RU2680834C1 - Antitumor composition of doxorubicin with atp inhibitor-dependent reverse cell transporter - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, in particular to an antitumor composition, which is an aqueous solution of doxorubicin and an inhibitor of ATP-dependent reverse cell transporters in a 1:15 mass ratio, where the inhibitor of ATP-dependent reverse cell transporters is a mixture of polyoxypropylenehexol of formula 4,where n=2–6, and polyoxypropylene glycol of the formula 5,where m=5–9, having a hydroxyl number of 215–240 mg KOH/g, compound 4 has a molecular weight from 1,000 to 1,500 Da, compound 5 has a molecular weight from 300 to 500 Da, the molar ratio of compounds 4 and 5 is 0.9–1.1.EFFECT: invention provides an increase in the efficacy and safety of the action of the antitumor drug doxorubicin, even for the most aggressive types of multi-drug resistant malignant tumors.1 cl, 16 dwg, 3 ex

Description

The invention relates to medicine, namely to pharmacology, and can be used to increase the effectiveness of antitumor chemotherapy by overcoming the multidrug resistance of tumor cells under the influence of a new structural class of inhibitors of ATP-dependent reverse cell transporters (ABC transporters, from the English ATP-binding cassette transporters). The invention can be used in the field of biology, pharmacology, pharmaceuticals and medicine to significantly enhance the effectiveness of antitumor drugs, including the antibiotic of anthracycline series doxorubicin widely used in chemotherapy of malignant tumors.

One of the most important problems of modern chemotherapy for malignant tumors is the multidrug resistance of tumor cells - the innate or acquired immunity of cells to antitumor drugs that differ in their mechanism of action and structure. One of the main mechanisms of drug resistance is the reuptake and release of xenobiotic molecules penetrating into the cell by ATP-dependent pumps of the ABC transporter family [1]. The ABC transporter family includes P-gp glycoprotein (P-glycoprotein), multi-drug resistance proteins of the MRP family (multidrug resistance-associated protein) and breast cancer resistance protein BCRP (breast cancer resistance protein). The activity of these ABC transporters, the expression of which increases significantly with malignancy of the cell (transition from normal to malignant cells), leads to a significant decrease in the effectiveness of antitumor chemotherapy. Similar effects have been experimentally shown and investigated on the example of most drugs for chemotherapy of tumors [2], including targeted ones. Inhibitors of ATP-dependent inverse cell transporters have a very broad substrate specificity, re-capturing and ejecting very many antitumor drugs from the cell, for example: 5-fluorouracil, actinomycin D, bisanthrene, chlorambucil, colchicine, cisplatin, cytarabine, daunorubicin, docetaxelicin epirubicin, etoposide, gefitinib, irinotecan, methotrexate, mitomycin C, mitoxantrone, paclitaxel, tamoxifen, teniposide, topotecan, vinblastine, vincristine, etc. [3]. For all these preparations, the effect of a significant decrease in their effectiveness, due to the activity of inhibitors of ATP-dependent inverse cell transporters, is shown. This problem is also very acute for the widely used preparation of the anthracycline series of doxorubicin.

Thus, the combined use of an antitumor drug with an effective and safe inhibitor of ATP-dependent reverse cell transporters is a promising approach to increasing the effectiveness of a wide range of antitumor drugs. The creation of such drugs will significantly reduce the therapeutic dose of active substances, and, as a result, their side effects, and thereby make a qualitative breakthrough in pharmacology and medicine.

A wide range of compounds are known from the prior art, including drugs approved for use that can inhibit ABC transporters. So, a large-scale research work was carried out to create inhibitors of ATP-dependent reverse cell transporters as drugs that increase the sensitivity of cancer cells to the action of antitumor drugs [3]. In particular, atorvastatin, amlodipine, cyclosporin A, disulfiram, nifedipine, verapamil, GF120918, LY475776, LY335979, MS-209, OC144-061, plonic, Ponic, were shown to be active and studied for combined antitumor chemotherapy 833, R101933, S9788, VX-710, XR-9576, V-104. Azithromycin, cyclosporin A, furosemide, glibenclamide, probenecid, MK-571 were studied as MRP2 inhibitors. Cyclosporin A, dipyridamole, elacridar, fumitremorgin C, novobiocin, ortataxel, reserpine, ritonavir, tarqvidar, GF120918, VX-710, XR-9576 were studied as BCRP inhibitors.

Numerous attempts are known to overcome the multidrug resistance of tumor cells to doxorubicin by its combined use with reverse transporter inhibitors. Thus, the combined use of inhibitors of ATP-dependent reverse transporters of the first generation, such as cyclosporin A and verapamil, with anthracycline drugs led to pronounced toxic effects without increasing efficacy [4, 5]. An attempt to encapsulate cyclosporin A in micelles in order to reduce toxic effects also did not increase the effectiveness of doxorubicin [6]. Second-generation inhibitors, such as PSC-833 and VX-710, have been developed to increase the inhibitory activity and reduce the toxic effects of first-generation inhibitors. It was shown that the analogue of cyclosporin A PSC-833 is able to increase the sensitivity of SK-MES-1 / DX1000 tumor cells to the action of doxorubicin, but its use was accompanied by serious undesirable pharmacotoxic effects in patients [7, 8]. Also, the use of VX-710 with doxorubicin [9] did not lead to significant positive effects, while experimental evidence appeared that VX-710 acts by more complex mechanisms associated not only with inhibition of ABC transporters [10]. It was shown that second-generation inhibitors are often able to enhance the toxic effects of antitumor drugs by changing the profile of their pharmacokinetics and biodistribution [11]. Third-generation drugs, such as GF120918, LY335979, R101933 and XR9576, became even more effective reverse transport inhibitors in in vitro models, however, their use with chemotherapeutic drugs also did not lead to significant clinical results [12]. Attempts have been made to enhance the antitumor efficacy of doxorubicin by combination with natural dietary supplements that inhibit P-gp [13].

In general, both for doxorubicin and other antitumor drugs, the strategy described so far has not led to significant clinical results. The current state of research in this area is characterized by local successes at the level of in vitro, however, the transition to in vivo, and especially clinical trials, as a rule, does not bring the desired effect, mainly due to undesirable side effects of antitumor combinations or insufficient breadth and effectiveness of action inhibitors of ATP-dependent inverse cell transporters.

Thus, the above-mentioned analogues of the claimed technical solution are combinations of doxorubicin and other antitumor drugs with inhibitors of ATP-dependent inverse cell transporters. Moreover, all of the above drugs are characterized by common disadvantages, consisting mainly of undesirable side effects of antitumor combinations or insufficient latitude and effectiveness of the action of inhibitors of ATP-dependent reverse cell transporters.

To identify these shortcomings in detail, the applicant provides a description of one of the above analogues, taken as a prototype according to the application for the invention [7], the essence of which is the development of inhibitors of the ABC transporter P-glycoprotein based on cyclosporin analogues, in particular, PSC-833. It was shown that the cyclosporin A analogue of PCS833 is able to increase the sensitivity of SK-MES-1 / DX1000 tumor cells to doxorubicin [7]. However, the use of PSC-833 in patients with acute myeloid leukemia, although it led to some increase in the antitumor effect, was accompanied by serious undesirable pharmacotoxic effects, which does not allow its use in the clinic [8].

Based on the foregoing, it is obvious that for the full realization of the prospects of this approach, more active and safer inhibitors of ATP-dependent reverse cell transporters with an optimal pharmacokinetic and pharmacotoxic profile, capable of synergistically interacting with an active antitumor drug are required.

In RF patent No. 2641304 [19] a new inhibitor of ATP-dependent reverse cell transporters is described, which belongs to the group of chiral conjugates (optically active hybrid molecules) of oligoester polyol nature (hereinafter referred to as OEP inhibitor), which is a mixture of polyoxypropylenehexol of formula 4

,

,

where n = 2-6, mainly n = 4,

and polyoxypropylene glycol of formula 5

,

,

where m = 5-9, mainly m = 7.

The inhibitor is a conjugate of polyoxypropylene glycol with a molecular weight of from 300 to 500 Da and polyoxypropylenehexol with a molecular weight of from 1000 to 1500 Da in an equimolar ratio, with a hydroxyl number in the range of 215-240 mg KOH / g.

The OEP inhibitor according to the patent of the Russian Federation No. 261304 significantly increases the absorption of drugs by living cells and tissues, is characterized by high safety and efficiency:

- has low cytotoxicity in relation to human cell cultures compared to most inhibitors of ATP-dependent inverse cell transporters described in the literature;

- does not affect the microviscosity of the cytoplasmic membranes of tumor cells of the MCF-7 line and MCF-7 / RES cells with acquired drug resistance;

- does not penetrate the cytoplasmic membrane into the tumor cells of MCF-7;

- in the concentration range of 8.7–870 μg / ml, it causes specific inhibition of P-gp-mediated reverse transport of doxorubicin, increasing its concentration in acceptor wells 1.9–3.8 times, respectively;

- eliminates the active glycosylated isoform of 190 kDa ABCB1, causing the accumulation in the cells of the inactive high mannose isoform of 175 kDa transporter;

- inhibits the ATPase activity of isolated Sf9 cell membranes with overexpression of human P-glycoprotein;

- does not change the intracellular level of ATP;

- when administered intragastrically in terms of toxicity, it refers to non-toxic substances, when administered intravenously - to low-toxic substances.

The aim of the technical solution is to obtain a composition of the antitumor drug doxorubicin and an EIA inhibitor, which provides an increase in the effectiveness of doxorubicin, including overcoming the multiple drug resistance of tumor cells, increasing the safety of tumor chemotherapy, reducing toxic reactions and creating effective dosage forms for chemotherapy of cancer patients with multiple drug resistant.

The technical result of the claimed technical solution is to create an effective antitumor composition of doxorubicin with a representative of a new class of EIA inhibitor, for which RF patent No. 2641304 has been issued, which allows to significantly increase the efficacy and safety of the antitumor drug doxorubicin.

The essence of the claimed technical solution is an antitumor composition, which is an aqueous solution of doxorubicin and an inhibitor of ATP-dependent reverse transporters of cells in a mass ratio of 1:15, where the inhibitor of ATP-dependent reverse transporters of cells is a mixture of polyoxypropylenehexol of formula 4

,

,

where n = 2-6,

and polyoxypropylene glycol of formula 5

,

,

 where m = 5-9,

having a hydroxyl number of 215-240 mg KOH / g, compound 4 has a molecular weight of from 1000 to 1500 Da, compound 5 has a molecular weight of from 300 to 500 Da, the molar ratio of compounds 4 and 5 is 0.9-1.1.

The object of the study in all of the examples below is an OEP inhibitor, an antitumor antibiotic, doxorubicin, and a composition of doxorubicin with an OEP inhibitor. To carry out the research, permission was obtained from the GUAZ Ethics Committee of the Republican Clinical Oncology Dispensary of the Ministry of Health of the Republic of Tatarstan (Minutes of the meeting dated 09/22/2014).

The goal and the claimed technical result is achieved by creating a fundamentally new composition unknown from the studied prior art: based on an inhibitor of ATP-dependent inverse transporters of oligoester polyol cells and the antitumor cytotoxic drug doxorubicin known per se.

The antitumor cytotoxic drug doxorubicin is used to treat a wide range of malignant tumors, including: breast cancer, lung cancer (small cell), mesothelioma, esophageal cancer, gastric cancer, primary hepatocellular cancer, insulinoma, carcinoid, head and neck cancer, thyroid cancer, malignant thymoma, ovarian cancer, germ cell tumors of the testis, prostate cancer, bladder cancer (treatment and prevention of relapse after surgery), endometrial cancer, cervical cancer, sarcoma m attacks, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, Wilms tumor, osteogenic sarcoma, soft tissue sarcoma, Kaposi’s sarcoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, Hodgkin’s disease and non-Hodgkin’s lymphoma instrukciya / doksorubitsin-ebeve-rastvor_11206 /).

A limitation for the therapeutic use of doxorubicin is its high toxicity to actively dividing normal body cells. An effective way to reduce it is considered to be the use of doxorubicin not in free form, but as part of medicinal compositions or in the form associated with transport systems (carriers).

The above technical result is generally achieved by the applicant in that the combination of doxorubicin with an EIA inhibitor in the form of an injection solution is prepared in the following way:

- prepare the concentrate of the EIA inhibitor by dissolving the EIA in water for injection;

- the prepared concentrate solution is thoroughly mixed and filtered through membrane filters with a pore diameter of 0.2 μm;

- prepare a solution of doxorubicin in water for injection according to the instructions for use;

- doxorubicin solution is added to the obtained OEP-inhibitor concentrate and everything is thoroughly mixed, as a result, the claimed composition is obtained.

This goal is achieved by the fact that the applicant has developed an antitumor pharmaceutical composition to increase the effectiveness of the antitumor drug, including an OEP inhibitor (an inhibitor of ATP-dependent inverse cell transporters), doxorubicin and water.

The pharmaceutical composition in accordance with the claimed technical solution can significantly increase the effectiveness and safety of the antitumor drug doxorubicin.

As a result of the creation and research of various compositions, the applicant found that the claimed technical result is achieved provided that the claimed pharmaceutical composition contains from 1.1 to 1.6 wt.% (In terms of 100 wt.% Of the pharmaceutical composition of the dosage form) of the EIA inhibitor, from 0.082 up to 0.100 wt.% doxorubicin, the rest up to 100 wt.% - water. It can be water for injection or saline.

According to the claimed technical solution, the proposed antitumor composition of doxorubicin with an EIA inhibitor, most preferably contains 1.36 wt.% (In terms of 100 wt.% Pharmaceutical composition of the dosage form) of an OEP inhibitor, as well as from 0.091 wt.% Doxorubicin.

The optimal form contains doxorubicin and an EIA inhibitor in a ratio by weight of 1:15. The ratio of 1:15 is dictated by the maximum possible concentration, which does not cause the death of animals. This ratio was calculated from the results of evaluating the effect of an OEP inhibitor on the transepithelial transport of doxorubicin through the plasma membrane of CaCo-2 cells (see RF patent No. 2641304, Example 12), where the activity of in vitro OEP inhibitor was studied at various concentrations from 0.087 to 870 μg / ml, with the same content of doxorubicin (1 μM or 0.58 μg / ml). In the invention according to the patent of the Russian Federation No. 261304 it is shown that the OEP inhibitor inhibits the reverse transport of doxorubicin, increasing it at a concentration of 8.7 μg / ml almost 2 times. This ratio of doxorubicin concentrations of 0.58 μg / ml and an OEP inhibitor of 8.7 μg / ml corresponds to a ratio of 1:15, which is optimal for in vitro cells studied in the known invention, because when experiments are conducted "in vitro" outside a living organism.

Moreover, in general, it is possible to draw a preliminary conclusion that when the doxorubicin-OEP inhibitor ratio is 5:50 mg / kg (1:10), the effectiveness of the composition when using is clearly reduced, and when the ratio is 5: 100 mg / kg (1:20 ) vascular embolism in animals can occur, which can lead to their death, moreover, in the known invention, the effect of the composition was studied in vitro, i.e. outside a living organism, and the action on a living organism was not the subject of invention, i.e. not studied.

An analysis of the literature data by the applicant [20] indicates that the therapeutic dose of doxorubicin in mouse xenograft models is 5 mg / kg. The doxorubicin-OEP-inhibitor ratio chosen by the applicant = 5:75 mg / kg (1:15) is associated with the highest possible concentration of the OEP inhibitor, which does not lead to embolism (blockage of the bloodstream).

In more detail, the process of creating the claimed composition is achieved as follows.

The antitumor composition is prepared by dissolving in water for injection or in physiological saline.

In a volumetric flask with a capacity of 10 cm ³ using a dispenser, pour water for injection in a volume of 5.0-5.5 cm ³ . The flask is placed on a magnetic stirrer. With stirring, the pharmaceutical substance EIA in the amount of 1.50 g is loaded and mixed for 5-10 minutes. Then bring the volume of solution in the flask to the mark with water for injection. Get 10 cm ^{3 a} solution of a concentrate of EIA inhibitor with a concentration of 150 mg / ml The prepared concentrate solution is thoroughly mixed and filtered through laboratory membrane filters with a pore diameter of 0.2 μm.

50 mg of doxorubicin dissolved in 50 ml of water for injection are added to 5 ml of the obtained EIA inhibitor concentrate and mixed thoroughly. Obtain 55 ml of a composition with a mass ratio of doxorubicin and an OEP inhibitor of 1:15. The final volume is adjusted to the necessary depending on the indications and the developed protocol of administration.

The claimed technical solution is illustrated in Fig.1 - Fig. 16:

Figure 1 in Table 1 shows the concentrations of half-maximal growth inhibition compounds for conditionally normal (SS ₅₀ , μM) and human tumor cells (IC ₅₀ , μM). Mean values ± standard deviation.

Figure 2 in Table 2 - shows a study of the effectiveness of the claimed technical solution on the model of xenografts of human breast cancer in three experimental groups of mice - control, using the claimed technical solution and doxirubicin.

Figure 3 in Table 3 shows the average size of the tumors and the inhibition of tumor growth (SRW) in the groups after the first (day 8) and second (day 12) intravenous administration of the studied drugs.

Figure 4 in Table 4 - shows the parameters of the inhibitory activity of the studied drugs.

Figure 5 in Table 5 shows the number of animals in the experimental groups in an experiment to study the effectiveness of the claimed technical solution on a model of xenograft myxofibrosarcoma in rabbits.

Figure 6 - shows the size of the tumors in animals at the time of treatment.

In FIG. 7 (7A, 7B, 7B) - the dynamics of the growth of breast cancer in the xenograft model is presented (mean ± standard error).

On Fig - shows the size of the breast tumors in the xenograft model on days 8 and 12 (mean ± senior error). On day 12, one animal remained in the Doxorubicin – OEP inhibitor 5/75 composition group.

In FIG. 9 - shows the exponential growth curves of breast cancer tumors in a xenograft model (mean ± SEM).

In FIG. 10 - shows the change in the size of xenograft myxofibrosarcoma under the influence of chemotherapy.

In FIG. 11 - a photograph of myxofibrosarcoma in the anterior chamber of the rabbit eye on the 2nd day after xenograft.

In FIG. 12 shows a photograph of a rabbit’s eye on the 30th day after xenograft of myxofibrosarcoma against the background of the action of the composition of doxorubicin with an OEP inhibitor. The tumor is not visualized.

In FIG. 13 - micrograph of myxofibrosarcoma on the 30th day of incubation in the anterior chamber of the eye. Control group. The red precipitate corresponds to a positive immunohistochemical reaction with antibodies against vimentin - shown by arrows.

In FIG. 14 - micrograph of myxofibrosarcoma on the 30th day of incubation in the anterior chamber of the eye against the background of the administration of doxorubicin. The arrow shows a vimentin-positive (tumor) cell.

In FIG. 15 - micrograph of myxofibrosarcoma on the 30th day of incubation in the anterior chamber of the eye against the background of the introduction of the composition of doxorubicin and oligoester polyol.

In FIG. 16 - a comparative analysis of the ratio of vimentin-positive (tumor) cells in xenografts of the control and experimental groups of animals on the 30th day after the operation.

Symbols and abbreviations

in vitro - technology for performing experiments in laboratory conditions, when experiments are conducted outside a living organism;

ATCC - (from the English American Type Culture Collection) - American collection of cell cultures;

IC ₅₀ — concentration of half-maximal inhibition of tumor cell growth;

CC ₅₀ — concentration of half-maximal inhibition of growth of conditionally normal cells;

μM - micromole per liter;

DMSO - dimethyl sulfoxide;

mQ - purified water;

MTT - 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide;

HSF - conditionally normal fibroblasts of human skin;

MCF-7 human breast adenocarcinoma, ATCC;

MCF-7 / Vin - human breast adenocarcinoma resistant to vinblastine and doxorubicin;

CaCo-2 - human colorectal adenocarcinoma, ATCC;

DOX - doxorubicin hydrochloride

HCT-15 - human colorectal adenocarcinoma, NCI-60;

HCT-116 - human colorectal carcinoma, NCI-60;

OVCAR-4 - human ovarian adenocarcinoma, NCI-60;

PC-3 - Human Prostate Adenocarcinoma, NCI-60;

A-498 — Human Kidney Carcinoma, NCI-60;

NCI-H322M - non-small cell lung carcinoma of the person,

 NCI-60;

M-14 - human skin melanoma, NCI-60;

SNB-19 - human glioblastoma, NCI-60;

SF-539 - Human Gliosarcoma, NCI-60.

Dox - doxorubicin.

Materials and reagents

L-glutamine, b-MEM culture media, DMEM culture medium, penicillin-streptomycin (cat. No. A065), Hanks solution, trypsin-EDTA solution, Versen solution, sodium pyruvate were purchased from PanEco (Russia). MTT 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) - from Promega. Doxorubicin hydrochloride (DOX) - from Sigma-Aldrich (USA) and Pharmstandard; Hanks solution, aktaza - from Gibco; tablets 17v-estradiol, 3.0 mm, 1.7 mg, 60 days release - from Innovative Research. FBS, Fetal Bovine Serum, from HyClone. Fetal calf serum - from the company RAA; trypan blue dye - from Dia-M company (Russia); Matrigel - from the company BD (cat. No. 356235).

Below, the applicant provides examples of the practical implementation of the claimed technical solution.

Example 1 The study of the cytotoxic activity of the claimed composition in vitro

The aim of the experiment was an experimental study of the cytotoxic activity of an OEP inhibitor, doxorubicin (Dox) and the composition of doxorubicin and an OEP inhibitor in vitro against tumor cell lines MCF-7, MCF-7 / Vin, HSF, CaCo-2, HCT-15, HCT -116, OVCAR-4, PC-3, A-498, NCI-H322M, M-14, SNB-19, SF-539 and conditionally normal cell lines of primary fibroblasts isolated from human skin. Studies included the construction of a dose-response curve and determination of the concentration of half-maximal inhibition of the growth of tumor cells (IC ₅₀ ) and conditionally normal cells (CC ₅₀ ).

The cytotoxicity of the OEP inhibitor, doxorubicin and the combination of doxorubicin and the OEP inhibitor in cell cultures was evaluated using the proliferative MTT test, the essence of which is as follows. Yellow MTT under the action of mitochondrial succinate-dehydrogenase cells turns into formazan, which has a purple color. During cell lysis, formazan crystals easily pass into a solution of organic solvents such as isopropanol, triton, or DMSO. According to the optical density of the formazan solution (500-600 nm), the activity of mitochondrial reductases and, accordingly, cell viability were determined (MTT Cell Proliferation Assay. Instruction Guide. American Type Culture Collection [Electronic resource]. 2011).

Before the experiment, stock solutions of the test compounds were prepared in 5 ml volumetric flasks in water [quality mQ] for injection. The concentration of stock solutions of the EIA inhibitor was 100 mg / ml; doxorubicin - 10 ^-4 M; the composition of doxorubicin and an EIA inhibitor: 10 ^-4 M DOX + 200 μg / ml EIA inhibitor (composition 1) and 10 ^-4 M DOX + 100 μg / ml EIA inhibitor (composition 2).

Stock solutions of the compounds were filtered using 0.22 μm syringe filters with a PTFE (Teflon) membrane. When preparing a composition of doxorubicin with an EIA inhibitor, a solution of an EIA inhibitor in water for injection was first prepared, then the resulting solution was added in the required volume to a weighed portion of doxorubicin.

Cells were cultured in b-MEM medium supplemented with 10% fetal calf serum, L-glutamine and 1% penicillin-streptomycin in an atmosphere of 5% CO ₂ at 37 ° C until a monolayer was formed. To obtain a cell suspension, a monolayer of cells was trypsinized, followed by trypsin inactivation by adding serum b-MEM medium. Cell counting was carried out in a Neybauer chamber by trypan blue exclusion. Cells were subcultured 2 times a week in a ratio of 1: 6 [14].

For a three-day test, 1000 cells in 90 microliters of culture medium were added to the wells of a 96-well plate. Before adding to the tablet, the cells were carefully suspended. A cell suspension was added to the wells of a plate using a multichannel dispenser and sterile tips. Next, the tablets with the cells were incubated for 24 hours in a CO ₂ incubator to adhere the cells to the substrate. Using a multichannel dispenser and sterile tips, aliquots of the prepared solutions of the test compounds in the volume of 10 microliters per well were added to the wells of the plate with cells.

The study was carried out in triplicates. Instead of the compounds to be analyzed, similar volumes of mQ water were added to the control wells of the plate. After introducing the test substances, the cells were cultured in a CO ₂ incubator under standard conditions for 72 hours.

On the third day of cultivation, the confluency of the cells in the control wells was 70-90% of the bottom surface.

After the incubation time, the culture medium with the test substances was removed from the tablet using a vacuum aspirator.

In the bath for a multi-channel dispenser, a mixture was prepared: 9 ml of culture medium + 1 ml of MTT reagent (5 mg / ml in Hanks solution) per 96-well plate. The prepared reaction mixture was added in a volume of 100 μl to each well of the plate and incubated in a CO ₂ incubator for 3.5 hours [15]. After the incubation time, the culture medium with MTT reagent was removed with a vacuum aspirator, 100 μl of DMSO was added to each well of the plate and incubated for 5-10 minutes. Appeared violet staining was detected on a Tecan plate reader at 555 nm (reference wavelength - 650 nm).

The results were processed using the OriginPro 8. software. The percentage of viable cells in each test well was calculated relative to the wells of positive control, the viability of which was taken as 100%. Then, cell viability curves were plotted, expressed as a percentage, relative to the decimal logarithm of the concentrations of added compounds. The resulting curve was subjected to “Fit Sigmoidal-DoseResp” analysis in OriginPro 8 software, and the concentration logarithm was found at 50% cell viability. The concentration of half-maximal inhibition of cell growth was calculated (IC ₅₀ ). The results of the studies described above are presented in Figure 1 in Table 1.

For most of the studied cells, the IC _{50 of the} OED inhibitor exceeds 1.5 mg / ml, which indicates its complete safety.

A certain specificity of the OEP inhibitor is shown to glioblastoma SNB-19 and glio sarcoma SF-539 cells - 0.46 ± 0.12 mg / ml and 0.30 ± 0.04 mg / ml, respectively, which indicates its some, albeit insignificant, potential effect in relation to the corresponding tumors . In this case, the composition of the OEP inhibitor with doxorubicin increases the cytotoxicity of doxorubicin for tumor cells by 1.1-6.6 times, and for CaCo-2 cells by 246 times at a constant concentration in the nutrient medium of 100 μg / ml.

With an increase in the composition of a constant concentration of an OEP inhibitor in a nutrient medium up to 200 μg / ml, an increase in the cytotoxicity of doxorubicin in the studied cells is observed by 1.5–54 times, for CaCo-2 by 307 times.

The cytotoxicity of doxorubicin for conditionally normal human skin fibroblasts does not change.

The results of the described experiment indicate that in the composition of the EIA inhibitor significantly, in some cases more than 300 times, increases the sensitivity of tumor cells to the cytotoxic effect of doxorubicin. At the same time, an EIA inhibitor does not increase the sensitivity of normal cells to doxorubicin. This ensures a significantly increased selectivity of the composition of doxorubicin and an EIA inhibitor compared to doxorubicin. The result obtained is not obvious to a specialist and in practice means a significant increase in the efficiency and safety of the claimed composition.

Example 2. The study of the effectiveness of the claimed composition on a xenograft model of human breast cancer in rodents

The study of the antitumor activity of the claimed composition was carried out on a xenograft model of human breast cancer. The model was formed on the basis of MCF7 / Vnb cells, which were preincubated for 90 days in the presence of vinblastine and doxorubicin to develop multidrug resistance (see Figure 2, Table 2).

MCF7 / Vinb human breast cancer cells were thawed in medium and cultured in T175 attached culture vials. As 100% confluency was reached, the cells were subcultured on average once every 3-4 days. With detachment, the culture fluid was removed, washed in phosphate-buffered saline and a nektaza solution was added. Incubated for 5 minutes at 37 ° C, medium was added and suspended. Next, the vials were dispersed onto T175.

The study was conducted on female SCID Beige mice. Animal supplier Charles River GmbH, the average age of the mice is 6-8 weeks. The choice of animals is based on the fact that immunodeficient mice are a convenient model for studying the antitumor activity of drugs. The lack of immunity allows them to transplant human cancer cells, which increases the prognostic reliability of the activity of the studied substances.

Since this model (human breast cancer xenograft) is hormone-sensitive, mice were implanted with capsules with the gradual release of 17c-estradiol [16, 17]. Before the start of the experiment, the model was tested - the dynamics of tumor growth in the presence of different doses of 17c-estradiol, the time of appearance and variability of tumor size after implantation were evaluated. Tumors appeared most evenly and grew upon implantation of 17b-estradiol at a dose of 1.7 mg / mouse and MCF7 / Vinb cells at a dose of 2 million cells / mouse. These conditions were chosen as a model for testing the antitumor activity of the claimed technical solution at the second stage of the study.

During the main experiment, the efficacy of the composition of doxorubicin and an OEP inhibitor was studied on the obtained xenograft model. The route of administration is clinical (intravenous). Solutions for drug administration were prepared using physiological saline as a solvent.

When preparing a composition of doxorubicin with an EIA inhibitor, a solution of an EIA inhibitor in physiological saline was first prepared, then the resulting solution was added in the required volume to a weighed portion of doxorubicin. The solutions were incubated at room temperature on a vortex for 20 minutes. After incubation, the substances completely dissolved, the precipitate did not precipitate.

First, a concentrated solution of the doxorubicin – OED inhibitor composition was prepared to administer the maximum dose, then part of it was diluted with physiological saline to obtain more diluted solutions for the administration of lower doses. The volume of administration was 10 ml / kg.

After the end of the adaptation period, capsules of 17b-estradiol were subcutaneously implanted into the animals, and after 2 weeks, MCF7 / Vinb cells in Matrigel (1: 1, 200 μl per mouse).

On the day of implantation in mice, MCF7 / Vinb cells were detached, washed with medium and suspended in saline. A suspension of cells with a concentration of 20 million / ml was prepared. Cells cooled in ice. A volume of Matrigel equal to the volume of the cell suspension was added with a pre-cooled pipette and mixed smoothly. Thus, the final cell concentration was 10 million / ml.

The cell suspension was injected into mice subcutaneously along the spine (in the region of the right scapula) in a volume of 0.2 ml (2 million cells) per mouse. Before implantation of the cells, the corresponding area of the mouse skin was shaved and treated with AHD-2000 disinfectant solution.

After the formation of tumors, animals were divided into 7 experimental groups, 6-7 animals each. The average size of the tumors in the groups was ~ 250 mm ³ .

Treatment of animals with the studied drugs was started the day after randomization (randomization is the process of randomly distributing experimental participants into groups or the order in which experimental conditions were presented to them) into groups. The drugs were administered intravenously 2 times, once every 5 days. The control group of animals was injected with solvent in an equivalent volume. Doses and groups of animals are shown in Figure 2 in Table 2.

Measurement of subcutaneous implanted tumors was performed 2 times a week. Tumor volume was determined by the formula:

p / 6 x L x W ² = mm ³ ,

where L - corresponds to the largest diameter of the tumor, W - to the smallest. Measurements were carried out using a caliper.

 The antitumor activity of the studied drugs was determined by the inhibition of tumor growth relative to tumors in the control group receiving the solvent. For this, the values of inhibition of tumor growth (SRW) were calculated:

SRW = (С-Т) х100 / С,%

where C is the average tumor size in the control group treated with the solvent, T is the tumor size in the experimental group.

The inhibitory effect of the drugs was also evaluated by the logarithm of dead cells. For this, the average doubling time in the control and experimental groups was estimated. The logarithm of the number of dead cells was estimated by the formula:

Lg (n) = (Tk-To) /3.32Td,

where (Tk-To) is the time delay of tumor growth in the experimental group; Td is the time of doubling the size of the tumor in the control; 3.32 - the number of doublings necessary to increase Lgn no less than 2-4 times.

We also monitored the health and behavior of animals, recorded the number of deaths. Inspection of animals was carried out at the time of administration of the compounds, and then twice a day (morning and evening) to register mortality and general condition. Measurement of the weight of animals was carried out 2 times a week. According to the rules of humane treatment of animals, when reaching a tumor volume> 2000 mm ³ in 50% of the animals in the group, the entire group was euthanized. At the end of the experiment, all surviving animals were euthanized by CO ₂ inhalation.

Data analysis was performed using Microsoft Office Excel 2007 and GraphPad Prizm 5.

In FIG. 6 shows the size of animal tumors in the experimental groups before treatment. The model is characterized by high heterogeneity in terms of tumor volume. At the time of randomization, the average tumor size between groups did not differ.

The studied drugs were administered 2 times during the study, once every 5 days. After administration, animals treated with doxorubicin at doses of 1 mg / kg and 5 mg / kg, as well as a combination of doxorubicin / OEP inhibitor with an equivalent dose of doxorubicin, showed a dose-dependent decrease in motor activity, a hunched posture, and a decrease in food intake were observed. There was no difference between the condition of the animals treated with doxorubicin and the combination of doxorubicin / EIA inhibitor with an equivalent dose of doxorubicin.

In FIG. 7 shows the curves of tumor growth in the control and experimental groups. Due to the large number of groups, to facilitate the perception of the results, the figure is divided into 3 parts (AB) in accordance with the doses of doxorubicin.

In FIG. Figure 8 shows the average sizes of the tumors in the groups on days 8 and 12.

The analysis of Figure 3 showed that doxorubicin in doses of 0.5 and 1 mg / kg practically does not affect the growth of multidrug-resistant breast tumors, while the addition of an OEP inhibitor leads to a noticeable increase in its antitumor activity by 30% compared to the free form doxorubicin and 43% and 30% relative to the control group, respectively.

The maximum effectiveness of the combination of doxorubicin / EIA inhibitor was shown at a dose of 5/75 mg / kg.

Based on the obtained tumor sizes, the values of inhibition of tumor growth (TPO) were calculated (Fig. 3, Table 3) and the logarithm of dead cells (Fig. 4, Table 4). The analysis of the tables showed that doxorubcin in doses of 0.5 and 1 mg / kg does not affect the growth of xenografts of the breast with acquired drug resistance. At a dose of 5 mg / kg, doxorubicin causes a slowdown in tumor growth.

The addition of doxorubicin to the OEP inhibitor at a ratio of 1:15 causes a marked increase in the antitumor effect of doxorubicin - the combination of doxorubicin / OEP inhibitor at doses of 0.5 / 7.5 and 1/15 mg / kg had an antitumor effect comparable to that of doxorubicin at a dose of 5 mg / kg. At the same time, it is necessary to note the almost complete absence of animal death in groups with a doxorubicin composition with an OEP inhibitor of 0.5 / 7.5 and 1/15 mg / kg (1 animal in both groups fell), while 50% of animals died in the 5 mg / kg doxorubicin group .

The greatest antitumor effect was achieved with the use of the doxorubicin / OED inhibitor composition at a dose of 5/75 mg / kg - on the 8th day of the study, the average tumor size in the group was statistically significantly lower than the control group, the TPO value was ~ 64% (see Fig. 3, Table 3).

On the 12th day of the study, only one animal remained in the group, which does not allow us to talk about the statistical reliability of the results at this time; however, it should be noted that the SRW value remained at the same level as on day 8. The doubling time of cells increased by 1.5 times compared to the control, the logarithm of dead cells - more than 5 times (see Fig. 4 Table 4).

Thus, the experiment described in Example 2 demonstrated that the addition of an OEP inhibitor to doxorubicin significantly enhances the antitumor effect of doxorubicin — the combination of doxorubicin / OEP inhibitor at doses of 0.5 / 7.5 and 1/15 mg / kg had an antitumor effect comparable to with doxorubicin at a dose of 5 mg / kg, while the toxicity of the combination was significantly lower

Thus, according to the applicant, it should be emphasized that the efficacy of the doxorubicin / OED combination was demonstrated in an experiment with an aggressive type of tumor with the multidrug resistance phenotype obtained by incubating MCF-7 human breast adenocarcinoma cells with known antitumor drugs vinblastine and doxorubicin in within 90 days. Such tumors are extremely difficult to treat, and therefore the results described above are extremely encouraging and obvious to a person skilled in the art.

Example 3. The study of the effectiveness of the claimed composition on a xenograft model in rabbits.

In this experiment, the effect of the claimed composition on tumor growth was studied on a xenograft model of human tumors in the anterior chamber of the rabbit eye, the effectiveness of which is described in detail in the RF patent for invention No. 2638285 [18].

During the experiment, a xyotransplantation of a human myxofibrosarcoma tumor into the anterior chamber of the rabbit eye was performed, then the change in the size of the transplanted tumor in the anterior chamber of the rabbit eye was assessed against the background of the administration of doxorubicin and an EIA inhibitor. For this assessment, tumor xenografts in the anterior chamber of the rabbit eye were morphologically characterized; in addition, a morphometric study was conducted of changes in the number of vimentin-positive (tumor) cells in sarcoma on the 30th day after xenotransplantation.

36 rabbits of both sexes weighing 2800-3500 g were used in the experiment. The animals were kept under standard conditions with free access to water and feed. The reaction of xenotransplanted myxofibrosarcoma to the administered pharmacological preparations was investigated. Animals were divided into 4 groups of 9 rabbits in each group (Figure 5, Table 5).

Starting the day after surgery, injections were given to all animals.

Control rabbits were injected with physiological saline in a volume of 1 ml / kg.

The rabbits of the experimental groups were injected intraperitoneally with a solution of doxorubicin in increasing concentration to exclude complications of healing of the injured cornea. The first two injections are 1 mg / kg, the next two are 2 mg / kg and then 3 mg / kg.

In the group, the doxorubicin / OED inhibitor composition was additionally administered with 1 ml of a solution of 0.8% OEP inhibitor per kilogram of rabbit weight.

Solutions of the starting components were prepared in water for injection.

In all animals, transplanted tumor fragments were photographed after 24 hours to evaluate their changes, and then tumor area was counted. Statistical processing was performed using Student's criterion.

After transplantation, the size of the tumors in all groups began to increase.

In the control group (without treatment), the tumor size by 30 days increased by 39 ± 4.2% (P <0.05).

By 12 days after transplantation in the group treated with doxorubicin, the increase in tumor size was 48 ± 4.7% (P <0.05). After this period, the size of the tumor did not significantly change.

In the group of animals treated with an EIA inhibitor, the tumor sizes increased by 9 ± 1.6% by 30 days (P> 0.05).

In the group of animals treated with the doxorubicin / OED inhibitor composition, the xenotransplanted tumor did not change its size until 12 days, but then began to decrease and decreased by 30 times compared to the initial size by an average of 2.5 times (P <0.05) (Fig. 10 )

Morphological examination in all groups showed tumor ingrowth in the area of the corneal injury, the absence of blood vessels and foci of necrosis (Figs. 11 and 12).

30 days after surgery, the animals were transplanted and fixed in 10% formalin. Paraffin sections were stained with hematoxylin or immunohistochemical detection of vimentin-positive (tumor) cells was performed.

To detect the expression of vimentin (1: 100 dilution), a marker of fibro-connective tissue, the indirect immunoperoxidase method and monoclonal rabbit (mouse) antibodies were used using the LSAB kit (DAKO, Denmark). Vimentin is expressed by connective tissue tumor cells (sarcomas), and given that antibodies binding only to human antigens were used in the experiment, vimentin is used to detect xenograft tumor cells. Immunohistochemical analysis was carried out on sections with a thickness of 4 μm. Paraffin sections were dewaxed and rehydrated, and then placed in a phosphate buffer solution (pH 7.4). Then, endogenous peroxidase activity was blocked by placing sections for 40 minutes in a 1% hydrogen peroxide solution. Washed in phosphate buffer in 2 shifts for 3-5 minutes.

The excess fluid around the sections was removed and the antibodies to vimentin corresponding to this group were dug into the sections of each group. Incubated in a humid cool chamber for 12 hours. Washed in phosphate buffer in 2 shifts for 3-5 minutes. Digested secondary antibodies conjugated with biotin. Incubated for 40 minutes. Washed in 2 shifts of phosphate buffer. Excess fluid around the sections was removed and streptavidin conjugated with horseradish peroxidase was dug up. Incubated 40 minutes. Washed in 2 shifts of phosphate buffer. The excess fluid around the sections was removed. Immunohistochemical reactions were visualized with a solution containing aminoethylcorbazole and hydrogen peroxide in acetate buffer (pH 6.4). As a result of the interaction of hydrogen peroxide with horseradish peroxidase, atomic oxygen is released, which, by oxidizing aminoethylcorbazole, transfers it to an insoluble state. Thus, the red precipitate (aminoethylcorbazole) indicates expression in vimentin sections. Sections were made in glycerol-gelatin. Vimentin-positive (tumor) cells were counted in 3 different fields of view and an average value was obtained. Statistical processing was performed using Student's criterion.

On xenograft sections, a positive immunohistochemical reaction with antibodies against vimentin is determined in the cytoplasm of the cells. Given that the antibodies used only react with human antigens, the identified vimentin-positive cells are transplanted tumor cells.

Against the background of the administration of doxorubicin by 30 days after xenotransplantation, the number of vimentin-positive cells decreases by more than 6 times (Figs. 13 and 14).

By this time, with the introduction of the doxorubicin / OED inhibitor composition in most histological preparations, vimentin-positive cells are not determined, and only one is present in the remaining ones (Figs. 15 and 16).

Within the framework of the described study, it was found that at the final stage of doxorubicin chemotherapy (30 days), the size of xenotransplanted myxofibrosarcoma does not differ from the control, but the number of tumor cells decreases by 84% compared with the control.

The composition of doxorubicin and an EIA inhibitor leads to a decrease in tumor size by a factor of 3.3 compared with the control. Moreover, according to the results of immunohistochemical studies, only some samples contain single tumor cells.

Thus, in the described experiment, the composition of doxorubicin and an EIA inhibitor significantly exceeds its antitumor effect on the xenograft tumor in the anterior chamber of the rabbit eye, doxorubicin, used separately.

The achieved result is not obvious for a specialist and is based on the synergistic interaction of the antitumor drug and the EIA inhibitor.

Thus, the results of the experiments described in Examples 1-3 allow us to make a logical conclusion that the applicant has achieved the set goals and the claimed technical result, namely, an effective antitumor composition of doxorubicin with a representative of a new class of EIA inhibitor (obtained by the patent applicant RF №2641304), which allows to significantly increase the effectiveness and safety of the antitumor drug doxorubicin.

The resulting composition of the antitumor drug doxorubicin and an EIA inhibitor ensures the implementation of the goals, namely, achieved:

- increasing the effectiveness of doxorubicin, including to overcome the multidrug resistance of tumor cells. The combination of doxorubicin / EIA inhibitor at doses of 0.5 / 7.5 and 1/15 mg / kg had an antitumor effect comparable to that of doxorubicin at a dose of 5 mg / kg (see Figure 3, Table 3). SRW on the 12th day of the study was 42.7%, 26.5% and 35.7%, respectively, and the logarithm of dead cells during this period was 0.224 in all three cases (see Figure 4, Table 4). Moreover, in groups with the free form of doxorubicin at doses of 0.5 mg / kg and 1 mg / kg, TPO was 11.4% and 1.1%, and the logarithm of dead cells was 0.075 and 0.037, respectively;

- increasing the safety of tumor chemotherapy. It should be noted the almost complete absence of animal death in the doxorubicin / OEP inhibitor groups 0.5 / 7.5 and 1/15 mg / kg (1 animal fell in both groups), while 50% of the animals died in the 5 mg / kg doxorubicin group. Death: control - 0/7, Dox 0.5 - 0/6, Dox 1 - 0/7, Dox 5 - 3/6, OEP + Dox 0.5 - 1/7, OEP + Dox 1 - 1/6, OEP + Dox 5 - 5/6.

- elimination of toxic reactions of cells and thereby ensuring the creation of a basis for creating effective dosage forms for chemotherapy of patients with multidrug resistance.

The claimed technical solution based on the use of the composition of doxorubicin and a new OEP inhibitor allows us to solve a range of problems in the chemotherapy of malignant tumors that could not be previously solved with doxorubicin taken separately or other chemotherapy agents existing at the time of application, due to which The claimed technical solution provides the possibility of resolving practically insoluble problems before the filing date of this application, namely, it is possible to increase the efficiency of treatment I cancer cells in vitro 1.5 - 54 times for 10 tumor lines, for CaCo-2 307 times, in vivo doxorubicin / EPA inhibitor combination - 5-10 times in comparison with the free form of doxorubicin, this combination toxicity much lower. This fact proves the existence of an inventive step.

The most important characteristic of the claimed solution is that the proposed composition can significantly increase the effectiveness and safety of the antitumor drug doxorubicin, even with respect to the most aggressive types of malignant tumors with multidrug resistance. At the same time, one should probably pay attention to the fact that based on the claimed technical solution, it seems possible to carry out various kinds of modifications and / or changes on the basis of the claimed solution, without going beyond the scope of patent claims.

The claimed technical solution meets the criterion of "novelty", presented to the invention, since the investigated prior art has not identified technical solutions that have the claimed combination of distinctive features that achieve the stated results.

The claimed technical solution meets the criterion of "inventive step" for inventions, as it is not obvious to a specialist in this field of science and technology.

The claimed technical solution meets the criterion of "industrial applicability", as it can be repeatedly repeated and used to create chemotherapeutic drugs and reproduced in a production environment using standard equipment and well-known technological processes.

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Claims (7)

The antitumor composition, which is an aqueous solution of doxorubicin and an inhibitor of ATP-dependent reverse transporters of cells in a mass ratio of 1:15, where the inhibitor of ATP-dependent reverse transporters of cells is a mixture of polyoxypropylenehexol of the formula 4

, where n = 2-6, and polyoxypropylene glycol of formula 5

, where m = 5-9, having a hydroxyl number of 215-240 mg KOH / g, compound 4 has a molecular weight of from 1000 to 1500 Da, compound 5 has a molecular weight of from 300 to 500 Da, the molar ratio of compounds 4 and 5 is 0.9-1.1.

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