RU2209078C1 - COMPOSITION FOR KILLING TNF-α-SENSITIVE TUMOR CELLS - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: composition has cytostatic agent and TNF-α. Preparations intercollating into DNA is taken as the cytostatic agent and biologically active mouramylpeptide is added. Mass proportions of the cytostatic agent, muramylpeptide and TNF -α is 1:0.06 - 300:0.00001 - 0.1. EFFECT: enhanced effectiveness of treatment.

Description

 The invention relates to medicine and veterinary medicine and can be used in the treatment of tumors in humans and animals.

 Widespread use in the treatment of tumors of cytotoxic drugs, such as cisplatin, doxorubicin.

 Cisplatin - cis-Platinum (II) -diamine, dichloride - a synthetic antitumor drug with a wide spectrum of action [Mashkovsky M.D. Medicines - M.: Medicine, 1993, p. 523].

 Doxorubicin is an antitumor antibiotic of the anthracycline series. It has the ability to block the matrix activity of DNA [Mashkovsky M.D. Medicines - M .: Medicine, 1993 p. 534-535].

 Also known is the use for the treatment of tumors of the antibiotic actinomycin-D-acrinocycline-di (threonine-D-valinyl-proinyl-sarcosyl-N-methyl-valinyl) -lactone [Mashkovsky M. D. Drugs. - M.: Medicine, 1993, p. 532-533].

 In recent years, active attempts have been made to use natural cytotoxic agents in the treatment of tumors, in particular, tumor necrosis factor-α (TNF-α). TNF-α - a protein having a molecular weight of 17.1 kDa, is a cytotoxic factor of activated macrophages and normal killers. Natural and recombinant TNF-α have shown good results when tested as an antitumor agent in animals [Agier et al., 1988, Tumor necrosis factors. Handbook of Cytolytic Lymphocytes and Components: Immune System Effects, E.R. Podak, ed., CRC Press Inc. Boca Raton, Fla. , p. 105]. They caused necrosis of a number of experimental tumors on syngeneic mice and have been shown to be highly effective on human tumors transplanted into athymic mice [Asher A. 1987, Studies on the anti-tumor efficacy of systemically administered recombinant tumor necrosis factor against several murine tumors in vivo. J. Immunol. 138 (3): 963-74 .; Kuroda K. et al., 2000, Human tumor necrosis factor-alpha mutant RGD-V29 (F4614) shows potent antitumor activity and reduced toxicity against human tumor xenografted nude mice. Cancer Lett 2000 Oct 16; 159 (1): 33-41]. Attempts have been made to use TNF-α for treatment of tumors in a clinic [Terlikowski S.J. 2001, Tumour necrosis factor and cancer treatment: a historical review and perspectives. Rocz Akad Med Bialymst. 46: 5-18; Lejeune F.J., et al., 2001, Isolated limb perfusion: the European experience. Surg Oncol Clin N Am, 10 (4): 821-32; Nashan D., Luger T.A. 2001, Cytokines: current status and prospects in the treatment of skin tumors. Hautarzt; 52 (8): 691-6].

 The use of the above cytotoxic drugs is complicated by side effects. To suppress the growth of tumor cells, high doses of these agents, toxic to normal cells and organs, are required. For example, cisplatin negatively affects the kidneys. Doxorubicin has cumulative cardiotoxicity. When using TNF-α, patients have hypotension, pulmonary edema, intravascular thrombi and other serious side effects. Another problem is the acquisition by tumor cells of resistance to cytotoxic agents.

 Immunomodulators include cytokines of the immune system, peptides of the thymus and bone marrow, low molecular weight bioregulators of bacterial origin. The latter include muramyl peptides [Ivanov V. et al., 1987, Structure, design, and synthesis immunoactive peptides. Pure & Appl. Chem. 59, 317-324].

 A known composition for the treatment of tumors containing muramyl peptide and cytostatic cisplatin or doxorubicin [MacEwen EG, Kurzman, Helfand S., Vail D., London C., Kisseberth W., Rosenthal RC, Fox LE, Keller ET, Obradovich J., et al. Current studies of liposome murarmyl tripeptide (COP 19835A lipid) therapy for metastasis in spontaneous tumors: a progress review. (1994) J. Drug Target, 2 (5), 391-6]. Osteosarcomas in dogs are treated by the intravenous separate and sequential administration of muramyl peptide (N-acetylmuramoyl-alanyl-D-isoglutaminyl-alanyl-2- (1 ', 2'-dipalmitoyl) -sn-glycero-3'-phosphoryl-ethylamide) and cisplatin : the course of repeated administration of cisplatin ends with the course of repeated administration of muramyl peptide. In the treatment of hemangiomas in dogs, repeated intravenous administration of doxorubicin with cyclophosphamide is used in parallel with repeated intravenous administration of muramyl peptide. In both cases described above, it is possible to achieve a significant increase in the life span of animals, significantly exceeding that in chemotherapy and monotherapy with osteosarcoma and hemangiomas in dogs using one muramyl peptide. The disadvantage of these formulations for treatment is primarily the need for repeated administration of drugs. Treatment is significantly complicated by the mandatory preliminary surgical removal of the underlying tumor.

 A known composition containing TNF-α (16,000 units / mouse) in combination with chemotherapeutic agents: doxorubicin (1-10 mg / kg), 5-fluorouracil (3-100 mg / kg) cyclophosphamide (50-200 mg / kg), for the treatment of Meth A tumors transplanted in syngeneic mice. The most effective combinations induce complete tumor regression. [Regenass U., Muller M., Curschellas E., Matter A. Anti-tumor effects of tumor necrosis factor in combination with chemotherapeutic agents. Int J Cancer. 1987; 39 (2): 266-73]. The disadvantages of this composition include intravenous infusion of the administered composition. This method of administration provides the maximum manifestation of cytotoxicity at high doses of chemotherapeutic agents 5 mg / kg of doxorubicin, 100 mg / kg of cytoxane and 100 mg / kg of fluorouracil.

 A known composition for the treatment of cancer patients, containing actinomycin D together with TNF-α [Seibel N.L., Dinndorf P.A., Bauer M., Sondel P.M. , Hammond G. D., Reaman G. H. Phase I study of tumor necrosis factor-alpha and actinomycin D in pediatric patients with cancer: a Children's Cancer Group study. (1994), J. Immunother Emphasis Tumor Immunol, 16 (2), 125-31, and Sella A., Aggarwal B. B., Kilboum R. G., Bui. CA., Zukiwski A. A., Logothetis C. J. Phase I study of tumor necrosis factor plus actinomycin D in patients with androgen-independent prostate cancer. (1995) Cancer Biother, 10 (3), 225-35]. However, the widespread use of actinomycin D in oncology is limited to severe adverse reactions in patients.

 Known closest to the claimed composition containing cisplatin and PHO-α, which can be successfully used to destroy cells of the passivated lines OVCAR-3 and SK-OV-3 of human ovarian adenocarcinoma, resistant to the cytotoxic effect of cisplatin and TNF-α, used individually [Mutch D., Powell C., Kao M., Collins J. In vitro analysis of the anticancer potential of tumor necrosis factor in combination with cisplatin. Gynecol Oncol. 1989 Sep .; 34 (3): 328-33].

 The disadvantages of the known composition include the limited cytolysis of tumor cells in culture, the maximum level of which does not exceed 56%.

 The invention solves the problem of increasing the effectiveness of the composition for the destruction of TNF-α-sensitive tumor cells and reduce the toxicity of the drug.

 The problem is solved due to the fact that the composition for the destruction of TNF-α-sensitive tumor cells containing cytostatic and TNF-α, as a cytostatic agent contains intercalating in DNA preparations and additionally biologically active muramyl peptide with a mass ratio of muramyl peptide, cytostatic and TNF-α 1 : 0.06-300: 0.00001-0.1.

 The inventive composition, intended for the destruction of TNF-α-sensitive cells, contains a cytostatic agent from the group of intercalating DNA preparations interacting with chromatin, in particular cisplatin, doxorubicin or actinomycin D, TNF-α cytokine and muramyl peptide: GMDP (N-acetylglucosaminyl-β1- -> 4-N-acetylmuramoyl-alanyl-D-isoglutamine), GMDP-K (N-acetylglucosaminyl-β1 -> 4-N-acetylmuramoyl-alanyl-D-isoglutamic acid) or another immunologically active muramyl peptide.

 The advantage of the proposed triple combination of drugs (cytostatic, TNF-α and muramyl peptide) is the use of reagents that mutually reinforce the antitumor effect of each of the components of the composition. This reduces the effective concentration of reagents. For example, in combination (cisplatin, TNF-α and muramyl peptide), 4 times lower doses of cisplatin and 5-20 times lower doses of TNF-α are used to achieve 100% survival of Balb / c mice with Ehrlich ascites carcinoma in the literature on monotherapy of transplantable tumors in mice with cisplatin (Landrito JE, Yoshiga K., Sakurai K., Takada K. Effects of intralesional injection of cisplatin dissolved in urografin and lipiodol on Ehrlich ascites tumor and normal tissues of CD-1 mice. (1994 ) Cancer Chemother Pharmacol; 34 (4): 323-30 and Krug H., Taubert G., Lenschow S. Proliferation kinetics of Ehrlich ascites tumors during cisplatin administration. (1990) Acta Histochem Suppl; 39: 61-65) and TNF -α (Obrador E, Navarro J, Mompo J, Asensi M, Pellicer JA, Estrela JM. Glutathione and the rate of cellular proliferation determine tumor cell sensitivity to tumor necrosis factor in vivo. (1997) Biochem J; 325 (Pt 1), 183-189 and Krosnick J ., Mule J., Mctosh J., Rosenberg S. Augmentation of antitumor efficacy by the combination of recombinant TNF and chemotherapeutic agents in vivo. (1989) Cancer Res., 49, 3729-3733). The undoubted advantage is a single injection of drugs. It should be noted that in the proposed antitumor combination, the available domestic drug GMDP and the cytostatics cisplatin and doxorubicin widely used in oncological practice are used.

In the described composition, a recombinant human TNF-α preparation is used (laboratory of gene chemistry of the IBCh named after M.M.Shemyakin and Yu.A. Ovchinnikov RAS) with an activity of 4x10 ⁷ units. per 1 mg of protein, GMDP (Peptek company), Russia, cisplatin, doxorubicin and actinomycin D- (Sigma company, USA), PBS-0.1 M phosphate buffer, pH 7.4, containing 0.15 M NaCl), prepared from commercial salts of the grade hc in bidistilled water.

 The invention is illustrated by examples.

 Example 1

A sample of cisplatin 1 mg is dissolved in 1 ml of physiological saline on a stirrer at room temperature and sterilized using a membrane filter with a pore diameter of 0.2 μm. The resulting cisplatin solution was diluted with sterile saline to a concentration of 9 μg / ml. A solution of recombinant TNF-α (0.6 mg / ml) in PBS with 0.1% bovine serum albumin is diluted with sterile saline to a concentration of 3.75 x 10 ^-3 μg / ml. A portion of GMDP (1 mg) is dissolved in 1 ml of physiological saline or PBS and sterilized using a membrane filter with a pore diameter of 0.2 μm. GMDP is diluted with sterile saline to a concentration of 30 μg / ml. The prepared preparations of cisplatin, TNF-α and GMDP are mixed in equal volumes and 100 μl are added to Petri dishes with tumor cells cultured in 900 μl of RPMI-1640 medium (Flow Laboratories, Scotland) containing 10% FCS (fetal calf Gibco BRL serum), thereby creating a concentration of GMDP of 5 μg / ml, cisplatin 0.3 μg / ml and TNF-α 0.125x10 ^-3 μg / ml, (mass ratio 1: 0.06: 0.000025) .

где А - число колоний в контроле (в чашке, в которую вместо препаратов вносят ЗФР)
В - число колоний в образце, обработанном препаратами.To test the cytolysis of L929 cells under the influence of the studied composition of the preparations, cells cultured in vials in an atmosphere of 5% CO ₂ at 37 ^° C are removed with trypsin solution (0.5%) and placed in 50 ml centrifuge glasses containing 30 ml of RPMI medium -1640 with FCS. After centrifugation at 300g, the cell pellet was suspended in a culture medium with 10% FCS. An aliquot was taken from the sample using a Goryaev camera, the concentration of cells was determined, the cell suspension was adjusted to a density of 500 cells / ml and placed in plates with a diameter of 30 mm. After 24 hours of cultivation, test preparations are added. Incubation is continued for 7 days in an atmosphere of 5% CO ₂ at 37 ^° C. After which the medium is removed using a Pasteur pipette, the cells attached to the plastic are washed 3 times with PBS (0.1 M phosphate buffer, pH 7.4, containing 0.15 M NaCl). The washed cells are fixed by pouring them with 0.5 ml of a mixture of methanol: acetic acid (volume ratio 3: 1) and leaving for 20 min at room temperature. Then the fixation solution was taken with a Pasteur pipette, 0.5 ml of a 0.5% solution of crystalline violet dye (commercial Sigma dye) in 20% methanol was added and incubated at room temperature for 20 minutes. Stained cells are washed from excess dye with distilled water, the washing procedure is repeated 7 times. The cups are dried and counted stained colonies visually in transmitted light. The percentage of dead cells is determined by the formula

where A is the number of colonies in the control (in the cup, in which instead of drugs add PBS)
B is the number of colonies in the sample treated with the preparations.

C = 87 ± 5%
The effect of the combination of drugs is compared with a number of control samples of cell cultures incubated with:
- saline or PBS C = 0%;
- one cisplatin at a concentration of 0.3 μg / ml C = 45 ± 2%;
- one TNF-α at a concentration of 0.125 x 10 ^-3 μg / ml C = 25 ± 2%;
- one GMDP in a concentration of 5 μg / ml C = 2 ± 2%;
- a combination of cisplatin at a concentration of 0.3 μg / ml with TNF-α at a concentration of 0.125 x 10 ^-3 μg / ml C = 64 ± 3%;
- a combination of cisplatin at a concentration of 0.3 μg / ml with GMDP at a concentration of 5 μg / ml C = 67 ± 3%;
- a combination of TNF-α at a concentration of 0.125 x 10 ^-3 μg / ml with GMDP at a concentration of 5 μg / ml C = 44 ± 2%.

 Example 2

The composition is prepared according to example 1, but using solutions of GMDP and TNF-α with concentrations of 30 μg / ml and 0.375x
10 ^-3 μg / ml, respectively. Mix in equal volumes of GMDP, cisplatin and TNF-α, add to the culture medium and get a solution containing 1 μg / ml GMDP, 0.3 μg / ml cisplatin and 0.0125x10 ^-3 μg / ml TNF-α (mass ratio 1 : 0.3: 0.0000125). The tests are carried out as in example 1. C = 72%.

 Example 3

The composition is prepared according to example 1, but using solutions of GMDP with a concentration of 3 μg / ml, cisplatin with a concentration of 900 μg / ml, TNF-α with a concentration of 0.0375 μg / ml and get a working solution containing 0.1 μg / ml of GMDP. 30 μg / ml cisplatin and 1.25x10 ^-3 μg / ml TNF-α (mass ratio 1: 300: 0.0125). The tests are carried out as in example 1. C = 100%.

 Example 4

The composition is prepared according to example 1, but doxorubicin is used as a cytostatic agent. A 1 mg doxorubicin preparation was dissolved in 1 ml of physiological saline on a stirrer at room temperature and sterilized using a membrane filter with a pore diameter of 0.2 μm. The resulting doxorubicin solution was diluted with sterile saline to a concentration of 30 μg / m. A solution of recombinant TNF-α (0.6 mg / ml) is diluted with sterile saline to a concentration of 7.5 x 10 ^-3 μg / ml. A portion of GMDP (1 mg) is dissolved in 1 ml of physiological saline or PBS and sterilized using a membrane filter with a pore diameter of 0.2 μm. GMDP is diluted with sterile saline to a concentration of 30 μg / ml. The prepared preparations of doxorubicin, TNF-α and GMDP are mixed in equal volumes and 20 μl are added to the wells of a 96-well plate with tumor cells cultured in 180 μl of RPMI-1640 medium containing 10% FCS, thereby creating a concentration of 1 μg / ml GMDP, 1 μg / ml of docorubicin and 0.25x10 ^-3 μg / ml TNF-α (mass ratio 1: 1: 0,00025).

где А - показатель экстинкции в контроле (в образце, в который вместо препаратов вносили ЗФР),
В - показатель экстинкции в образце, обработанном препаратами.Assessment of the level of cytolysis is performed in a test with crystal violet: cells (2x10 ⁴ cells / well) in 100 μl of RPMI-1640 containing 10% FCS are placed in a 96-well plate. After 48 hours of incubation of cell cultures with the composition of the preparations in an atmosphere of 5% CO ₂ at 37 ^° C, the medium is removed, the cells are washed 3 times with PBS, fixed in a mixture of methanol / acetic acid 3/1 for 20 minutes and stained with a 0.5% crystalline solution purple in 20% methanol for 20 minutes. Then the tablet cells are washed 7 times with water and dried. The dye that bound to the cells was dissolved in 1% SDS and its absorbance at 540 nm was measured on a Titertek Multiskan MCC / 340 spectrophotometer. The percentage of cytotoxicity is determined by the formula

where A is the extinction index in the control (in the sample, in which instead of drugs were added PBS),
B is the extinction index in the sample treated with the preparations.

 C = 90 ± 3%.

 Example 5

The composition is prepared according to example 1, but actinomycin D is used as a cytostatic agent. Actinomycin D preparation 1 mg is dissolved in 1 ml of physiological saline on a stirrer at room temperature and sterilized using a membrane filter with a pore diameter of 0.2 μm. The resulting actinomycin D solution was diluted with sterile saline to a concentration of 30 μg / ml. Solutions of actinomycin D (30 μg / ml), TNF-α and GMDP are mixed in equal volumes and 20 μl are added to the wells of a 96-well plate with tumor cells cultured in 180 μl of RPMI-1640 medium with 10% FCS and a solution is obtained containing 0.1 μg / ml GMDP, 1 μg / ml, actinomycin D and 0.01 μg / ml TNF-α (mass ratio 1: 10: 0.1). The tests are carried out as in example 4. C = 100%
Example 6

 The composition is prepared according to example 1, but is used as muramyl peptide GMDP-K at a concentration of 300 μg / ml, cisplatin is taken at a concentration of 90 μg / ml, TNF-α - 0.375 μg / ml and a solution containing 10 μg / ml of GMDP-K is obtained 3 μg / ml cisplatin and 0.0125 μg / ml TNF-α (weight ratio 1: 0.3: 0.00125). The tests are carried out as in example 4. C = 100%.

 Example 7

A sample of cisplatin 1 mg is dissolved in 1 ml of physiological saline on a magnetic stirrer at room temperature and sterilized using a membrane filter with a pore diameter of 0.2 μm. Cisplatin is diluted with sterile saline to a concentration of 30 μg / ml. A solution of recombinant TNF-α (0.6 mg / ml) is diluted with sterile saline to a concentration of 37.5 x 10 ^-3 μg / ml. A portion of GMDP (1 mg) is dissolved in 1 ml of physiological saline and sterilized using a membrane filter with a pore diameter of 0.2 μm. GMDP is diluted with sterile saline to a concentration of 30 μg / ml. The prepared preparations of cisplatin, TNF-α and GMDP are mixed in equal volumes. Add 20 μl of the mixture to the wells of a 96-well plate with MCF7 tumor cells cultured in 180 μl of RPMI-1640 with 10% FCS, thereby creating a concentration of GMDP of 1 μg / ml, cisplatin 1 μg / m and TNF-α 0, 0125 μg / ml (mass ratio 1: 1: 0.0125).

Testing the effect of a combination of drugs on the viability of tumor cells of the MCF7 human breast adenocarcinoma cell line cultured in vials in an atmosphere of 5% CO ₂ at 37 ^° C. Cells are removed from the surface of the vials with trypsin solution (0.5%) and placed in 50 centrifuge glasses ml containing 30 ml of RPMI-1640 medium with 10% FCS. After centrifugation at 300g, the cell pellet was suspended in RPMI-1640 culture medium with 10% FCS. An aliquot was taken from the sample using a Goryaev camera, the cell concentration was determined, the cell suspension was adjusted to a density of 1x10 ⁵ cells / ml and placed on tablets. After 24 hours of cultivation, test preparations are added. Cells are incubated with a combination of drugs for 120 hours in an atmosphere of 5% CO ₂ at 37 ^o C. Then determine the viability of cells in experimental and control cultures.

где А - показатель экстинкции в контроле (в образце, в который вместо препаратов вносили ЗФР), "
В - показатель экстинкции в образце, обработанном препаратами.Cell viability is evaluated by colorimetric method according to the degree of restoration of MTT dye - [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide] - stained substrate for mitochondrial dehydrogenases (Sigma USA) - to formazone. For this, 2.5 μl of MTT paint with a concentration of 2.5 mg / ml is added to each well 2.5 hours before the end of the cultivation. After incubation, the plates are centrifuged at 300 g for 10 minutes. The supernatant is removed from the wells, the precipitate is dissolved in 100 μl of dimethyl sulfoxide with continuous shaking for 15 minutes. The optical density of the contents of the wells was measured on a Titertek Multiskan MCC / 340 instrument at a wavelength of 540 nm. The percentage of cytotoxicity is determined by the formula

where A is the extinction index in the control (in the sample into which PBS was added instead of drugs), "
B is the extinction index in the sample treated with the preparations.

 C = 69 ± 3%.

The effect of the combination of drugs is compared with a number of control samples: cell cultures incubated with
- saline or PBS. C = 0%;
- one cisplatin at a concentration of 1 μg / ml. C = 28 ± 1%;
- one TNF-α at a concentration of 0.0125 μg / ml. C = 42 ± 2%;
- one GMDP in a concentration of 1 μg / ml. C = 2 ± 1%;
- a combination of cisplatin at a concentration of 1 μg / ml with TNF-α at a concentration of 0.0125 μg / ml. C = 61 ± 3%;
- a combination of cisplatin at a concentration of 0.3 μg / ml with GMDP at a concentration of 1 μg / ml. C = 32 ± 2%;
- a combination of TNF-α at a concentration of 0.0125 μg / ml with GMDP at a concentration of 1 μg / ml C = 60 ± 3%.

 Example 8

(массовое соотношение 1:4:0,0125). Испытания проводят по примеру 7. Ц=82±3%.The composition is prepared according to example 7, but doxorubicin at a concentration of 120 μg / ml is used as a cytostatic agent and a solution containing 1 μg / ml of GMDP, 4.0 μg / ml of doxorubicin and 0.0125 μg / ml of TNF-α is obtained

(mass ratio 1: 4: 0.0125). The tests are carried out as in example 7. C = 82 ± 3%.

 Example 9

 The composition is prepared according to example 7, but using cisplatin at a concentration of 90 μg / ml and get a solution containing 1 μg / ml GMDP, 3.0 μg / ml cisplatin, 0.0125 μg / ml TNF-α (mass ratio 1: 3: 0.0125). The tests are carried out on cells of the histiocytic lymphoma cell line U937 as on the MCF7 line with the following changes. To spread cells in a 96-well plate, U937 cells from vials are collected by pipetting them in culture medium. The incubation time of cells with drug combinations is 72 hours. C = 90 ± 3%.

 Example 10

The composition obtained in example 4, add 20 μl of the mixture to the wells of a 96-well plate with tumor cells taken from cancer patients. Exudates from the pleural cavity of cancer patients are obtained from ONC RAMS, Moscow. The exudate cells are centrifuged at 300 g and the pellet is resuspended in RPMI-1640 medium in a volume 10 times smaller than the original. The resulting suspension (7 ml each) is carefully layered on a lymphocyte separating medium (ficoll / diatrizoate with a density of 1.077 g / ml), poured into 4 ml in 15 ml centrifuge tubes. Then the suspension is centrifuged for 25 min at 600g at room temperature. Tumor cells and white blood cells are localized in the interphase of the gradient. The interphase is taken with a Pasteur pipette. Cells are washed twice by centrifugation at 300g for 10 min in culture medium without FCS. Then the cells are suspended in RPMI-1640 medium with 10% FCS at a density of 1x10 ⁵ cells / ml and 180 μl of the suspension are placed in the cells of a 96-well plate. The incubation time of the cells with the composition of the preparations is 72 hours.

где А - число мертвых клеток в образце, "
В - общее число клеток.The assessment of the level of cytolysis is carried out in a test with trypan blue: the cells are removed with a rubber scraper, washed 2 times with medium without serum. The removed cells and swabs are combined into one sample. An aliquot of 50 μl was taken from the sample, 10 μl of a 0.4% trypan blue solution was added to it and immediately placed in a Goryaev chamber to count living and dead cells (dead cells, unlike living cells, included dye). At the same time, at least 100 tumor cells are considered. The percentage of cytotoxicity is determined by the formula

where A is the number of dead cells in the sample, "
B is the total number of cells.

 C = 100%.

 Example 11

где А - число живых мышей через 7 мес. после перевивания им опухолей,
В - первоначальное число мышей в группе (10 особей).A sample of cisplatin 1 mg is dissolved in 1 ml of physiological saline on a stirrer at room temperature and sterilized using a membrane filter with a pore diameter of 0.2 μm. Cisplatin is diluted with sterile saline to a concentration of 200 μg / ml. A solution of recombinant TNF-α (0.6 mg / ml) is diluted with sterile saline to a concentration of 0.625 μg / ml. A portion of GMDP (1 mg) is dissolved in 1 ml of physiological saline and sterilized using a membrane filter with a pore diameter of 0.2 μm. GMDP is diluted with sterile saline to a concentration of 750 μg / ml. The prepared solutions of cisplatin, TNF-α and GMDP are mixed in equal volumes and the resulting preparation of 0.6 ml is injected intraperitoneally into the mouse. Thus, in each specimen with Ehrlich ascites carcinoma implanted intraperitoneally to Balb / c mice, a composition comprising 41.7 μg / ml GMDP, 66.7 μg / ml cisplatin and 0.210 μg / ml TNF-α (mass ratio 1 : 1.6: 0.0050). By changing the final volume of the composition of the preparations from 0.04 to 4 ml, the concentration of the introduced agents is proportionally changed, while maintaining their mass ratio. Drug dose is 25 ug / mouse GMDP (6.25 mg / m ^2), 0.125 .mu.g / mouse of TNF-α (0,031 g / m ²⁾ and 40 ug / mouse of cisplatin (10 mg / m ^2). A group of mice consists of 10 individuals. Estimated average life expectancy and survival. Life expectancy is defined as the sum of life expectancy in days of all deceased in a group of animals divided by the number of these animals. Survival is determined by the formula

where A is the number of live mice after 7 months. after transplanting tumors
In - the initial number of mice in the group (10 individuals).

 In the group of mice in which the described therapy was carried out, the survival rate was 100%.

The effect of the combination of drugs, evaluated by mouse survival and longevity, is compared with the same parameters of the animals of the control groups, each of which consists of 10 individuals with implanted Ehrlich carcinoma ascites cells. Control groups consist of tumor carriers that were given the following drugs:
- saline solution - all mice develop ascites and they die. Life expectancy is 32 days.

 - cisplatt at a dose of 40 μg / mouse - the survival rate of mice is 30%.

- TNF-α at a dose of 0.125 μg / mouse - all mice develop ascites and they die. The average life expectancy of mice is 37 days
- GMDP at a dose of 25 μg / mouse - survival rate of 20%.

 - a combination of cisplatin at a dose of 40 μg / mouse with TNF-α at a dose of 0.125 μg / mouse - 80% survival rate.

 - a combination of cisplatin at a dose of 40 μg / mouse with GMDP at a dose of 25 μg / mouse - 80% survival rate.

 - a combination of TNF-α at a dose of 0.125 μg / mouse with GMDP at a dose of 25 μg / mouse - survival rate of 30%.

 It should be noted that in the group of mice that did not implant tumor cells, but were administered a combination of drugs, the survival rate is 100%.

Claims (1)

 Composition for killing TNF-α-sensitive tumor cells containing cytostatic and TNF-α, characterized in that the composition contains intercalating drugs in DNA and additionally biologically active muramyl peptide in a weight ratio of muramyl peptide, cytostatic and TNF-α 1: 0 06-300: 0.00001-0.1.