RU2530592C2 - Method of suppressing tumour growth - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to biology and medicine and is aimed at the increase of efficiency of the tumour growth suppression with the application of recombinant human proteins, which bind with a receptor of cytokine TRAIL/Apo2L and trigger apoptotic death of tumour cells, without damaging normal cells. In particular the invention relates to a method of suppressing the tumour growth. The invention consists in the fact that the said recombinant proteins, binding with receptors of cytokine TRAIL/Apo2L, are applied in a combination with substances, which suppress resistance of the tumour cells to TRAIL proteins, and in addition to it cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys is applied. It is supposed that the said peptide enhances the penetration of TRAIL from the vessels to the tumour cells.

EFFECT: application of a combination of recombinant TRAIL proteins and substances, which increase the sensitivity of the tumour cells to them, in a combination with the said peptide in accordance with the claimed invention, makes it possible to increase efficiency of the tumour growth suppression.

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Description

The present invention relates to biology and medicine, and in particular to methods of suppressing tumor growth using a recombinant protein that binds to the receptors (DR4 and DR5) of the TRAIL / Apo2L cytokine and starts the apoptotic death of tumor cells without damaging normal cells.

Human recombinant proteins developed on the basis of the TRAIL / Apo2L cytokine, as well as monoclonal antibodies to the DR4 and DR5 receptors of this cytokine, selectively cause the death of tumor cells without damaging normal healthy tissue cells. This provides a unique opportunity to create antitumor drugs based on such proteins that selectively damage tumor cells and do not have a toxic effect on the patient's body (see Srivastava RK, Neoplasia 2001, 3 (6), 535-546; Wang S., Oncogene, 2008, 27, 6207-6215).

A known method of suppressing tumor growth by using recombinant peptides that bind to the receptors (DR4 and DR5) of the TRAIL / Apo2L cytokine and trigger apoptotic death of tumor cells (US Patent No. 6046048, 04-04-2000).

The disadvantage of this method is that the cells of some tumors have a weak sensitivity to the action of TRAIL / Apo2L. In addition, tumor cells of various origins can become resistant to TRAIL / Apo2L proteins as a result of an adaptive response to worsening conditions of the pericellular microenvironment.

A known method of suppressing tumor growth by using recombinant peptides that bind to the receptors (DR4 and DR5) of the TRAIL / Apo2L cytokine and trigger apoptotic death of tumor cells, in which to overcome the resistance of tumor cells to TRAIL-mediated apoptosis, mutation is performed in the recombinant TRAIL protein, which decreases the efficiency of protein binding to DcR1 and DcR2 trap receptors for the TRAIL / Apo2L cytokine, which reduce the sensitivity of cells to this cytokine. As a result of this mutation, the efficiency of binding of the recombinant protein to the death receptors of DR4 and DR5 is increased, which may contribute to increasing the effectiveness of the damaging effect of the protein on tumor cells (RF Patent 24005038, 29-07-2009).

The disadvantage of this method is that the resistance of tumor cells to TRAIL-induced apoptosis is due to various mechanisms, and an increased number of receptors is only one of such mechanisms, which is not the main one for most tumor cells. This reduces the possibilities of using the mutant recombinant protein TRAIL, which has low affinity for the DcR1 and DcR2 trap receptors, to suppress tumor growth.

The closest adopted for the prototype is a way to increase the efficiency of the use of recombinant TRAIL proteins by combining them with substances that reduce the resistance of tumor cells by affecting the molecular targets of the cells responsible for their survival, in particular with the nexavar drug, which is an inhibitor of RAF kinase and tyrosine receptor kinases (Clin Cancer Res., 2010, 16 (21); 5189-99).

The disadvantage of the method adopted for the prototype is the limited penetration of the recombinant TRAIL protein into the tumor tissue, which reduces the antitumor effect of the combination of TRAIL proteins with sensitizing substances in vivo. Insufficiently effective penetration of substances to tumor cells is one of the main problems of oncology, which is caused by rapid multiplication of tumor cells, outstripping vascular growth, and increased interstitial pressure in the tumor tissue parenchyma. Particularly significant is the problem of limiting the rate of transfer to tumor cells for protein substances having a low diffusion coefficient and a quick time to remove them from the bloodstream.

The objective of the invention was to develop a method for increasing the antitumor effect of combinations of recombinant TRAIL proteins with substances that suppress the resistance of tumor cells to damaging effects due to their use with substances that increase the efficiency of their penetration into tumor tissue.

To solve this problem, a method for suppressing tumor growth using recombinant TRAIL proteins in combination with substances that suppress the resistance of tumor cells to the apoptogenic effect of TRAIL proteins is proposed, in addition to the indicated combination of substances, the cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly is used -Pro-Asp-Cys. The use of this peptide provides an increase in the efficiency of suppressing tumor growth by the indicated combinations of the recombinant protein TRAIL with substances that increase the sensitivity of tumor cells to it.

The problem of increasing the efficiency of penetration of antitumor drugs into the parenchyma of tumor tissue is one of the most relevant in oncology. Allegations that the vessels in the tumor tissue are significantly fenestrated, that is, there are gaps between the endothelial cells, and therefore are highly permeable to the preparations according to numerous experimental studies, are not true. The use of specific peptides has previously been proposed to increase the efficiency of drug penetration into tumor tissue (US Patent Application 20090246133). The method is based on the use of the Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptide incorporating the Arg-Gly-Asp motif (amino acid sequence arginine-glycine-aspartic acid), which provides its primary binding to endothelial vascular cells in the tumor tissue, and the Cys-Arg-Gly-Asp-Lys-Gly motif, which is released after proteolysis of the peptide and, by binding to the neuropilin 1 receptor on the surface of endothelial cells, increases the efficiency of the penetration of substances from blood vessels to tumor cells. Using model human tumors in immunodeficient mice, it was shown that the introduction of the peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys in addition to doxorubicin enhanced the penetration of cytostatic into the tumor tissue. Also, inhibition of the growth of experimental tumors in animals was shown, but the effect was small. The authors obtained a more significant effect with the introduction of the peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys with the drug Bortuzomab, which is an antibody against the receptor Не2 new. However, the mechanism for increasing the efficiency of drug penetration through the vessels into the tumor tissue using the Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptide remains unknown, as the authors write about it. Moreover, it remains unknown in combination with what antitumor drugs this peptide is able to increase the efficiency of their penetration into the tumor and, accordingly, antitumor activity. Accordingly, it is not known whether the use of Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptides in addition to the recombinant TRAIL proteins will be effective. Below are examples of the implementation of the proposed method on models of human tumors in immunodeficient mice.

Materials and test methods.

The test of the proposed method was carried out on models of human tumors in immunodeficient mice BALB / c nude. For this, mice of 7-8 weeks of age weighing 20-22 g were taken from the Pushchino laboratory animal nursery (FIBH RAS, Pushchino). To induce tumors, fibrosarcoma cells (NT-1080 line) or human lung carcinomas (A 549 line) were grown in vitro and injected under the skin of mice with 0.3 ml of DMEM nutrient medium, 1 × 10 ⁶ NT-1080 cells per mouse or 3 × 10 ⁶ A 549 cells per mouse. The test report was approved by the ethics committees of the Moscow Research Institute of Oncology. P.A. Herzen and the Institute of Theoretical and Experimental Biophysics RAS.

The recombinant human protein izTRAIL, modified with isoleucine zipper to increase activity, was obtained at the ITEB RAS. The izTRAIL protein caused apoptotic death of A 549, HT-1080 cells and many other tumor cells in vitro, starting from a concentration of 0.1-5 ng / ml, and did not cause the death of normal mesenchymal stem cells from human bone marrow in vitro at concentrations of 20 μg / ml.

To suppress the resistance of tumor cells, the chemotherapeutic drug doxorubicin and the targeted antitumor drug Nexavar were used. The active substance of the Nexavar preparation, sorafenib, is an inhibitor of intracellular kinases (Raf, tyrosine kinase receptors) and, according to our data, inhibits the resistance of tumor cells A 549, HT-1080 to recombinant TRAIL proteins in vitro

The cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys was obtained at the FIBCH RAS. The peptide at a concentration of 4 μg / ml was non-toxic to the used in vitro tumor cells.

The antitumor effect was evaluated by inhibition of tumor growth. Tumor growth inhibition (TPO) was calculated by the formula:

SRW (%) = [(V control-V experiment) / V control] × 100, where V is the tumor volume in mm ³

The minimum significant activity criteria for SRW were considered in excess of 50% (Guidelines for the study of the antitumor activity of pharmacological substances. / Treschalina EM, Zhukova OS, Gerasimova GK and others // Guide to experimental (preclinical) study new pharmacological substances. M: IIA Remedium, 2000, p. 319) Statistical processing of the results was carried out using the Whitney – Mann test (p <0.05).

Example 1

Testing of the proposed method was carried out on a model of a human tumor initiated by the introduction of human fibrosarcoma cells under the skin of mice, the NT-1080 line. 7 days after the injection of tumor cells, the animals were randomly divided into the following groups:

1 - a control group in which, starting from the 7th day of tumor growth, was injected intraorbitally (intravenously) with physiological saline (0.3 ml) at the same time as the izTRAIL protein and the Cys-Arg-Gly-Asp-Lys-Gly- peptide Pro-Asp-Cys.

2 - Nexavar was administered orally in mice of this group in a single dose of 50 mg of sorafenib (active ingredient of Nexavar) per 1 kg of animal weight daily for 24 days.

3 - to mice of this group, the recombinant izTRAIL protein was administered intraorbitally (intravenously) in a single dose of 15 mg / kg at the same time as Nexavar.

4 - in this group, the recombinant izTRAIL protein was administered intraorbitally (intravenously) in a single dose of 15 mg / kg 3 hours after the administration of the Nexavar preparation.

5 - to mice of this group, the Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptide was administered intraorbitally (intravenously) in a single dose of 4 mg / kg 3 hours after the administration of the Nexavar preparation.

6 - the mice of this group were injected with the peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys intraorbitally (intravenously) in a single dose of 4 mg / kg together with izTRAIL protein (15 mg / kg) 3 hours after administration the drug "Nexavar".

Data characterizing the antitumor efficacy of the complex treatment of BALB / c nude mice with human NT-1080 sarcoma using the izTRAIL recombinant protein, the Nexavar targeted preparation, as well as the Cys-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp peptide -Cys are shown in FIG.

As can be seen from figure 1, representing the growth of tumors in these groups, a recombinant antitumor protein at a dose of 15 mg / kg, 15,000 times higher than the effective concentration of this protein on NT-1080 cells in vitro, was not effective in the monovariant and the TPO was about 10% . The Nexavar preparation in concentrations toxic to these cells in vitro was poorly effective (TPO about 35%) and the peptide in non-toxic concentration did not enhance its effect. The recombinant izTRAIL protein in combination with the Nexavar preparation nonadditively inhibited tumor growth to 55%, and the additional use of the Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptide enhanced tumor growth inhibition by 80%. The increase in the tumor growth inhibition coefficient due to the addition of the cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys to the combination of the recombinant izTRAIL protein and the Nexavar preparation was significant according to the Whitney-Mann test (p <0.05).

Example 2

The test of the proposed method was carried out on a model of a human tumor initiated by the introduction of A 549 carcinoma cells under the skin. 21 days after the injection of the tumor cells, when it became possible to identify the tumors and determine their size (about 4 mm in diameter), the animals were randomly divided into the following groups :

1 - control group, in which, starting from the 21st day of tumor growth, saline (0.3 ml) was injected intravenously (tail vein) at the same time as the izTRAIL protein.

2 - the Nexavar preparation was administered to mice orally in a single dose of 50 mg of sorafenib (the active substance of the Nexavar preparation) per 1 kg of animal weight daily.

3 - to mice of this group, the recombinant izTRAIL protein was administered intravenously (tail vein) in a single dose of 10 mg / kg at the same time as Nexavar.

4 - recombinant izTRAIL protein was administered intraorbitally (intravenously) in a single dose of 15 mg / kg 2 hours after the administration of the Nexavar preparation.

5 - Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptide was administered intravenously in a single dose of 4 mg / kg 2 hours after the administration of the Nexavar preparation.

6 - in this group, the peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys was administered to mice intravenously in a single dose of 4 mg / kg together with izTRAIL protein (10 mg / kg) 2 hours after the administration of the drug " Nexavar. "

As can be seen from figure 2, which represents the growth of tumors in these groups, a recombinant antitumor protein at a dose of 10 mg / kg, 1,500 times higher than the effective concentration of this protein on A549 cells in vitro, was not effective in the monovariant to suppress tumor growth (TPO - 15 -twenty%). The drug Nexavar at a concentration of 10 μg / ml was toxic to these cells in vitro, but had a weak antitumor effect (TPO was about 35%). The peptide in non-toxic concentration did not enhance its effect. The recombinant izTRAIL protein in combination with the Nexavar preparation nonadditively inhibited tumor growth by up to 50%, and the additional use of the Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys peptide enhanced tumor growth inhibition by 75%. The increase in the tumor growth inhibition coefficient due to the addition of the cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys to the combination of the recombinant izTRAIL protein and the Nexavar preparation was significant according to Whitney-Mann statistics (p <0.05).

Example 3

The test of the proposed method was carried out on a model of a human tumor initiated by the introduction of A 549 carcinoma cells under the skin. 21 days after the injection of the tumor cells, when it became possible to identify the tumors and determine their size (about 4 mm in diameter), the animals were randomly divided into the following groups :

1 - control group, in which, starting from the 21st day of tumor growth, saline (0.3 ml) was injected intravenously (tail vein) at the same time as the izTRAIL protein.

2 - a group in which the drug doxorubicin was administered, starting from 21 days of tumor growth, intravenously (tail vein) in a single dose of 2 mg per 1 kg of animal weight with an interval of 7 days.

3 - recombinant izTRAIL protein was administered in a single dose of 10 mg / kg 2 hours (intraperitoneally), 24 and 48 hours (intravenously) after administration of doxorubicin according to the scheme of group 2.

4 - recombinant izTRAIL protein was administered in a single dose of 10 mg / kg at the same time and in the same manner as in group 3, but without the administration of doxorubicin.

5 - cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys was administered in a single dose of 4 mg / kg together with izTRAIL protein (10 mg / kg) according to the group 3 scheme after administration of doxorubicin.

6 - cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys was introduced according to the same scheme as in group 5, after administration of the Nexavar preparation, but without izTRAIL.

From figure 3, representing the growth of tumors, it is seen that a recombinant antitumor protein at a dose of 10 mg / kg, 1,500 times higher than the effective concentration of this protein on A549 cells in vitro, was not effective in the monovariant (TPO - 5-10%). Doxorubicin at a therapeutic dose caused significant inhibition of tumor growth (TPO reached 60%). The peptide in non-toxic concentration did not enhance the antitumor effect of doxorubicin. The recombinant izTRAIL protein in combination with doxorubicin non-additively suppressed tumor growth (increase of TPO up to 75%). The additional use of the peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys in combination with izTRAIL and doxorubicin enhanced tumor growth inhibition by 90%. The increase in the tumor growth inhibition coefficient due to the addition of the cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys to the combination of the recombinant izTRAIL protein and doxorubicin was significant according to Whitney-Mann statistics (p <0.05)

Thus, the proposed method of suppressing tumor growth, in which the use of the recombinant protein TRAIL in combination with antitumor drugs and targeted substances supplemented with the cyclic peptide Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, allows to increase the efficiency of inhibition of tumor growth .

Claims (1)

A method of suppressing tumor growth using recombinant proteins that bind to TRAIL / Apo2L cytokine receptors and have selective toxicity against tumor cells, in combination with substances that suppress the resistance of tumor cells to the damaging effects of these proteins, characterized in that they additionally use the cyclic Cys-Arg peptide -Gly-Asp-Lys-Gly-Pro-Asp-Cys.

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