EA023383B1 - Method for suppressing tumour growth and treating diseases by selective inhibition of receptor sites with tyrosine kinase activity - Google Patents

Abstract

The invention relates to medicine and includes a method for suppression of tumour growth, based on selective inhibition of specific receptor sites with tyrosine kinase activity, consisting of amino acids, present on cells of tumour or vessels. Selective inhibition of receptor sites with tyrosine kinase activity, described in the disclosed invention, without inhibition of the remaining part of receptor and/or other receptors in case of multi-kinase inhibitors leads to reliable increase of antitumor efficiency of applied tyrosine kinase inhibitors, reduction of toxicity of administered treatment, possibility to apply low agent concentration for complete blocking, to conduct long-term treatment. The advantage of the invention consists in development of novel class of medications for treatment of malignant tumours and other diseases.

Description

(57) The invention relates to medicine, and includes a method for suppressing tumor growth based on the selective inhibition of specific sites of receptors with tyrosine kinase activity, consisting of amino acids located on tumor cells or blood vessels. Selective inhibition of the sites of tyrosine kinase receptors described in the present invention without inhibiting the rest of the receptor and / or other receptors in the case of multikinase inhibitors leads to a significant increase in the antitumor efficacy of the tyrosine kinase inhibitors used, to reduce the toxicity of the treatment, and to use a low concentration of the agent to completely block carry out long-term treatment. An advantage of the invention is the development of a new class of drugs for the treatment of malignant neoplasms and other diseases.

SUMMARY OF THE INVENTION

The invention relates to medicine and includes a method for suppressing tumor growth, based on the selective inhibition of specific sites of receptors with tyrosine kinase activity, consisting of amino acids located on tumor cells or blood vessels. Selective inhibition of the sites of tyrosine kinase receptors described in the present invention without inhibiting the rest of the receptor and / or other receptors in the case of multikinase inhibitors leads to a significant increase in the antitumor efficacy of the tyrosine kinase inhibitors used, to reduce the toxicity of the treatment, and to use a low concentration of the agent to completely block carry out long-term treatment. An advantage of the invention is the development of a new class of drugs for the treatment of malignant neoplasms and other diseases.

Full Description of the Invention

It is known that the development of malignant neoplasms is based on excessive proliferation of cells, as well as the formation of blood vessels in the tumor through which it is fed (angiogenesis) (ΐ. Рол1тап с1. А1. ШШгс: 339, 58 (1989). Formation of new blood vessels comes from an existing endothelium and is an important component of many diseases and disorders, including such as tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovask lar glaucoma, hemangiomas, immune rejection of transplanted corneal and other tissues, and chronic inflammation.

Proliferation of tumor cells, as well as endothelial cells, can be caused by various factors that naturally occur in nature. These factors bind to receptors on the surface of tumor, endothelial and other cells, which leads to activation of the receptors and the signal inside the cell, followed by division.

According to numerous sources, receptors with tyrosine kinase activity are often expressed on tumor cells, which leads to proliferation of tumor cells and endotheliocytes, and contributes to tumor progression. Often, activating mutations of receptors with tyrosine kinase activity also alter the course of the disease and sensitivity to the therapy.

This group of receptors includes receptors for vascular endothelial growth factor (UEORK), epidermal growth factor receptors (EORK), insulin-like growth factor receptors (1ORK), fibroblast growth factor receptors (RORK). For example, the role of the latter has been proven in the development of a number of malignant neoplasms, in particular renal cell carcinoma, lung cancer (indicated as one of the causes of resistance to EOPC inhibitors), breast cancer, pancreatic cancer, stomach cancer, bladder cancer, and prostate cancer , ovarian cancer, endometrial cancer, colorectal cancer.

Receptors with tyrosine kinase activity are united by a common feature - the intracellular part is represented by tyrosine kinase. Phosphorylation of tyrosine kinase leads to receptor activation and signal propagation into the cell. Other parts of the receptors include the supramembrane and intram Membrane (transmembrane) parts. The supmembrane and intram Membrane parts of different types of receptors with tyrosine kinase activity differ from each other: both in the primary amino acid sequence and in the secondary, tertiary structure. In some cases, homology of the structure of receptors is noted. The transmembrane part of receptors with tyrosine kinase activity contains 1 transmembrane domain. Currently, a large number of targeted drugs have been created that block / inhibit the activity of various types of receptors with tyrosine kinase activity. These include tyrosine kinase inhibitors, as well as monoclonal antibodies. Existing drugs reduce the activity of intracellular tyrosine kinase by blocking the various active parts of one or, as a rule, several receptors. Indiscriminate blocking is associated with a decrease in the efficiency of action on tumor cells, vascular cells, as well as other structures expressing the corresponding type of receptor.

The present invention describes a method for selectively inhibiting certain regions of receptors with tyrosine kinase activity without affecting the rest of the receptor and / or other receptors in the case of multikinase inhibitors, which leads to a significant increase in the antitumor efficacy of the tyrosine kinase inhibitors used, the ability to use a low concentration of the agent to completely block, and therefore, to reduce the toxicity of the treatment, to conduct long-term treatment. The selective effect described in the application on receptors with tyrosine kinase activity is also different in that it is directed against specific amino acid sequences, which are often not active binding sites of a receptor with a specific ligand (growth factor), which leads to destruction of the receptor and loss of its activity. The described selective effect also implies that the ligand can bind to the receptor, but the activation of the intracellular part - tyrosine kinase - still does not happen. In addition, the selective inhibition described in the invention may be carried out against certain amino acids of tyrosine kinase receptor active centers. The present invention shows that inhibition of growth, for example, of tumor cells, can be caused by inhibition of a specific amino acid sequence and is independent of the presence of certain receptors UEORK, PORK and others. That is, all receptors can be presented on the cells, an inhibitor against each is not required - growth inhibition can be achieved by inhibiting several amino acid sequences.

The main object of the present invention is a method for selectively inhibiting the amino acid sequence that can occur in the structure of receptors with tyrosine kinase activity, which leads to the above effects. The basic amino acid sequence is shown in FIG. 1. This amino acid sequence may differ by several amino acids, and this will not affect the effectiveness of the created inhibitors. For example, in FIG. 2 and 3 show possible amino acid sequences in which amino acids from one of the ends are added.

The inventive inhibitors of the main subject matter of the invention can block any fragments of the amino acid sequence indicated in FIG. 1, which does not lead to a change in efficiency. For example, sections of the amino acid sequence may be subject to blocking as indicated in FIG. 4-10.

A distinctive feature of the present invention is the destruction of not only the active centers of the receptor, but also its other components. Thus, the effect on the amino acid sequence indicated in FIG. 11, can lead to inactivation of receptors and can be used in treatment.

Therapeutic use of the method

To use the method described in the present invention, in therapeutic practice, any antagonists of these amino acid sequences by the method are administered to a mammal, preferably to a human, in a pharmaceutically acceptable form, including intravenous, as well as the following routes: intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, inturtisinovial , intracranial, oral, local or inhaled.

Antagonists can also be administered by intratumoral, peritumoral, intrafocal and focal pathways to provide local action along with a systemic therapeutic effect. Such administration forms include pharmaceutically acceptable carriers that are inherently neither toxic nor therapeutic. Examples of such carriers are ion exchange agents, alum, aluminum stearate, lecithin, plasma proteins (such as human plasma protein), buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon oxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances and polyethylene glycol. Carriers for local or gel-based antagonist forms include polysaccharides, such as sodium carboxymethyl cellulose or methyl cellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene polyoxypropylene block polymers, polyethylene glycol and alcohols. For administration in all cases, conventional dosage forms obtained from warehouses are used. Such forms include, for example, microcapsules, nanocapsules, liposomes, patches, inhalation preparations, aerosols, sublingual tablets, and sustained release preparations. The antagonist in such preparations will usually be contained in a concentration of from about 0.001 to 100 mg / ml.

Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing an antagonist; such matrices have a certain shape, for example, it can be films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels [for example, poly (2-hydroxyethylmethacrylate)] described by Langer et al. (1. Wuschet D. Mae. Kek. 15, 167 (1981) and Langer (Cbet. Tesb. 12 ( 1982), or poly (vinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl L-glutamate, described by Sidman et al. (Wurutheck 22, 547 (1983), non-degradable ethylene vinyl acetate (Langer et al. see above), degradable copolymers of lactic and glycolic acids, such as Lirgoi Eero1 (injectable microspheres consisting of polymers lactic and glycolic acids and leuprolide acetate), and poly-E - (-) - 3-hydroxybutyric acid, while polymers such as ethylene vinyl acetate and a copolymer of lactic and glycolic acids are capable of continuously releasing molecules for more than 100 days Certain hydrogels release proteins in shorter periods of time.When the encapsulated polypeptide antagonists remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at a temperature of 37 ° C, which leads to the loss of biol cal activity and possible changes in immunogenicity. In order to stabilize, reasonable strategies may be developed, depending on the mechanism in place. For example, if an aggregation mechanism is found, which manifests itself in the formation of an intermolecular δ-δ bond via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization to remove acidic solutions, control humidity, use appropriate additives and develop specific polymer

- 2 023383 matrix compositions.

Antagonistic sustained-release antagonist formulations also include antagonistic antibodies contained in liposomes. Liposomes containing antagonists can be obtained by methods known in the art, for example, those described by Epstein et al. (Pr. ΝηΙ. Asai. 8c1. 82, 3688 (1985); Huang et al. (Prgoss. Baa. Asai. 8c1. 77, 4030 (1980); US Patent No. 4,445,045 and US Patent No. 4,544,445. Liposomes are generally small (about 200-800 A) and belong to a single-layer type in which the lipid content is higher than 30 mol% cholesterol; the selected ratio may vary to select the optimal treatment conditions.Liposomes with a long circulation are covered by patent Ohm No. 5013556.

Another way of using this invention is the incorporation of the antagonist into products having a specific shape. Such products can be used to modulate endothelial cell growth and angiogenesis. In addition, such products can be used to modulate the invasion of tumors and metastases.

Possible conjugation of the antagonist by the method and other therapeutic agents. When preventing or treating a disease, the required dose of an antagonist will depend on the type of disease, on its severity and course, on whether antibodies are administered for prophylactic or therapeutic purposes, on previous therapy, on the patient’s medical history and his response to the antagonist, and on the instructions of the patient a doctor. The antagonist can be administered to the patient in various ways, at a time, or as a series of appointments.

Antagonists that selectively affect amino acid sequences of the present method can be used to treat various neoplastic and non-neoplastic diseases and disorders. Tumors and related conditions that can be treated with this include breast cancer, non-small cell and small cell lung cancer, tracheal tumors, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial hyperplasia, endometriosis, fibrosarcoma, chondrosarcoma, head and neck tumors, hepatoblastoma, Kaposi’s sarcoma, melanoma, skin cancer, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic cancer, adrenal cancer, retinoblastoma, astrocytoma, glioblastoma, annomu, oligodendroglioma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, renal cell carcinoma, bladder cancer, cancer of the penis, prostate cancer, Wilms' tumor, abnormal vascular proliferation associated with phakomatoses.

It is possible to use the method for non-cancer diseases that can be treated, including such as rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, fibroplasias, neovascular glaucoma, thyroid hyperplasias (including Grave’s disease), transplantation of the cornea and other tissues, chronic inflammation, pneumonia, nephrotic syndrome, ascites, preeclampsia, pericardial effusion (for example, associated with pericarditis) and pleural effusion. Depending on the type of disease and the severity of the disease, the initial dose for administration to the patient will be from 1 μg / kg to 30 mg / kg and may be administered by one or many separate prescriptions / administrations or by continuous infusion. A typical daily dose may vary from about 1 μg / kg to 100 mg / kg or more, depending on the above factors. For reappointment for several days or more, depending on the conditions, treatment is repeated until the desired suppression of the symptoms of the disease is achieved. However, other dosage regimens may also be used. The success of treatment is easily determined by conventional methods and analyzes, for example, X-ray imaging of tumors.

According to another application of the invention, the effectiveness of an antagonist in preventing or treating diseases can be improved by administering the antagonist serially or in combination with another substance effective for a given purpose, such as, for example, tumor necrosis factor, interferons, interleukins; antibodies and small molecule inhibitors capable of neutralizing or inhibiting the activity of vascular endothelial growth factor and / or its receptors, and / or hepatocyte growth factor and / or its receptors, and / or epidermal growth factor and / or its receptors, and / or placental growth factor and / or its receptors, and / or epidermal growth factor and / or its receptors, and / or insulin-like growth factor and / or its receptors, and / or growth factor of fibroblasts and / or its receptors, and / or tTOC, and / or other intracellular kinases, or one or more its usual therapeutic substances, such as, for example, alkylating compounds, antibiotics, antimetabolites, anthracyclines, vinca alkaloids, epipodophyllotoxins, and other cytostatics. Such substances may be present in the composition to be administered or may be administered separately. In addition, the antagonist of the method can be administered serially or in combination with radiological treatment.

One or more antagonists are administered to a patient with a tumor in therapeutically effective doses determined, for example, by observing tumor necrosis or its metastatic foci, if any. Such therapy continues until further improvement ceases to be observed, or clinical examination shows that the tumor or its metastases have disappeared. With the progression of the disease, one or more of the substances described above (o) are introduced or other treatment methods are used. Since the effectiveness of additional substances will vary, it is advisable to compare them

- 3 023383 effect on the tumor by standard matrix screening. Repeated administration of the antagonist and additional agent is performed until the desired clinical effect is achieved. Alternatively, the antagonist (s) of the method are administered together and, if desired, together with additional substances.

The antagonists of the method can be used with supportive and accompanying therapy drugs, for example, with erythropoietins, drugs that stimulate leukopoiesis or increase the number of platelets and / or neutrophils, macrophages, with nutritional support, non-steroidal anti-inflammatory drugs, blood components, dexamethasone, zoledrone against KAKIK.

Below, as examples, some evidence is provided of a method for suppressing tumor growth by blocking the amino acid sequences provided in the present invention. The following examples are offered by way of illustration only and should not be construed as limiting the present invention in anything.

Example 1. Research results: analysis of the survival or proliferation of tumor cells ίη νίΐτο with the addition of a monoclinal antibody blocking the amino acid sequence shown in FIG. one.

The following cell lines were selected for the study:

1) human non-small cell lung cancer;

2) human prostate cancer;

3) human breast cancer;

4) human renal cell carcinoma;

5) human stomach cancer;

6) human nasopharyngeal cancer;

7) human ovarian cancer.

On the cells of these cell lines in the standard Western blot analysis, overexpression of receptors of various growth factors was determined, namely UEORK, PORK, EORK, URK, as well as platelet growth factor (ΡΌΟΡΚ). The overall level of expression of factors on tumor cells ranged from 30-100% depending on the type of receptor and cell line. Using sequencing, it was proved that the receptor regions with the amino acid sequence shown in FIG. 1-11 are present on cells of all lines except the line of human nasopharyngeal cancer. Based on the findings, human lines of renal cell cancer, breast cancer, prostate cancer, non-small cell lung cancer, stomach cancer, and ovarian cancer were selected for further research. As an antagonist blocking the amino acid sequence shown in FIG. 1, a specific high-affinity (Kb = 1.7 nM) mouse monoclonal antibody 10-10 was used. The term monoclonal antibody is used here to refer to an antibody obtained from a population of sufficiently homogeneous antibodies, i.e. the individual antibodies that make up the population are identical in their specificity and affinity, with the exception of possible naturally occurring mutations that may be present in small amounts. The 10-10 blocking murine antibody was specifically developed using the hybridoma method (O. Kbyob, S. Μίΐδίοίη. Ы ааЩЩг 256 256, 495 (1975) for the present studies. The mouse was immunized with an antigen representing the peptide from the amino acid sequence shown in Fig. 1 .

Tumor cell lines were seeded at a density of 10 ⁴ cells / ml in 6-well plates. A 10-10 blocking monoclonal antibody was added to each well at a concentration of 100 μg / ml and in equal volume. Also, as a control, an extraneous monoclonal antibody without neutralizing ability was added to a portion of the culture (acquired in Lacanth, concentration 100 μg / ml). After incubation, a combination of growth factors (UEOR, POP, 1OP, EOP) of 1 ng / ml was added to each well. As an additional control, part of the cells was grown in the absence of both antibodies and growth factors. After culture growth for 3 weeks, the cells in each well were counted using a computer program on a NeShey Raskagb 5> sap) e1 analyzer (USA).

As shown in FIG. 12, monoclonal antibody 10-11 completely inhibited the ability of the added growth factors to support the growth and survival of cells of all these tumor lines (more than 90%). A monoclonal antibody without neutralizing ability (ABcat) did not cause changes in cell survival. In the experiment, it was found that the suppression of proliferative activity does not depend on the dose of the agent that blocks the amino acid sequence (the effectiveness of several doses was the same).

The general conclusion of this example: when selectively exposed to a blocking agent, in this case an antibody, on the amino acid sequence indicated in FIG. 1, strong inhibition of tumor cell growth is achieved with respect to several cell lines.

Example 2. Research results: study and comparison of the effectiveness of isolated blocking of the amino acid sequence shown in FIG. 1, and blocking several receptors with tyrosine kinase activity with standard tyrosine kinase inhibitors.

- 4 023383

In the study, a line of human renal cell carcinoma of the Sai type was selected as an experimental cell line, the cells of which express various receptors with tyrosine kinase activity, in particular in 80-100% - UEORP, POPC and RIOPP, which was revealed in a previous study. In this regard, this cell line was used in experiments as a model.

Cells were seeded in 96-well plates with a volume of 90 μl / well. After 24 hours of incubation in a humidified incubator at a temperature of 37 ° C, 5% CO ₂ and 95% air, test dissolved agents were added to the cells in an amount of 100 μg / ml. As the test agents, low molecular weight inhibitors were used: 1) the ΙΟ -11 inhibitor obtained by chemical synthesis for this study, purposefully blocking the amino acid sequence shown in FIG. 1, and 2) commercially available ΒΙΒΡ 1120, which is an inhibitor of UEORP, RIORR, PORK, showing 1C50 in the range (20-100 nmol / l).

Inhibitors were added to the cells; some of the cells were left without the addition of any agent (control group). A day after incubation with inhibitors, a mixture of growth factors UEOR, RIOR, POP in an amount of 10 nmol / L was added to the cells. In the event that inhibitors blocked the activity of receptors with tyrosine kinase activity, the added growth factors should not have their effect. The cells of the control group were divided into two subgroups, growth factors were added to one, the other was left without stimulation.

Incubation with growth factors lasted for 2 days, after which all cells were counted using a computer program on an analyzer Нс \ у1сО Raskagb 5> sap] e1 (USA).

The percentage inhibition was estimated as the ratio of the number of cells in the group with inhibitors to cells that were not treated and were stimulated by growth factors. The inhibition level for ΙΟ-11 was 95%, the inhibition level for B1BP 1120 was 54%. Statistical differences in the number of cells for ΙΟ-11 and B1BP 1120 were significant (P = 0.003), which means advantages in inhibition for ΙΟ-11. The number of cells in the control group without stimulation was significantly lower than in the control group with stimulation by growth factors (P = 0.001). The ΙΟ11 inhibitor significantly inhibited the effects of added growth factors; the number of cells was significantly lower compared to the stimulated control (P <0.001). The results are graphically shown in FIG. thirteen.

General conclusion: selective inhibition of the amino acid sequence indicated in FIG. 1 leads to better efficacy results than non-selective inhibition of the UEORP, POPP, and RIOPP receptors. Growth factors have a stimulating effect on the proliferation of tumor cells; their effects are significantly inhibited by inhibiting the amino acid sequence shown in FIG. one.

Example 3. Test results: inhibition of tumor growth ίη νίνο while blocking the amino acid sequence shown in FIG. 1, 2, 4 and 11.

Female mice (Thin / Peebe) 8 weeks old (purchased from Nag1ap Zrgadie JaMeu, 1ps. (Indianapolis, USA) were injected subcutaneously with 2x10 ⁶ human renal cell carcinoma (Cact) tumor cells in 100 μl of phosphate buffered saline (PBS). After the growth of the tumor was established (1 mm ³ ), the mice were divided into 7 groups.

The first (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with the monoclonal antibody ΙΟ-10 described in the previous examples at a dose of 10 mg / kg.

The second (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with the ΙΟ-11 inhibitor described in the previous examples at a dose of 10 mg / kg.

The third (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with a ΙΟ-11/2 inhibitor obtained by chemical synthesis and blocking the amino acid sequence shown in FIG. 2, at a dose of 10 mg / kg.

The fourth (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with a ΙΟ-11/4 inhibitor obtained by chemical synthesis and blocking the amino acid sequence shown in FIG. 4, at a dose of 10 mg / kg.

The fifth (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with the ΙΟ-11/11 inhibitor obtained by chemical synthesis and blocking the amino acid sequence shown in FIG. 1, at a dose of 10 mg / kg.

The sixth (control) group of mice (N = 10) did not receive treatment.

The seventh (control) group of mice (N = 10) was treated with non-specific immunoglobulin (1dO), intraperitoneally, at a dose of 10 mg / kg.

Tumor size and animal weight were measured every 3 days. Mice underwent euthanasia upon reaching a tumor of 2000 mm ³ or on day 60 of the experiment.

The graph of tumor growth in groups is shown in FIG. 14. The figure shows that in those mice that were introduced antagonists (monoclonal antibody or low molecular weight inhibitors) of the amino acid sequence shown in figures 1,2,4,11, tumor growth was significantly slowed down than in mice from the control groups.

The median tumor volume of the mice of the control groups was significantly larger compared to the mice of the treatment groups (P <0.001).

- 5 023383

In more than 60% of mice treated with antagonists, the tumor volume did not reach a critical value of 2000 mm ³ during the control period of the study - 60 days, in contrast to the mice of the control groups, where the last mouse was euthanized on day 32, as the tumor volume reached 2000 mm ³ .

General conclusion: selective antagonists (both monoclonal antibodies and inhibitors obtained chemically), amino acid sequences (Figs. 1, 2, 4, 11) have a significant inhibition of tumor growth, thereby affecting life expectancy.

Example 4. Research Results: Comparison of the effectiveness of selective inhibition of the amino acid sequence shown in FIG. 1, and general blocking of receptors with tyrosine kinase activity against tumor growth ίη νίνο.

Female mice (Vads / Pibs) 6-8 weeks old (purchased from Nag1ap 8gradie Oau1eu, 1ps. (Indianapolis, USA) were subcutaneously injected with 2x10 ⁶ human renal cell carcinoma cancer cells (Sacl) in 100 μl of physiological saline with phosphate buffer (RFPB) ) After tumor growth was established (1 mm ³ ), the mice were divided into 6 groups.

The first (control) group of mice (N = 10) did not receive treatment.

The second (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with the ΙΟ-11 inhibitor described in the previous examples at a dose of 10 mg / kg.

The third (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with the ΙΟ-11 inhibitor described in the previous examples at a dose of 50 mg / kg.

The fourth (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with a ΒΙΒΡ 1120 inhibitor blocking UEORK, ΡΌΟΡΚ, PORK, at a dose of 10 mg / kg.

The fifth (treatment) group of mice (N = 10) was injected intraperitoneally 2 times a week with a ΒΙΒΡ 1120 inhibitor blocking UEORK, ΡΌΟΡΚ, PORK, at a dose of 50 mg / kg.

Tumor size and animal weight were measured every 3 days. Mice underwent euthanasia upon reaching a tumor of 2000 mm ³ or on day 60 of the experiment.

The change in the median tumor volume and the time to reach a tumor size of 2000 mm ³ are shown in the table.

As can be seen from the table, after 15 days of treatment, significant differences were achieved in the median of the tumor volume between groups 2 and 4. An increase in the dose of the ΙΟ-11 inhibitor did not affect the effectiveness, and, therefore, even a small dose is accompanied by a pronounced effect. On day 20, significant differences in tumor size were obtained in favor of ΙΟ-11 for all treatment groups.

A slowdown in tumor growth was observed in groups of mice treated with ΙΟ-11.

General conclusion: inhibition of the amino acid sequence shown in FIG. 1 is more effective in suppressing tumor growth than inhibition of receptors UEORK, ΡΌΟΡΚ, PORK. The use of inhibitors that selectively block this amino acid sequence can reduce the dose of the drug without loss of effectiveness, which will be accompanied by less toxicity.

Example 5. The results of the study: analysis of the survival or proliferation of eidothelial cells ίη νίίτο when adding monoclonal antibodies ΙΟ-10.

To analyze the survival of endothelial cells in the environment of vascular endothelial growth factors (UEORA) and fibroblast growth factor (LOBP) and when the amino acid sequence shown in FIG. 1, a similar experiment was conducted as when blocking the vascular endothelial growth factor described in US Pat. No. 2,900,222,716 (Koskue 11; RGPOa ea a1. I8 Kaίeηί 20090022716).

As a model of endotheliocytes, bovine cells of the capillary endothelium of the adrenal cortex (ECCN) were used (Reggaga ea a1. Proc. No. Asab. 8sk 84: 5773 (1987).

In the beginning, we revealed a high expression of UEORK and RORK on ECEC in the standard Western blot analysis.

Then EECs were seeded with a density of 5x10 ⁴ cells / ml in 12-well plates. UEOR-A 10 ng / ml and LOBP 10 ng / ml were added to each well in the presence or absence of monoclonal antibody ΙΟ-10 (10 μg / ml), as well as an extraneous monoclonal antibody without neutralizing activity (ABcat (10 μg / ml )). After culture growth for 5 days, the cells in each well were counted using a computer program on a Neuben Raskagb 8sap] e1 analyzer (USA). As an additional control, ECECs were grown in the absence of growth factors.

As shown in FIG. 15, the monoclinal antibody ΙΟ-10 inhibited the ability of the added growth factors to support the growth and survival of bovine ECEC.

A monoclonal antibody without neutralizing activity (ABcat) had no effect on cells.

Thus, the example demonstrates that when blocking the path indicated in FIG. 1 amino acid sequence, endothelial cells stop multiplying and lose mitogenic activity, which can lead to a violation of angiogenesis in the tumor.

- 6 023383

The list of amino acid sequences

Amino acid sequence (8EO GO ΝΟ: 1)

KMEKKNAURAAYUTUKGKSRASSIRMRTMKIKSKERK0ENK1SSUKUKI0NI81.1MB5

UUR $ OKZMUTS7UBNEU $ GMNTUNSU7EK $ РНРР1Ь0АСЕРАМА5ТУУ5еОУУГУСКУ

U5OASRN10M1KNUEKMSZKUSR0SRUKUKAAOTTMTTOKE1EU ¥ 1KMUTGEOASEU

TSJAC№TS15EN5AIEETUARARSR.EKETTA5R0UEETAGUS16 UREGASM7UTUT1ES

Amino acid sequence (§Εζ> ΙΟ ΝΟ: 2).

RUMTMTEKMEKRYNAURAATSUKEXRA (ZSKRMRTME№EKMSKERK0ENK113SUKUR

Ν0ΗΜ5ΕΙΜΕ2ννΡ50Κ0ΝΥΤ € ννΕΝΕΥ68ΙΝΗΤΥΗΕΌννΕΗ5ΡΗΚΡΙΕ0Α8ΕΡΑΝΑ5Τνν

ΟαϋνΕΓνσκνΥδΟΑΟΡΗΙΟ ^ ΚΗνΕΚΝΟδΚΥαΡΟαΕΡΥΕΚνΕΚΑΑσνΝΤΤΟΚΕΙΕνί-ΥΙΡ

MUTGEOAASEUTSEASN51S12ENZAI1TUARARSKEKE1TAZRO ¥ ЬЕ1А1УС1С7ГЫАС

Μνντνιι, σ

Amino acid sequence (8E () GO ΝΟ: 3).

KMKENAUR ΑΑΝΤνΚΕΡΟΡ ASSYRMRTMK IKYSKERKS) ENK1SSUKUK IONIZYMBZ ννΡ $ ΡΚ (5ΝΥΤ 0ννΕΝΕΥ23ΕΝΕΥ ΝΗΤΥΗΣΟννΕ KZRNR1Y2A ZRAMAZTUU SZBUEGUSKU

ΥδϋΑΩΡΗΙΟΜ ΙΚΗνΕΚΝΰδΚ HARNESS UKAASUYT ΌΚΕΙΕνΣΥΙΚ ΝνΤΕΕΌΑΰΕΥ

TSEASYZKP ZENZAIT ΡΑΡ6ΚΕΚΕΙΤ Α3ΡΌΥΕΕΙΑΙ USUTYAS ΜνντνίΙΌΚΜ

Amino acid sequence (8E (? GO ΝΟ: 4).

ΚΜΕΚΒΕΗΑνΡΑΑΝΤνΚΓΡ0ΡΑΰ2ΝΡΜΡΤΜΚ1 ^ ΚΝ3ΚΕΓΚζ> ΕΗΚΙ33Υϊ <νΚΝΐ2ΗΗ3ΕΙΜΕ3 ννΡ5ΕΚ5ΝΥΤ 0ννΕΝΕΥ35Ι ΝΗΤΥΗΙΌ К5РНВР1Ы2А 3 [, ΡΑΝΑ5Τνν ССОУЕЕУСКУ

ΥδΌΑίΡΗΙΟΜ ΙΚΗΛΈΚΝΰδΚ USROSRYRUK νΕΚΑΑΰνΝΤΤ ΟΚΕΙΕνΣΥΙΕ ΝνΤΕΕΌΑΰΕΥ

TSASM3131 ZENZAIET

Amino acid sequence (8Ε <^ GO ΝΟ: 5).

ΙΕ £ Α0ΕΡΑΝΑ $ Τνν6ΰ0νΕΕνςκνΥ50Ας> ΡΗΐς) ΝΐΚΗνΕΚΝ <35ΚΥ3Ρ0 <3ΕΡΥΣΚ

7ЬКААСЫЫТТ0КЕ1ЕУЬ ¥ 1ЕМУТРЕ0АСЕУСЬАСЫ5ТС12 ГНЗАЙТ ¥ Ъ

RARSREKE1TA5R0UIE1A1US1S7EYASMU7T71S

Amino acid sequence (8E <2 GO N0: 6), km ьн ν ν ν ν ΡΑί ΡΑί ΡΑί ΡΑί ΡΑί ΡΑί ν ν

Amino acid sequence ($ E <3 GO N0: 7).

ΚΜΕΚΕΕΗΑνΡΑΑΝΤνΚΓΡΑΙΝΗΊΪΗί0ννΕΚ3ΡΗΒΡΓΕ2Α3ΕΡΑΝΑ3ΤννΰΟ0νΕΕνσΚνΥ3ΟΑζ) ΡΚΙ2Μ

ΙΚΗνΕΚΝΰ5ΚΥΰΡΌ3ΑΡΥΣΚνΓΚΑΑ3νΝΤΤ0ΚΕΙΕνΣΥΙΡΝντΕΕϋΑ (3ΕΥΥ2ΕΑ (5Ν5Ι (3Γ5ΕΗ5ΑΝΕΤ

Amino acid sequence (8E <3 GO ΝΟ: 8).

ΚΜΕΚΚΕΗΑνΡΑΑΝΤνΚΓΚ € ΡΑ6ΟΝΡΜΡΤΜΚΝΕΚΝ6ΚΕΓΚ0ΕΗΚΙ00ΥΚνΒΝ0ΗΜ5ΕΙΜΕ5ννΡ5ϋΚ <3Ν ¥ Τ

ΟννΕΝΕΥΰδΙ ^ ΤΥΗΕΌννΕΗδΡΗΗΡΙΕφΑΰΕΡΑΝΑδΤννΰΟΏνΕΓνΟΚνΥδΏΑίΡΗΙΰΜΙΚΗνΕΚΝΰδΚ

Υ3ΡΟ2ΕΡΥΕΚνΕΚΑΑ3νΝΤΤ0ΚΕΙΕνΕΥΙΡΝνΤΡΕ0Α2ΕΥΤ0ΕΑ2Ν8Ι0Ι3ΡΗΞΑΗΕΤ

Amino acid sequence (5E <2 GO N0: 9).

ΚΜΕΚΕΕΗΑνΡΑΑΝΤνΚΕ3ΡΗΕΡΙΕΟΑ3ΕΡΑΝΑ3Τνν £ ΰθνΕΕνθΚνΥ3 & ΑΟΡΗΙΏΝΙΚΗνΕΚΝ65ΚΥ (: ΐ <3νΓΕΙΑΟ

Amino acid sequence (8E <? GO ΝΟ: 10).

ννΕΚ3ΡΗΚΡΙ3ΓΗ3ΑΝΕΤνΕΡΑΡ3ΚΕΚΕΙΤΑ3Ρ0ΥΕΕΙ

Amino acid sequence (5E <5 GO ΝΟ: 11).

ΑΙ USSUGETASMUUTUTES

Claims (3)

CLAIM 1. A method of suppressing tumor growth or treating diseases associated with tyrosine kinase receptor activity, comprising administering inhibitors that selectively affect the amino acid sequence of §EC EC GO N0: 1 in the structure of a tyrosine kinase receptor or to a sequence different from §EC EC GO N0: 1 by inserting several amino acid residues, or on a fragment of the sequence §EC EC GO N0: 1. 2. The method according to claim 1, where the sequences that differ from §ETS GO N0: 1 by the insertion of several amino acid residues are §ETS GO N0: 2 and 3. 3. The method according to claim 1, where fragments of the sequence §ETS GO N0: 1 are §ETS GO N0: 4-11. Change in median tumor volume and time to reach a tumor size of 2000 mm ³ in groups of mice Median tumor volume, mm ³ Days from start of experiment 10 fifteen twenty 60 1. Control group (N = 10) 950 1200 1800 ΝΑ 2.10-11.10 mg / kg (N = 10) 350 400 400 800 3.10-11.50 mg / kg 300 350 400 950 4. ΒΙΒΡ 1120, 10 mg / kg (N = 10) 450 1000 1500 ΝΑ 5. ΒΙΒΡ 1120, 50 mg / kg (N = 10) 350 600 1050 1800 The number of animals whose tumor reached 2000 mm ³ , Ν (%) Days of the start of the experiment fifteen twenty 34 60 1. Control group (N = 10) 3 (30) 6 (60) 10 (100) - 2.10-11, 10 mg / kg (N = 10) 0 (0) 1 (10) 3 (30) 4 (40) 3.10-11.50 mg / kg 0 (0) 0 (0) 2 (20) 4 (40) 4. ΒΙΒΡ 1120, 10 mg / kg (N = 10) 1 (10) 4 (40) 10 (100) - 5. ΒΙΒΡ And 20.50 mg / kg (N = 10) 0 (0) 2 (20) 8 (80) 9 (90) NΑ - impossible to evaluate (all animals were euthanized earlier, since the tumor size was more than 2000 mm ³ ) Amino acid sequence (8E () 1B ΝΟ: 1) ΚΜΕΚΕΙ, ΗΑνΡΑΑΝΤνΚΓΜ; ΡΑε5ΝΡΜΡΤΜΗΗΙ.Κ№3ΚΕΓΚΐ3ΕΗΕΐεθΥΚνΐΤΝ (2ΗΗ3Ι, ΙΜΕ3 UUR30KSN ¥ TSUUENE ¥ S31MNTUN1ZHUUEK5RKR1TS2ASYRAYAZTUU0S0UERUSKU ΥΒΟΑΟΡΗΙΟΜΙΚΗνΕΚΝΟΒΚΥΕΡΕΧΠ, ΡΥΙ, κνΑΚΑΑΟνΝΤΤΟΚΕΙΕνί, ΥΙΚΝνΤΕΈΟΑσΕΥ TSVAEZYLZENZANTURARECEKETTAZROUETMUSUTEASMUUTUPH FIG. one Amino acid sequence (8E <3 GO ΝΟ: 2). RUITMTEKMEKEYNAURAAMTUKERSRABSNRMRTMNMKYSEKOENKTSSUK LOW MESUURZ ϋΚαΝΥΤσννΕΝΕΥΕΒ I ΝΒΤΥΗΕθννΕΚ3ΡΗΚΡΙΕ0ΑΌΕΡΑΚΑ3Τνν ΟΟΟνΕΓνςΚνΥδΟΑΟΡΗΙΟΜΙΚΗνΕΚΝεδΚΥΟΡΟεΕΡΪΕΚνΕΚΑΑσνΜΤΤΟΚΕΙΕνΕΥΙΕ MUTEE0ASEUTSASR31S13GNZAITUEREEREZE1TAZR0UIE1A1US1eURYAS ΜννΤνίΤ, Ε FIG. 2 Amino acid sequence (8E0 Ιϋ N0: 3). KMEKKNAUR AAKTUKRESR ASZMRMRTMK IKYASKEGKS ENRTSSSU / V. No. 2 LOWMESIS ννΡ30Κι3Ν ¥ Τ € ννΕΝΕΥ03Ι ΝΗΤΥΗΕΟννΕ К5РНКР1Ь £ А СРАМАЗТУУ ССОУЕР / СУ / Υ50Ας) ΡΚΙζ> Μ TKNUEKNbZK SIMPLIFY νΐ, ΚΑΑΟνΝΤΤ ΟΚΕΙΕνίΑίΙΒ. ΝνΤΕΕΩΑΰΕΥ tsiasmz1S1 zrnzantu RARSKEKE1T azrouje1A1 usyutryas ΜνντνιυΕΚΜ FIG. 3 - 8 023383 Amino acid sequence (8EO GO ΝΟ: 4). ΚΜΕΚΚηΗΑνΡΑΜΤνΚΓΚΟΡΑεεΝΡΜΡΤΜΚΗΙ, ΚΝΟΚΕΓΚ0ΕΗΚΙ (30ΥΚνΚΝ (2ΗΗΕΙ.ΙΜΕ5 ννΡΕϋΚεΚΥΤ SUUENEUS £ 1 FIG. 4 Amino acid sequence (8 RA) 1ϋ ϋ: 5). IZASARAIAZT UUSSIUBGUSKU Υ 3 OJSC PH I I ОI I КН УЕ КИСЗ К Υ3 Р axis Ρ ΥI. TO UKAAUMTTOKE К ΕνίΛΙΚΗνΤΕΈΟΑΟΕΥΤΟΤ, ΑΟΝΞΙΟΙ Ξ GNZAITU RARSKEKEKHTAZROUYEHA! USGOURYASMUUTU1ES FIG. 5 Amino acid sequence (8Εί) No. ΝΟ: 6). KMEKKIAIAUR ΑΑΝΤνΚΓΚΑΤ USTEURUYS MUTUTES FIG. 6 Amino acid sequence (ZE <) GO ΝΟ: 7). KMEKKNAURAAITUKRVASHNTUNSUUEKERNKRIDAGRANAZTUUSSOUEGUSKUUZOAORNTSGI 1KNUEKNSSKUSR0SPR ¥ ЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬЬУЖУМУТУАЕУУСЬАТСМ £ 1С13РН5АНЕТ FIG. 7 Amino acid sequence (5E <) GO ΝΟ: 8). ΡΑΑΝΤνΡΑΑΝΤνΚΓΙίΟΡΑ5ΰΝΡΜΡΤΜΒ№ΚΜΟΚΕΓΚ0ΕΗΚΙ0σΥΚν№Κ) ΗΜ £ υΜΕ3ννΡ30ΚεΝΥΤ θννΕΝΕΥΟ3ΙΝΗΤΥΗΕθννΕΚ5ΡΗΚΡΙΙ, ΟΑεΐ, ΡΑΝΑ3Τννθ0ΕνΕΕ · ν (: ΚνΥ5ΟΑι2ΡΗΙ FIG. 8 Amino acid sequence | 3E <) GO N0: 9). KMEKKNAURAAYUTUKKZRNKRVDASLRANANTUUSSOUEGUSKUUZOA'ZRNZHTKNUEKNESKUSUSHUSHUS FIG. nine Amino acid sequence (8E <) C> ΝΟ: 10), UUEK5RNE.R15GNZAN1, TURAREKEKE1TA8R0U1, E1 FIG. 10 Amino acid sequence (8E <) Yu ΝΟ: 11). ΑΙ USISURYΑΟΜνντνιс FIG. eleven - 9 023383 Ipnbiroyans of the growth of colonies of cells of iol by the action of monoclonalind about antibodies 1010 against amino acid nasledovatelnoye! N XE (} 11) N0: 1 NSCLC RPZh RMZh RKR RZh Rya Designations: NSCLC - non-small cell lung cancer 11 lung cancer ΡΙ GH - cancer of the flying gland Breast cancer - cancer of the smaller gland PC!' renal cell carcinoma RJ - stomach cancer 1 * I am ovarian cancer Schedule: Column 1 Control: cells without lobes | cge111Sh of any agents Column? - Monoclonal antibody 10-10. 1 SJ mcg / ml (.. ‘sausage? - 1 ^ specific! Cs, kh) Column 4 - Control; cells stimulated by growth factors, beta addition of monoclonal antibodies or 1gS 12 Inhibition of colony growth to lightly by leukemia of the GTN of the pathway 10-11 prog amn1H) K1 (c. || relative | sequence 8EP II) N0: 1 by another ......