhibitor against HT-29 colon adenocarcinoma cells. The administration doses for three independent experiments were: 0.2 mg / mouse (10 mg / kg) and 0.4 mg / mouse (20 mg / kg) of the Ras-GTPase inhibitor in 0.2 ml of a 0.9% NaCl solution. The contents of the vial were pre-diluted with sterile saline (0.9% NaCl) to the required concentration (1 - 200 μl of the NaCl solution contained 0.2 mg; 2 - 200 μl of the NaCl solution contained 0.4 mg of the Ras-GTPase inhibitor).

[0063] Then the drug is administered according to the scheme:

[0064] In BALBc (NUDE) mice, HT-29 transplant line, intraperitoneal multiple administration every other day (three times a week) at doses of 0.2 mg / mouse (10 mg / kg) and 0.4 mg / mouse (20 mg / kg).

[0065] An equal group of mice with transplanted HT-29 tumor cells was selected as a control group, receiving injections of 0.2 ml of a 0.9% NaCl solution as a control.

[0066] A series of experiments on the cytotoxic effect of the Ras-GTPase peptide inhibitor sequence were performed on HT-29, OAW-42 and OVCAR-3 cell cultures. The cytotoxic effect was assessed using several methods. First of all, experiments were performed in which the number of living cells remaining in the culture after exposure to the K26K sequence in different concentrations was assessed using the MTT test. The incubation time of the studied cell cultures with the Ras-GTPase peptide inhibitor was 24 and 48 hours. The effect of the K26K drug on the cultures was studied in different concentrations from 2 to 40 μM.

[0067] When studying the effect of the K26K drug on HT-29 cell cultures, it was shown that the number of living cells decreases with an increase in the K26K concentration in the range from 5 to 40 μM; after 24 hours of incubation at a concentration of 40 μM K26K, the number of living cells decreases to 51%. It was also shown that with an increase in the incubation time to 48 hours, the number of living cells decreases by more than 50% already at a K26K concentration of 5 μM. When studying the cytotoxic effect of the K26K drug on OAW-42 cells, it was also shown that a decrease in the number of living cells occurs proportionally to an increase in the K26K concentration in the range from 2 to 40 μM, however, a reliable decrease in the number of living cells relative to the control samples is observed with an increase in concentration of more than 20 μM. At a concentration of 40 μM, a decrease in the number of living cells by 48% occurs.

[0068] Similar results of changes in the number of living cells were obtained when studying the effect of K26K on the OVCAR-3 cell culture. Significant decreases in the number of living cells were observed when exposed to K26K at concentrations greater than 30 μM; with an increase in the exposure time to 48 hours, no reliable decrease in the number of living cells was obtained.

[0069] Based on the conducted studies of the antitumor effect of the drug K26K by the MTT method, it was shown that the compound has a pronounced cytotoxic effect on the studied colon adenocarcinoma line HT-29, and to a lesser extent affects the ovarian cancer lines (OVCAR-3, OAW-42). The effect is proportional to the concentration of the drug, and increases with a longer exposure time; for HT-29, the differences during incubation for 24 and 48 hours are reliable. Figure 7 shows a comparison of the cytotoxic effect of the K26K sequence relative to the three studied lines during incubation for 48 hours (Fig. 1).

[0070] The study of the cytotoxicity of the peptide sequence of the Ras-GTPase inhibitor K26K using the LDH test was carried out after 24-hour and 48-hour incubation of cell cultures with K26K at concentrations of 5, 10, 20 and 40 μM.

[0071] It was shown that the LDH level increased for all the studied cultures proportionally to the K26K concentration and also increased with an incubation time of 48 hours. However, as in the assessment of the effect using the MTT test, the effect was more pronounced for the HT-29 culture and to a lesser extent for ovarian cancer cultures, OVCAR-3, OAW-42. The peak height in Figure 1 corresponds to the relative LDH level and is proportional to the number of dead cells. Using the LDH test method, it was shown that the studied drug based on the peptide inhibitor of Ras-GTPase - K26K, is capable of affecting cancer cells of various localizations, but when comparing the effects, it has a more pronounced cytotoxic effect relative to human colon adenocarcinoma cells. The cytotoxic effect linearly depends on the concentration of K26K added to the culture medium and on the incubation time (Fig. 2).

[0072] In the study of the antitumor effect of the K26K sequence by the CFDA-SE / PI staining method, the effects of K26K on HT-29, OVCAR-3, and OAW-42 cultures were compared. The results were assessed after 24 hours of cell incubation with the peptide Ras-GTPase inhibitor, with the effect on the cells of its various concentrations (10, 20, 30, and 40 μM). Three consecutive identical experiments were carried out for each of the studied cell lines.

[0073] It was shown that when exposed to the Ras-GTPase inhibitor K26K, there is an increase in the number of dead cells for the three cultures studied, however, the effect is maximal in the colon adenocarcinoma culture HT-29 (FIG. 3).

[0074] The study of the proapoptotic and cytotoxic activity of the Ras-GTPase inhibitor K26K was carried out using the flow cytometry method with Annexin V / PI staining.

[0075] The cytotoxic and proapoptotic effects were assessed for HT-29, OVCAR-3 and OAW-42 cell cultures at concentrations of K26K -5, 10, 20, 30 and 40 μM; the incubation time was 24 hours.

[0076] It was shown that for the studied cell cultures, the introduction of the drug of the K26K sequence causes the induction of apoptosis. The level of apoptosis depends on the concentration of K26K, as does the level of cells that died by necrosis. For antitumor therapy, the most preferable option is the death of tumor cells by apoptosis, since it does not cause extensive inflammation and the release of cell decay products and inflammatory response inducers into the interstitial space. Also, the presence of a high level of apoptosis indirectly indicates the specificity of the effect of the drug K26K, which is an inhibitor of Ras-GTPase. Figure 4 shows a comparison of the level of apoptosis induced by the introduction of the K26K sequence into the culture medium; it is shown that the maximum effect is observed when affecting the HT-29 cell line culture.As can be seen from the presented results, the dependences of the levels of apoptosis and necrosis for the HT-29, OVCAR-3, OAW-42 cell lines when exposed to K26K have large differences. The level of apoptosis when exposed to the drug K26K on the HT-29 culture increases significantly compared to the control already at a K26K concentration of 10 μM, and at a concentration of 40 μM it reaches 43.2%. While for the OVCAR-3 and OAW-42 cultures, a higher percentage of cell death by necrosis is observed. Thus, the conducted study of the types of cell death (apoptosis and necrosis) upon exposure to human lung cancer cell lines by flow cytofluorometry using Annexin V-FITC / PI double staining showed that the drug K26K, which is a Ras-GTPase inhibitor, has a pronounced specific antitumor effect on the human colon adenocarcinoma cell culture.This effect is characterized by the induction of cell death mainly via apoptosis. The level of apoptosis induced by the introduction of K26K into the culture medium depends on the cell type and is apparently due to the molecular genetic characteristics of the cell line. Ovarian cancer cell lines were less sensitive to the effects of the peptide inhibitor of Ras-GTPase - K26K.

[0077] For dynamic study of the effect of the peptide inhibitor of Ras-GTPase - K26K on the growth of cells of the cell lines HT-29, OVCAR-3, OAW-42, the RTCA iCELLIgence device by ACEA Biosciences (USA) was used. For each cell line, a series of experiments were conducted to study the effect of K26K in the concentration range of 10 - 40 μM. During the experiments, the cells were added to the plate in the amount of 20,000 per well, incubated for 3 hours for initial fixation on the bottom of the plate, and then the test substance was added to the culture medium in various concentrations.

[0078] When studying the cytostatic effect of the drug K26K on HT-29 cells, it was shown that at a concentration of 10 μM, a delay in proliferation is observed, the cellular index decreases by 1.5 times. At concentrations of the peptide inhibitor Ras-GTPase over 20 μM, a complete cessation of cell division is observed, i.e. a persistent cytostatic effect.

[0079] For the OVCAR-3 culture (ovarian adenocarcinoma), it was shown that the effect of delayed proliferation has a concentration dependence, however, the effect is reversible, and after 70 hours of incubation, complete restoration of the proliferative activity of cells with a K26K concentration of 10 and 20 μM is observed.

[0080] Changes in the proliferative activity of OAW-42 culture cells were observed at K26K concentrations greater than 20 μM and were defined as a complete and irreversible cessation of cell growth; however, at low concentrations (10 μM and less), a slight increase in the proliferative activity of OAW-42 cell lines was observed.

[0081] Thus, when studying the antitumor effect of the Ras-GTPase peptide inhibitor sequence - K26K in relation to cultures of human colon cancer - HT-29, ovarian cancer - OAW-42 and OVCAR-3, it was shown that the studied drug is a potential antitumor drug affecting tumor cells of various localizations. It has pronounced cytotoxic activity, determined by an increase in the number of dead cells and a decrease in the number of living cells in the cultures of the studied lines. The studied drug induces apoptosis in human tumor cells, and this effect depends on the type of cell line. Cytotoxic and proapoptotic effects have a direct concentration dependence. The cytostatic effect of the drug K26K, determined by a decrease in the proliferative activity of cells when K26K is introduced into the culture medium, also has a concentration dependence and is different for different types of cells.It was shown that stronger antitumor activity was observed against human colon adenocarcinoma cells, and specific activity was less pronounced against ovarian cancer cells.

[0082] Example 2. Generation of a biological tumor model in mice for further in vivo efficacy testing against colon adenocarcinoma cells

[0083] Cultivation and manipulations with cell lines were carried out under sterile conditions. Cells were cultured at 37°C and 5% CO2 in DMEM (Biolot, Russia) or RPMI (PanEco, Russia) medium supplemented with fetal bovine serum to 10% (HyClone, USA), streptomycin (to 100 μg / ml), penicillin G (to 100 U / ml) and panethicin (G420) 1 mg / ml. The number of viable cells was determined by staining with trypan blue solution.

[0084] The culture of tumor cells, HT-29 (human colon adenocarcinoma), stored at liquid nitrogen temperature, was defrosted and planted under standard conditions: culture medium: DMEM, 10% fetal bovine serum, penicillin-streptomycin antibiotics (50 U / ml, 50 μg / ml); high humidity at 37°C and 5% CO2. For transplantation, the cells were diluted in sterile Hank's solution to a concentration of 5x106 cells / ml.

[0085] To obtain standard grafting material, the HT-29 tumor cell strain was passaged twice in the standard manner, by subcutaneous transplantation.

[0086] On study day "0", animals of the control and experimental groups were given a subcutaneous injection (BALBc (NUDE) mice) of a suspension of HT-29 tumor cells, previously washed (10 ml PBS with centrifugation for 5 min at 300g) in 0.2 ml phosphate-buffered saline (PBS). Tumor growth was determined by the appearance of a palpable node

[0017] .

[0087] Stages of the experiment:

[0088] 1. Study of the growth dynamics of a subcutaneously transplanted tumor - human colon adenocarcinoma in BALBc mice against the background of the introduction of a Ras-GTPase inhibitor at two doses of application.

[0089] Administration scheme: every other day (three times a week) starting from the 6th day after tumor transplantation and the appearance of a palpable tumor node. The administration doses for three independent experiments were 0.2 mg / mouse (10 mg / kg) and 0.4 mg / mouse (20 mg / kg) of Ras-GTPase inhibitor in 0.2 ml of 0.9% NaCl solution. The control group consisted of animals with a subcutaneously transplanted tumor from HT29 cells, intraparietal administration of 0.2 ml of 0.9% NaCl solution once every two days. The number of administrations was 12. (Table 1)

[0090] The administration of the Ras-GTPase inhibitor is intraperitoneal.

[0091] 2. Study of survival of BALBc mice with subcutaneously transplanted tumor from HT29 cells during treatment with a drug inhibitor of Ras-GTPase (according to the same scheme).

[0092] Table 1. Experimental design for studying the specific pharmacological activity of the Ras-GTPase inhibitor in vivo in BALBc mice with repeated intraperitoneal administration

[0093] The total number of animals is 30.

[0094] Evaluation of antitumor activity in vivo was carried out on the basis of:

[0095] 1. Estimating the lifespan of animals. When assessing the lifespan of animals, the last day of life is considered to be the previous day before the day of death. The percentage increase in the lifespan of animals is calculated using the formula:

[0096] UPZh=(SPZhexperience - SGOCcontrol) / SGOCcontrol X 100%

[0097] 2. Evaluation of the size of the palpable subcutaneous tumor. The larger size of the tumor (length) and the smaller size perpendicular to it (width) are measured. The tumor volume is calculated in mm 3 according to the generally accepted formula:

[0098] V = L x W 2 / 2 , where eV is the tumor volume, L is the tumor length, \¥ is the tumor width.

[0099] 3. Inhibition of tumor growth was calculated using the formula:

[0100] TPO = (V control - V EXPERIMENT) / V control X 100%

[0101] Results

[0102] When studying the dynamics of tumor growth and survival of BALBc (NUDE) mice with a subcutaneously transplanted HT-29 tumor culture, it was shown that in both groups with intraperitoneal administration of the studied peptide inhibitor, inhibition of tumor node growth is observed (Figure 5). It was also found that increasing the concentration of the administered Ras-GTPase inhibitor from 10 mg / kg to 20 mg / kg does not lead to an increase in the effect.

[0103] Analysis of HT-29 tumor growth inhibition showed reliable (p<0.05) and significant (>50%) inhibition of tumor node growth at both doses of Ras-GTPase inhibitor. However, as shown in Figure 6, there was a decrease in the TPO value after two weeks of Ras-GTPase inhibitor use and a significant decrease on days 24-27 of the experiment.

[0104] The survival analysis showed that the use of both doses of treatment (0.2 mg / mouse (10 mg / kg) and 0.4 mg / mouse (20 mg / kg)) with the administration regimen every other day increased the survival time of the experimental animals. However, in the case of tumors from HT-29 cells, low mortality was observed, so in the control group the first mouse to die was on day 15, and on day 30 of the experiment the number of dead animals was 4 pcs. (40%). In the experimental groups, one mouse in group 1 died on day 32 of the experiment. Thus, due to the low mortality, it is not possible to draw a reliable conclusion about the effect of using the Ras-GTPase inhibitor on the survival of mice in these groups.

[0105] Thus, the conducted additional study of the specific pharmacological activity of the drug based on the peptide inhibitor of Ras-GTPase (K26K) showed that the compound exhibits antitumor activity against tumor cells of various localizations. The studies have shown high activity against colon adenocarcinoma cell lines HT-29 in experiments in vitro and in vivo. Reliable inhibition of tumor growth was shown when using intraperitoneal administration in doses of 0.2 mg / mouse (10 mg / kg). The use of a dose of 0.2 mg / mouse (10 mg / kg) at intervals of every other day is the most effective for this route of administration. Increasing the dose to 0.4 mg / mouse, as shown in the HT-29 model, does not lead to a reliable increase in efficiency.An additional study of the effectiveness of a drug based on a peptide inhibitor of Ras-GTPase has proven its antitumor activity against human and mouse colon adenocarcinoma cells.

[0106] Example 3 Acute toxicity study of the peptide inhibitor of RAS-GTPase (K26K) in Sprague-Dawley rats

[0107] A study of the acute toxicity of the test drug was conducted with its single intravenous and intraperitoneal administration to male and female SD rats in five experimental doses.

[0108] The minimum dose was equivalent to the expected therapeutic dose for humans. The maximum dose was selected based on preliminary studies showing that this dose can cause significant toxic effects.

[0109] Intravenous and intraperitoneal administration were used since this is the planned route of application in clinical practice.

[0110] The test drug was administered to animals once intravenously in a volume of 1 ml / kg or intraperitoneally in a volume of 2 ml / kg in five different doses (Table 2). The control animals were administered physiological saline in the same volumes. During the study, clinical signs of deviations in health, body weight and food consumption were recorded in the animals. On the 15th day of the study, the animals were euthanized and necropsied with an examination of macrolesions of organs and their weighing. Animal groups and doses in the study of acute toxicity of the peptide inhibitor of RAS-GTPase (K26K) in rats.

[0111] Table 2. Intravenous administration of K26K to rats Table 3. Intraperitoneal administration of K26K to rats

[0112] With intravenous administration of the test drug, 100% mortality was observed in male animals at doses of 120 and 90 mg / kg on the first day of the study for 3 hours after administration. In the group receiving a dose of 120 mg / kg, 2 males and 2 females were used, the remaining animals were excluded from the experiment, since administration of the drug to them would have caused inevitable death. In females, 100% mortality was also observed with intravenous administration at doses of 120 and 90 mg / kg, however, with the administration of a dose of 90 mg / kg, some animals died on the second day of the study. In addition, the death of one female was observed after receiving a dose of 60 mg / kg. With intraperitoneal administration, the death of 2 males from the group receiving a dose of 250 mg / kg was observed on the 12th day of the study. No deaths of females were recorded with this method of administration.

[0113] The main clinical manifestations of the toxic effect of the test drug when administered intravenously were blue ears and tail, red eyes, lateral posture, gait disturbance, and labored breathing, observed in all groups receiving the drug. The degree of manifestation of these signs was directly dependent on the administered dose. In addition, in animals receiving the drug at doses of 60 mg / kg and higher, blood was present in the urine on the first day after administration. On subsequent days of the study, necrosis of the tail below the injection site was observed in all groups except the one receiving the lowest dose. With the intraperitoneal route of administration, the main clinical signs on the first day after administration were lateral recumbency, gait disturbance, and sunken sides, and on the following days - chromodacryorrhea, hunching, decreased muscle tone, abdominal distension, and intestinal upset. The described signs were observed only at doses above 100 mg / kg.

[0114] Analysis of body weight gain revealed a statistically significant decrease in this parameter in male animals with intravenous administration in the groups receiving the test drug at doses of 60 and 30 mg / kg on the 7th and 14th days of the study, both relative to the control group and the group receiving the test drug at a dose of 15 mg / kg. With intraperitoneal administration, a tendency toward a decrease in body weight gain was observed in males, but no statistically significant differences were found. In females, no statistically significant differences in body weight were observed between the experimental groups with either route of administration.

[0115] No statistically significant intergroup differences in feed consumption were found.

[0116] Autopsy of animals that died after intravenous administration of the drug revealed multiple hemorrhages in the thymus, lungs, and heart, as well as foamy discharge from their lungs and bleeding from the urinary tract. Some animals showed enlarged mesenteric lymph nodes and hemorrhages in intestinal plaques.

[0117] During routine necropsy of surviving animals, during visual examination of the body integument, as well as body cavities and their contents, no pathology was noted in animals with intravenous administration of the test drug, except for necrosis of the lower part of the tail in the groups receiving the drug in doses higher than 15 mg / kg. In one animal that received a dose of 60 mg / kg, deformation of the testicle was noted, however, this sign was not observed at higher doses, therefore, it cannot be unambiguously associated with the action of the test drug. In animals with intraperitoneal administration of the drug, intestinal distension, deformation of the liver, spleen and thymus with a change in their consistency and color, as well as a decrease in size were noted. Also, in some animals, pathological changes were observed in the testicles, kidneys and adrenal glands. The degree of manifestation of the described signs was directly dependent on the administered dose.No abnormalities were detected only in animals receiving the lowest dose (15 mg / kg) of the drug.

[0118] Analysis of organ weights revealed no statistically significant differences between groups, except for an increase in the relative weight of the adrenal glands in males receiving an intravenous dose of 60 mg / kg relative to the control group, and a decrease in the relative weight of the kidneys in males receiving an intraperitoneal dose of 250 mg / kg.

[0119] Thus, based on the results of the study, it was concluded that the dose that does not cause adverse effects (NOAEL) for intraperitoneal administration can be considered a dose of 15 mg / kg, and with intravenous administration, even the minimum dose used caused short-term toxic effects.

[0120] Example 4. Acute toxicity study of the RAS-GTPase peptide inhibitor (K26K) in mice

[0121] A study was conducted to investigate the toxic effects of the test drug upon its single intravenous and intraperitoneal administration to male and female CD-1 mice in five experimental doses.

[0122] The test drug was administered to animals once intravenously in a volume of 5 ml / kg or intraperitoneally in a volume of 10 ml / kg in five different doses (Table 4). The control animals were administered saline in the same volumes. During the study, clinical signs of deviations in health, body weight and food consumption were recorded in the animals. On the 15th day of the study, the animals were euthanized and necropsied with an examination of macrolesions of organs and their weighing. Table 4. Animal groups and doses in the study of acute toxicity of the peptide inhibitor of RAS-GTPase (K26K) in mice with intravenous administration

[0123] Table 5. Animal groups and doses in the study of acute toxicity of the peptide inhibitor of RAS-GTPase (K26K) in mice with intraperitoneal administration Intravenous administration of the test drug resulted in the death of 1 female receiving the drug at a dose of 75 mg / kg on the first day of the study and one male receiving a dose of 50 mg / kg on the 13th day of the study. Intraperitoneal administration resulted in the death of 5 males from the group receiving a dose of 500 mg / kg, 4 males from the group receiving a dose of 250 mg / kg, and 2 males from the group receiving a dose of 150 mg / kg. In females, 5 animals died in each of the above groups.

[0124] The main clinical effect of the test drug when administered intravenously was the blueing of the tail, observed in all groups receiving the drug. However, this effect was short-term and reversible. With the intraperitoneal route of administration, the main clinical signs in the first day after administration of the drug were a decrease in general tone, cyanosis, piloerection, recumbent posture, gait disturbance, and hunching.

[0125] During the analysis of the animals' body weight and its increase, no statistically significant intergroup differences were found in either males or females for either administration method. No statistically significant intergroup differences in feed consumption were found either.

[0126] Autopsies of the dead animals revealed no obvious signs of pathology.

[0127] During routine necropsy of surviving animals, during visual examination, animals with intravenous administration of the test drug showed hemorrhages in the thymus, which were observed in all experimental groups, regardless of the dose. In addition, one male receiving the test drug at a dose of 75 mg / kg showed a decrease in testicular weight. No pathology was detected in animals with intraperitoneal administration of the drug.

[0128] Analysis of organ weights revealed a statistically significant increase in absolute and relative thymus weights in males given an intravenous dose of 75 mg / kg relative to the control group. No statistically significant intergroup differences in organ weights were found with the intraperitoneal route of administration.

[0129] Based on the results of the study, it was concluded that the maximum tolerated dose (MTD) of the test drug for mice is 50 mg / kg.

[0130] In the course of the study conducted to study the acute toxicity of the drug Ras-GTPase inhibitor when administered intravenously and intraperitoneally to mice and SD rats, the following was established:

[0131] - death of rats with intravenous administration was observed starting from a dose of 60 mg / kg, death of mice starting from a dose of 75 mg / kg, death of rats with intraperitoneal administration was observed starting from a dose of 250 mg / kg, death of mice starting from a dose of 150 mg / kg;

[0132] - autopsy of surviving animals with intravenous administration did not reveal changes in the internal organs; with intraperitoneal administration, hemorrhages of internal organs were revealed, which indicates a local irritant effect of the drug;

[0133] - clinical signs observed upon administration of the drug were short-term and reversible. Thus, based on the results of the acute toxicity study, it was concluded that the maximum tolerated dose (MTD) of the test drug for mice can be considered a dose of 60 mg / kg, for rats 50 mg / kg.

[0134] Example 5. Toxicological studies of subacute (subchronic) toxicity of a drug when administered intravenously

[0135] The aim of the study was to determine the toxic effects of a drug based on a peptide inhibitor of Ras-GTPase upon its repeated intravenous administration to two types of animals (male and female SD rats and rabbits) for 28 days followed by a 14-day withdrawal period at three experimental doses.

[0136] The studies of subacute (subchronic) toxicity of the medicinal product, the peptide inhibitor of Ras-GTPase K26K, were conducted in accordance with the “Protocols for preclinical studies of subacute (subchronic) toxicity of the medicinal product, the peptide inhibitor of Ras-GTPase (K26K)”.

[0137] In a study of subacute toxicity of the drug - peptide inhibitor of RAS-GTPase on rats, 4 groups of male and female CD rats with 12 animals in each were used. The test drug or saline solution was administered to the animals intravenously into the tail vein every other day for 28 days, in a volume of 2 ml / kg in three experimental doses (8.6 mg / kg, 17.2 mg / kg and 34.4 mg / kg) (Table 6). During the study, the body weight, food consumption and the manifestation of clinical signs of toxicity of the test drug were recorded in the animals. In the final week of the study, the animals underwent functional tests to assess the state of the central nervous system and cardiovascular systems. On the 29th day of the study, some animals were euthanized and necropsy with organ collection for subsequent histological analysis. During necropsy, the weight of internal organs was recorded and blood samples were collected for biochemical and hematological parameters, as well as bone marrow samples.The day before necropsy, animals were placed in metabolic cages to record diuresis and collect urine for clinical study. The remaining animals were necropsied after a 14-day withdrawal period (on day 43 of the study).

[0138] Table 6. Groups and doses in subacute toxicity studies in rats

[0139] In a study of the subacute toxicity of the drug - a peptide inhibitor of RAS-GTPase on rabbits, 12 male and 12 female rabbits of the Gray Giant breed were used.

[0140] The test drug or saline solution was administered to animals intravenously into the ear vein every other day for 28 days, in the volume and doses indicated in Table 7. The control group of animals (Group 1) was administered the vehicle (saline solution). During the study, the animals' body weight, food consumption, and signs of health deviations were recorded. On the 29th day after administration, the animals were euthanized and underwent a detailed necropsy with examination of the internal organs, their weighing, and fixation for subsequent histological analysis. During necropsy, blood samples were taken for biochemical and hematological analysis.

[0141] Table 7. Groups and doses in subacute toxicity studies in rabbits

[0142] No clinical signs of toxicity of the test drug were observed in rats during the study, except for necrosis of the tail region below the injection site in animals receiving the drug at the maximum dose. The death of one male (No. 32) was recorded on the 14th day of the study from the group receiving the test drug at a dose of 8.6 mg / kg. However, given that no deaths were observed in animals receiving higher doses of the drug, it can be assumed that the single case of death is associated with an individual reaction of the organism.

[0143] There were no statistically significant intergroup differences in body weight and weight gain in female animals receiving the test drug at the three doses studied during the study. In males, there was a statistically significant decrease in body weight gain throughout the study, starting from day 7 in the group receiving the maximum dose, on days 7, 14, 21, and 28 of the study in the group receiving the medium dose, and on days 14, 21, and 28 in the group with the minimum dose.

[0144] No statistically significant deviations in feed consumption were found in animals receiving the test drug.

[0145] Results of serum biochemistry showed that in males, when administered the test drug, there was a statistically significant increase in total cholesterol and total protein levels in the 34.4 mg / kg group, and in triglyceride levels in the 17.2 mg / kg group. These changes were reversible and were not observed after the withdrawal period. However, in the 17.2 mg / kg group, there was a statistically significant increase in glucose levels relative to the control on day 43 of the study. In females in the low-dose group, there was a statistically significant increase in calcium levels on day 29 of the study, which was reversible by the end of the withdrawal period.

[0146] In hemostasis indices in male rats receiving the test drug in three experimental doses, no statistically significant intergroup differences were found on the 29th and 43rd days of the study. In females receiving the test drug at a dose of 34.4 mg / kg, a statistically significant increase in fibrinogen concentration was found on the 29th day of the study, reversible by the 43rd day of the study.

[0147] In rats of both sexes that received the study drug at doses of 8.6 mg / kg, 17.2 mg / kg and 34.4 mg / kg, no statistically significant deviations from the control level were found in the hemogram on the 29th day of the study, but a slight tendency toward a decrease in the number of erythrocytes (RBC), hemoglobin (Hb) and hematocrit (HCT) concentrations was observed. At the end of the 14-day withdrawal period, a slight tendency toward hypochromic anemia (\RBC, \Hb, \HCT, \MCH, 7RDW) persisted in males that received the drug at all studied doses (8.6 mg / kg, 17.2 mg / kg and 34.4 mg / kg), with no dose-dependent effect observed. Statistically significant changes relative to control animals in males were recorded in the form of a decrease in hemoglobin levels when the drug was administered at a dose of 17.2 mg / kg, and an increase in the level of anisocytosis (RDW) when a dose of 34.4 mg / kg was administered.In females, a slight tendency toward hypochromic anemia also persisted after the end of the withdrawal period, regardless of the dose of the drug. At the same time, the average platelet volume increased statistically significantly in response to the administration of a dose of 34.4 mg / kg.

[0148] During the planned necropsy at the end of the study, no macroscopic changes in the internal organs were detected. Analysis of the organ weight on the 29th day of the study in female animals revealed a tendency toward an increase in the relative values ​​of the spleen weight when the drug was administered at all experimental doses. A statistically significant increase in this indicator relative to the control values ​​was recorded in the groups receiving doses of 8.6 and 34.4 mg / kg. In addition, a statistically significant increase in the relative kidney weight was noted in females when a dose of 17.2 mg / kg was administered. Deviations in these indicators were reversible and were not observed by the 43rd day of the study. In males, no deviations in organ weight indicators relative to the control group were detected on the 29th day of the study.However, on the 43rd day of the study, statistically significant increases in relative brain mass were observed in the groups of males that received the test drug at doses of 8.6 and 34.4 mg / kg compared to the group that received saline.

[0149] Thus, as a result of the conducted study, it was established that the test preparation in all studied doses caused some toxic effects. In the lowest used dose of 8.6 mg / kg, toxic manifestations were minimal, reversible and clinically insignificant. The observed effects largely depended on the rate of intravenous administration of the preparation and the concentration of the active substance. Therefore, the use of the test preparation in a dose equivalent to the therapeutic dose for humans can be recommended for clinical studies using slow (drip) administration.

[0150] In a study of subacute toxicity of the drug - peptide inhibitor of RAS-GTPase on rabbits, no mortality was observed in animals during the study. No signs of intoxication of the animal body were detected throughout the study. Throughout the study, no statistically significant differences were observed between the groups of animals that received the test drug in two different doses and the control animals that received the vehicle in terms of body weight gain. No statistically significant differences were observed between the groups of animals that received the test drug in two different doses and the control animals that received the vehicle in terms of feed consumption. During the analysis of hematological parameters and serum biochemistry parameters of animals euthanized on day 29 of the study, no statistically significant differences were found between the animals that received the test drug in two different doses and the control animals that received the vehicle.During routine necropsy of animals, during visual inspection of the external condition of the body, internal surfaces and passages, cranial cavity, thoracic, abdominal and pelvic cavities with organs and tissues located therein, neck with organs and tissues, frame and musculoskeletal system, morphological signs of deviation from the generally accepted norm associated with the action of the studied drugs were not revealed. Statistically significant differences in organ weight between animals receiving the test drug in two different doses and control animals receiving the vehicle were not revealed on the 29th day of the study. During the microscopic analysis of histological samples of the internal organs of animals, no damage to the histostructure of the organs was found.

[0151] During the study, no signs of toxicity associated with the action of the test drug were observed. No statistically significant differences were found between the animals that received the test drug in two experimental doses and the control animals in terms of body weight gain and food consumption. Based on the study, it was concluded that the drug based on the peptide inhibitor of Ras-GTPase does not cause obvious toxic effects when administered multiple times intravenously to male and female rabbits, is safe in the range of therapeutic doses, and can be recommended for further studies.

[0152] Thus, in the course of the study on rats it was established that the test preparation in all the studied doses caused some toxic effects. In the lowest used dose of 8.6 mg / kg toxic manifestations were minimal, reversible and clinically insignificant. The observed effects largely depended on the rate of intravenous administration of the preparation and the concentration of the active substance. Therefore, the use of the test preparation in a dose equivalent to the therapeutic dose for humans can be recommended for clinical studies using slow (drip) administration. The results of the preclinical study of subacute toxicity in rabbits indicate that the test preparation does not cause toxic effects in relation to rabbits in a dose equivalent to the human therapeutic dose, taking into account the recalculation for this species.

[0153] Based on the conducted study, it can be concluded that the medicinal product based on the peptide inhibitor of Ras-GTPase does not cause toxic effects when administered intravenously multiple times, is safe in the range of therapeutic doses and can be recommended for further research.

[0154] Example 6. Toxicological studies of subacute (subchronic) toxicity of the drug K26K upon its intraperitoneal administration

[0155] The aim of the study was to determine the toxic effects of a peptide inhibitor of Ras-GTPase upon its repeated intraperitoneal administration to male and female SD rats once every three days for 16 days followed by a 14-day withdrawal period at three experimental doses.

[0156] The study used outbred Sprague-Dawley (SD) rats aged 6–7 weeks, weighing 146 ± 10 g (males) and 126 ± 12 g (females).

[0157] The test peptide or control substance (0.9% NaCl) was administered to animals intraperitoneally once every three days for 2 weeks (6-fold administration for 16 days) in a volume of 5 ml / kg in three doses: 10, 30 and 90 mg / kg. The minimum dose used in the study was 10 mg / kg, close to the human therapeutic dose, taking into account the metabolic coefficient for rats. The maximum dose was 90 mg / kg, close to the minimum dose causing adverse effects according to the acute toxicity study. The third dose of 30 mg / kg was intermediate. During the study, the animals' body weight, food consumption, manifestation of clinical signs of toxicity of the test object were recorded, functional tests were performed to assess the state of the central nervous system, respiratory and cardiovascular systems. On the 17th day of the study, some animals were euthanized and necropsied with organ collection for subsequent histological analysis.At necropsy, the weight of internal organs was recorded, and blood samples were taken for hemostasis, biochemistry, and hematology parameters, as well as bone marrow samples. The day before necropsy, the animals were placed in metabolic cages to record diuresis and collect urine for clinical examination. The remaining animals were necropsied after a 14-day withdrawal period (on day 31 of the study). During the study, the potential local irritant effect of the test drug was assessed based on the results of clinical examination throughout the study, as well as the presence of macroscopic and microscopic tissue changes in the area of ​​administration.

[0158] Table 8. Groups and doses in the study of subacute toxicity in rats with intraperitoneal administration of the drug

[0159] Starting from the first day of the study, all animals, both males and females, who received the maximum dose of the test drug (90 mg / kg), showed signs of abdominal discomfort, hunching, decreased mobility, tucking of the toes of the hind legs, and gait disturbance. The described signs were observed within 24 hours after administration and were not observed the following day.

[0160] No deaths were observed during daily inspection of the animals in the cages. Female #55 from the group receiving the test peptide at a dose of 10 mg / kg was subjected to early necropsy on the 8th day of the study with signs of peritonitis and due to progressive deterioration of the condition.

[0161] In males receiving the test peptide at a dose of 90 mg / kg, a statistically significant decrease in body weight gain was observed relative to animals receiving 0.9% NaCl (on the 7th and 14th days of the study), as well as relative to the group of animals receiving the test peptide at a dose of 10 mg / kg (day 14 of the study). In females, a similar tendency to decrease in body weight gain was observed, which did not reach statistical significance.

[0162] Table 9. Body weight and body weight gain

[0163] By the 14th day of the study, statistically significant reduction in food intake was observed in males receiving the test peptide at a dose of 90 mg / kg relative to animals receiving 0.9% NaCl. A similar trend was observed in females when administered a dose of 90 mg / kg, but the reduction in food intake was not statistically significant.

[0164] Table 10. Feed consumption

[0165] * - P < 0.05 based on the Kruskal-Wallis criterion relative to the group receiving 0.9% NaCl.

[0166] Analysis of the parameters of locomotor activity, cardiovascular and respiratory systems of animals receiving the test peptide in the studied doses did not reveal statistically significant intergroup differences on the 24th day of the study.

[0167] Neither male nor female rats showed statistically significant changes in hemostasis parameters relative to animals receiving the control substance (0.9% NaCl) on the 17th day of the study.

[0168] In rats of both sexes given the peptide Ras-GTPase inhibitor at doses of 10 mg / kg, 30 mg / kg and 90 mg / kg, no statistically significant deviations in hemogram indices were found at the end of the administration course relative to the level in the control group given 0.9% NaCl. In males given the test peptide, a slight tendency towards leukocytosis was noted due to absolute lymphocytosis regardless of the dose of the drug. In females, such an effect was observed only after administration of a dose of 10 mg / kg. During the biochemical analysis of blood serum on the 17th day of the study, a statistically significant increase in the urea level was found in males given the RAS-GTPase inhibitor at a dose of 10 mg / kg relative to the control group given 0.9% NaCl. However, the observed changes in indices were not dose-dependent and therefore cannot be unambiguously considered to be associated with the action of the test peptide.

[0169] In both male and female rats, no statistically significant changes were found in the studied parameters of diuresis and general clinical urine analysis relative to the control group receiving saline solution on the 17th day of the study.

[0170] During the routine necropsy on the 17th day of the study, a number of animals, regardless of the group and the dose of the test peptide administered, showed changes in the color of the thymus and submandibular lymph nodes, a decrease in the size of the adrenal glands, and kidney deformation, not associated with the administration of the test peptide. In a number of animals receiving the RAS-GTPase Inhibitor, signs of peritonitis were observed with the administration of all three doses. The manifestation and frequency of these signs did not have a dose dependence, however, given that they were not observed in the animals of the control group, a connection between their occurrence and the administration of the test peptide cannot be ruled out.

[0171] Table 11. Necropsy results, day 17

[0172] In both male and female rats, no statistically significant intergroup differences in absolute and relative organ weights were found on day 17 of the study. The exception was an increase in the relative and absolute adrenal weight in males on day 17 in the group receiving the test peptide at a dose of 30 mg / kg, relative to the control group. When a dose of 90 mg / kg was administered, a similar tendency to increase adrenal weight was observed, which was not statistically significant.

[0173] Table 12. Organ weight (absolute values), day 17

[0174] Table 13. Organ weight (relative values), day 17

[0175] Thus, based on the conducted safety study of the peptide inhibitor of Ras-GTPase with its repeated intraperitoneal administration to male and female SD rats once every three days for 16 days followed by a 14-day withdrawal period in three experimental doses, it can be concluded that the studied drug does not have a pronounced general toxic effect in the doses used. The observed minor deviations of some indicators from the values ​​​​in the control group did not have a dose dependence and were recorded starting from the minimum dose. However, given that they were not observed in the animals of the control group, a connection between their occurrence and the administration of the test peptide cannot be ruled out.

[0176] When the studied peptide was administered at the maximum dose, signs of local irritant action were observed, recorded during a clinical examination

[0019] .

[0177] As a result of the assessment of the pharmacological safety of the test drug, a conclusion can be made about the absence of undesirable side effects in relation to the central nervous system, cardiovascular and respiratory systems in rats when the test peptide is administered at doses from 10 to 90 mg / kg.

[0178] LIST OF USED SOURCES

[0179] 1. Ahmadian MR, Stege R, Scheffzek K, Wittinghofer A. Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nat Struct Biol. 1997 Sep; 4(9): 686-9.

[0180] 2. 2. Scheffzek K, Ahmadian MR, Kabsch W, Wiesmuller L, Lautwein A, Schmitz F, Wittinghofer A. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science. 1997 Jul 18; 277(5324): 333-8.

[0181] 3. Colombo S, Peri F, Tisi R, Nicotra F, Martegani E. Design and characterization of a new class of inhibitors of ras activation. Ann N Y AcadSci 2004; 1030: 52-61.

[0182] 4. Hocker HJ, Cho KJ, Chen CY, Rambahal N, Sagineedu SR, Shaari K, et al. Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function. ProcNatlAcadSci USA 2013; 110: 10201-6

[0183] 5. Cortes J., Albitar M., Thomas D., Giles F., Kurzrock R., Thibault A., et al., Efficacy of the farnesyltransferase inhibitor R1 15777 in chronic myeloid leukemia and other hematologic malignancies, Blood, 2003, 101, 1692-1697

[0184] 6. Alsina M., Fonseca R., Wilson E.F., Belle A.N., Gerbino E., Price-Troska T., et al., Farnesyltransferase inhibitor tipifarnib is well tolerated, induces stabilization of disease, and inhibits farnesylation and oncogenic / tumor survival pathways in patients with advanced multiple myeloma, Blood, 2004, 103, 3271-3277 7. Kim E.S., Kies M.S., Fossella F.V., Glisson B.S., Zaknoen S., Statkevich P., et al., Phase II study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in patients with taxane-refractory / resistant nonsmall cell lung carcinoma, Cancer, 2005, 104, 561-569

[0185] 8. Upadhyaya P, Qian Z, Seiner NG, Clippinger SR 1 , Wu Z, Briesewitz R, Pei D. - Inhibition of Ras signaling by blocking Ras-effector interactions with cyclic peptides. - AngewChemlnt Ed Engl. 2015 Jun 22; 54(26): 7602-6. doi: 10.1002 / anie.201502763. Epub 2015 May 7

[0186] 9. Fawell S., Seery J. et al., 1994; Vives E., Brodin P., Lebleu B. 1997; Kaplan IM et al., 2005; Gupta B. et al., 2005; Fernandez-Carneado J. et al., 2005 10. Georgiev GP. 2000; Wender PA et al., 2000; Baryshnikov A.Yu. 2004

[0187] 11. Schutze-Redelmeier M.R. et al., 2004; Dr Chin R., MCtkl D D. D, 2004; DChg-Mei Wu et al., 2004

Claims

FORMULA INVENTION 1. A polypeptide capable of inhibiting RAS GTPase activity, represented by the amino acid sequence SEQ ID NO:

1.

2. A polypeptide according to claim 1, characterized in that it is intended for the treatment of oncological diseases.

3. The polypeptide according to claim 1, characterized in that the oncological disease is represented by stomach cancer or rectal cancer.

4. The polypeptide according to I. 1, intended for the treatment of gastric cancer or rectal cancer at a dose of 0.81-3.00 mg / kg.

5. A medicinal product based on the polypeptide according to I. 1, intended for the treatment of gastric cancer or rectal cancer.

6. The medicinal product according to item 5, presented in the form of a lyophilisate.

7. The medicinal product according to item 5, intended for intraperitoneal administration to a subject in a volume of 150 ml twice with an interval of 7 days.