RU2250775C2 - Means stimulating chemopreparation anti-tumor activity and method for treating oncological diseases - Google Patents

Abstract

FIELD: medicine; veterinary medicine.

SUBSTANCE: method involves using biologically active substances obtainable by means of bacteriological synthesis. Individual avermectines or their complexes are applied in vitro in concentrations of 0.01-10 ng/ml and in vivo at a dose of 0.01-1 mg/kg.

EFFECT: enhanced effectiveness of treatment; reduced toxic effect.

4cl, 2 tbl

Description

Technical field

The invention relates to pharmacology, namely to biologically active substances obtained by microbiological synthesis, which can be used in medicine and veterinary medicine as a means of enhancing the antitumor activity of chemotherapy drugs for the treatment of cancer.

State of the art

For the chemotherapy of various cancers, a large number of drugs of natural or artificial origin are used, belonging to different classes of chemical compounds and differing in their mechanism of action on tumor cells. Examples of such cytostatic drugs are cyclophosphamide, doxorubicin, vinblastine, etoposide, fluorouracil, and many others. However, in some cases, cells of malignant neoplasms exhibit high resistance to many chemotherapy drugs, which in other cases are quite effective in practical oncology (Stavrovskaya A.A. Cellular mechanisms of multidrug resistance of tumor cells. “Biochemistry”, 2000, Volume 65, Issue 1, p. 112-126).

Modulators are known - substances that enhance the effectiveness of known antitumor agents and help overcome the multiple drug resistance of tumors. Such substances include verapamil, cyclosporin A, nifedipine and its derivatives, amiodarone, trifluorkerazine, quinine and others (Ford J.M., Halt W. N. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Res. 1990, 42, 155-199).

The disadvantage of these compounds is the presence of serious side effects in doses in which they enhance the positive effect of antitumor drugs. In particular, verapamil causes heart failure and brain disorders, and cyclosporin A has immunosuppressive activity.

Didier A.D. and Loor F. (Didier A.D. and Loor F. The abamectin derivative ivermectin is a potent P-glycoprotein inhibitor. Anti-Cancer Drugs, 1996, 7, 745-751) conducted a comparative study of the action of cyclosporin and ivermectin, a semisynthetic derivative of avermectins. Ivermectin has been shown to be a better inhibitor of P-glycoprotein than cyclosporine. However, these studies have not received further development.

SUMMARY OF THE INVENTION

The aim of the invention is the search for low-toxic modulators of P-glycoprotein and other transport proteins that enhance the antitumor activity of known chemotherapeutic agents for the treatment of tumors, mainly resistant ones.

The present invention is based on previously unknown properties of individual avermectins, mainly of natural origin, and mixtures thereof.

In particular, it was found that avermectins enhance the antitumor activity of chemotherapy drugs.

In the treatment of cancer, including the use of chemotherapy drugs, additionally take drugs that enhance the antitumor activity of chemotherapy drugs. As chemotherapeutic agents, vincristine, taxol, etoposide, pharmacorubicin, doxorubicin are used, and avermectins are used as a means of enhancing the antitumor activity of chemotherapy drugs. Comparative experiments, in which, ceteris paribus, changed only the sequence of taking the drugs, showed that the greatest effect is achieved if avermectins are taken after taking chemotherapy drugs.

Avermectins prevent the pumping of drugs (chemotherapeutic agents) from tumor cells, chemotherapeutic drugs enter the cell in large quantities, which significantly increases the effectiveness of treatment.

Information confirming the possibility of carrying out the invention

Example 1. Breast carcinoma cells MCF-7 were plated in 96-well plates, 40,000 cells / well. The culture medium is DMEM supplemented with 10% fetal calf serum and 10 μg / ml gentamicin antibiotic. After the cells were attached to the substrate, vincristine (50 pg / ml) was added to the culture medium and, against its background, one of the avermectins A ₁ , A ₂ , B ₁ , B ₂ at a concentration (10 ng / ml). Then the cells were incubated at 37 ° C under standard conditions for 72 hours. To assess the synergism of the effect of the compounds on cell survival and proliferation, an MTT test and visual microscopy were used. After 72 hours, 20 μl of a solution of MTT dye 3- (4,5-dimethylthiazolyl 2) -2,5-diphenyltetrazolium bromide (Sigma) was added to the wells and the plates were cultured in a CO ₂ incubator for 2 hours until the formation of needle formazan granules. Then the dye medium was removed and 50 μl of DMSO was added to each well. The plate was vigorously shaken and the optical density of the samples was determined at a wavelength of 550 nm by scanning on a special device (Reader ANTOS 2020, BioRad).

To make the right choice of concentrations, the dose-response curves of the effects of vincristine and avermectins on the survival of MCF-7 cells were previously taken. Vincristine at a concentration of 50 pg / ml and each of avermectins at a concentration of 1 ng / ml do not affect the optical density (and, accordingly, the percentage of surviving cells). With the combined action of vincristine and avermectins A ₁ and B _{1, the} optical density drops sharply. Moreover, Avermectin A ₁ is the most effective (optical density drops by more than 4 times (P <0.05)). Avermectin B ₁ in the presence of vincristine reduces the optical density by 1.5-2 times.

Example 2. In the same manner as in example 2, the synergism of the cytotoxic effect of vincristine and avermectins on human lung carcinoma A549 and mouse melanoma B-16 cells was evaluated. Cells were plated in 96-well plates (40,000 cells / well). The culture medium is DMEM supplemented with 10% fetal calf serum and 10 μg / ml gentamicin antibiotic. After the cells were attached to the substrate, vincristine (50 pg / ml) was added to the culture medium and, against its background, one of the avermectins A ₁ , A ₂ , B ₁ , B ₂ in concentrations from 0.01 to 10 ng / ml. Then the cells were incubated under standard conditions for 72 hours. To assess the synergism of the effect of the compounds on cell survival and proliferation, an MTT test and visual microscopy were used. After 72 hours, 20 μl of a solution of MTT dye 3- (4,5-dimethylthiazolyl 2) -2,5-diphenyltetrazolium bromide (Sigma) was added to the wells and cultured at 37 ° C in a CO ₂ incubator for 2 hours until needle-shaped granules formed formazan. Then the dye medium was removed and 50 μl of DMSO was added to each well. The plate was vigorously shaken and the optical density of the samples was determined at a wavelength of 550 nm by scanning on a special device (Reader ANTOS 2020, BioRad).

Previously, dose-response curves of the effects of vincristine and avermectins A ₁ , A ₂ , B ₁ , B ₂ on these cells were recorded. Under the action of each individual compound (50 pg / ml vincristine and 0.01-10 ng / ml of any of avermectins), the optical density and, accordingly, the percentage of surviving cells practically does not change. At the same time, on the cells of A549 carcinoma with the combined action of vincristine and avermectins, the optical density decreases for A ₁ by 50 ± 8%, A ₂ - 40 ± 7%, B ₂ - 35-40%, B ₁ - is not effective.

On B16 mouse melanoma cells, only avermectin A ₁ acts. The optical density in the presence of A ₁ decreases by 40 ± 4.5%. Other avermectins on B16 mouse melanoma cells are not effective.

Examples 2-3 show that the effectiveness of avermectins depends on the type of cell. Different avermectins have specific activity for different types of cancer cells.

Example 3. Resistant to vincristine lymphocytic leukemia strain P388 was obtained by repeated administration of vincristine, 1 μg / g to mice with DBA ₂ vaccinated with lymphocytic leukemia. 1 million P388 cells were inoculated intraperitoneally with mice and vincristine was also intraperitoneally administered one day later. After the growth of tumor cells (after 7-10 days), the cells were transplanted into other mice and vincristine was again administered in a day. The procedure was repeated up to 6 times. For in vitro operation, cells were isolated, washed and placed in RPM1-1640 medium with serum and antibiotics. Agents were added before incubation, and after 22 hours of incubation at 37 ° C, the number of living cells was determined using trypan blue staining. Survival was calculated as the ratio of the number of living cells in the experiment (N ₀ ) to the control (N _k ): In% = (N ₀ / N _k ) · 100%. The IC ₅₀ for P388 cells was 14 ng / ml, and for vincristine-resistant cells (P388BP), 724 ng / ml. Avermectin B ₁ (10 ng / ml) increased the sensitivity to P388BP cells of vincristine by 35 times. Vincristine at a dose of 100 ng / ml did not affect the survival of P388BP cells, but in combination with avermectins, vincristine became toxic to cells, and avermectins significantly varied in effectiveness. Doses of avermectins that reduce the survival of P388BP cells to 50% (IC ₅₀ ) were for A ₁ , A ₂ , B ₂ , B _1, respectively, 0.01; one; 4 and 6 ng / ml. Avermectins per se at these concentrations are not toxic to cells. Thus, the resistance of P388BP cells to vincristine is removed by avermectins. In this case, A _{1 is} most active and B ₁ is least active.

Example 4. To obtain a taxol-resistant strain, Hep-2 cells were seeded into 100 ml / ml culture bottles (V = 6 ml) in DMEM medium supplemented with 10% fetal calf serum and 80 μg / ml gentamicin. After a day of incubation at 37 ° C, taxol was added at a final concentration of 10 nM. After 2-3 days, dead cells were removed by changing the medium. Surviving cells gave colonies, after their growth the cells were detached, reseeded and expanded for experiments. To determine the cytotoxicity of cell agents (50 thousand / ml) in a DMEM medium supplemented with serum (10%) and gentamicin (80 μg / ml), 0.1 ml were scattered into a 96-well plate. After a day of incubation in a CO ₂ incubator at 37 ° C, the studied agents were added and incubation continued for another two days. At the end of the incubation, the cells were stained with 0.02% violet crystalline in 20% ethanol, the paint was extracted with 1% SDS, the optical density was determined at 570 nm on a Multiscan Plus spectrophotometer. Cell survival (N / N ₀ %) was determined by the formula:

N ₀ / N _k = [{(D ₀ -D _background ) - (D _k -D _background )} / (D _k -D _background )] × 100%,

where D ₀ , D _k , D _background are the optical densities in the experiment, in the control and background, respectively. The IC _{50 of} taxol for the taxol-resistant strain of Hep-2TP increased to 1660 pM, which is 55 times greater than the IC ₅₀ for the original Hep-2 cells. Avermectin B _{1 was} added simultaneously with taxol. B _{1 itself} did not affect cell survival. The IC _{50 of} taxol in the presence of 0.01 ng / ml B ₁ was 1.4 pM for the strain Hep-2TP. Comparing this value with the IC _{50 of} taxol without B ₁ (1660 pM), we obtain the coefficient of modification of sensitivity to taxol with avermectin B ₁ equal to 1186.

Example 5. The effect of avermectins on multidrug resistance (MDR) of tumors was determined by increasing the fluorescence of calcein in P388BP cells, which was pumped out of the cells by transport proteins responsible for MDR. Cells (2 mln / 2 ml) were incubated with calcein ether (1 μM) for 10 minutes at 37 ° C in RPMI-1640 medium with 10% serum, then the cells were precipitated by centrifugation (12000 g, 3 min). The precipitate was resuspended in saline pH 7.4 with a Ca ²⁺ content of 40 μM. Calcein fluorescence was measured on a fluorimeter (excitation wavelength 493 nm, emission wavelength 515 nm). Avermectins were added 20 minutes before calcein ester. Avermectins increase the accumulation of calcein in the cells, which indicates their inhibition of MDR. The concentration that doubles the fluorescence varies for different avermectins from 0.01 to 10 ng / ml.

Example 6. Ehrlich carcinoma was inoculated intramuscularly (in the thigh) of 2 million cells per mouse in white mouse mice CH ₃ . Five days after vaccination, vincristine, aversectin C (avermectin mixture, see RU 2182174) were intraperitoneally administered, or both. Control mice were injected with 0.2 ml of 4% ethanol (control on aversectin C solvent). 60 animals were used in the experiments: 10 mice per option. The effect of the effects was determined by inhibition of tumor growth (SRW). Aversectin C (0.2-0.5 mg / kg) significantly increased the TPO of vincristine in the dose range from 30 to 300 ng / g. At a dose of 300 ng / g vincristine, TPO was 30–40%, and the addition of aversectin C increased TPO to 70–80%.

Example 7. Lewis carcinoma cells (LLC) in an amount of 30-40 mg / mouse suspended in 0.3 ml of culture medium (or 250-300 thousand / mouse of cultured LLC cells in suspension) were inoculated subcutaneously in male C57B1 breed (LLC strain was maintained on the same breed of mice). The effects of the combined effects of vincristine (200 ng / g) and avermectin B ₁ (0.1 mg / kg) were analyzed. Two groups of animals served as a control: only LLC (group K ₀ ) were administered to the first control group, LLC and vincristine (group K _v ) to the second. Vincristine (200 ng / g) and avermectin (0.1 mg / kg) were administered intraperitoneally once. Estimated increase in life expectancy and inhibition of tumor growth. Statistically significant (P <0.05) shows the positive effect of avermectin B ₁ both on the life expectancy of experimental animals and on the inhibition of tumor growth. In 2 series of experiments in the groups of animals that were injected with vincristine and avermectin B ₁ , the tumors simply did not develop or disappeared barely, while in the control group K ₀ after 35-45 days at 100%, and in the group K _v at 80 ± 20% of animal tumors reached critical sizes (22 ± 3.6 mm - 24 ± 2.8 mm). Long-term observation (60 days) showed that the surviving animals of the experimental group were no different from animals that were not injected LLC cells.

In other series of experiments, 60 ± 10% of the animals survived (their tumor did not appear at all or, appearing, disappeared within 10-14 days). In the groups K ₀ and K _v , the same picture was observed in these experiments as in the first series of experiments.

In experimental animals with a tumor, inhibition of tumor growth was statistically significant by the 20th day of observation by 65 ± 17% of the control To _v . Subsequently, the inhibition of tumor development decreased and amounted to 32 ± 9% by day 30. It should be noted that with an increase in the number of LLC grafted cells to 700 thousand / mouse, the results deteriorate sharply: animal survival drops to 20 ± 10%, and inhibition of tumor growth to 40 ± 13%.

Summary data on the effect of avermectins on the activity of antitumor drugs in vitro and in vivo are shown in tables 1 and 2, respectively.

Claims (4)

1. The use of avermectins as a means of enhancing the antitumor activity of chemotherapeutic drugs in vitro at concentrations of 0.01-10 ng / ml and in vivo at doses from 0.01 to 1 mg / kg. 2. A method of treating cancer, including the introduction of chemotherapeutic agents and agents that enhance the antitumor activity of chemotherapeutic agents, characterized in that individual avermectins or their complexes in doses from 0.01 to 1 mg / kg are used as agents that enhance the antitumor activity of chemotherapy drugs. 3. The method according to claim 2, characterized in that vincristine, or doxorubicin, or pharmacorubicin, or taxol, or etoposide are used as chemotherapeutic agents. 4. The method according to claim 2, characterized in that the avermectins are taken after taking chemotherapy drugs.