Pharmaceutical composition for the prevention and treatment of cancer

 

(57) Abstract:

The subject invention are pharmaceutical compositions for the prevention and treatment of cancer and the way they are received. The compositions include at least three compounds present in crowney system: at least one amino acid, at least one vitamin and at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, malic acid, salewoman acid and adenosine triphosphate. The compositions have reduced toxicity and greater efficacy in the prevention of cancer, suppress the formation of tumors in the case of AIDS and transplant, prevent the formation of metastases. 8 C.p. f-crystals, 9 Il, 2 tab.

The subject of this invention are pharmaceutical compositions for the prevention and treatment of cancer and the way they are received.

The most frequently used in cancer treatment methods are surgery, chemotherapy and irradiation treatment [1-3]. In the treatment by irradiation, in addition to the widely used ionizatsii, in special cases, such as carcinoma of the skin, apply phototherapy and localnode and structure, among the means of chemotherapy can also meet alkylating agents, plant alkaloids, antibiotics, antimetabolites, other medicines (for example asparaginase) and often used different hormones. New used in chemotherapy are the following: combination chemotherapy; long-term venous and arterial infusion of low doses of chemotherapeutic drugs to reduce toxicity; high doses of chemotherapy to overcome drug resistance and therapy of the same type in combination with autologous bone marrow transplantation: a chemotherapeutic agent in combination with biological response modifiers; applying a greater degree of adjuvant and neoadjuvant chemotherapy. The most commonly used biological response modifiers are the following: interferons, tumor necrosis factor, lymphokines, such as interleukin-2 and monoclonal antibodies. Various diet methods, studied serum preparations and method Simonton'using a psychogenic effects belong to the currently used methods in the treatment of neoplastic diseases, the effectiveness of which is not yet established.Unfortunately, treatment with the use of biological response modifiers through its own protective mechanism of the body has similar disadvantages, since it was found that it is only effective against several types of tumors and, in addition, it also has toxic side-effects [2]. Interferons also has numerous and severe side effects and, among other things, cardiotoxicity [6]. Similarly, were not justified the hopes associated with monoclonal antibodies [7]. Despite the fact that since 1980 there were more than 400 registered clinical trials of immunotherapy, none of them have still not been accepted as a treatment for cancer of any type [8].

Known [9] the drug used in the treatment of cancer and containing L-cysteine, L-methionine, L-histidine, L-phenylalanine, L-lysine, L-tryptophan, L-valine, L-leucine, L-threonine, L-malic acid and L-ascorbic acid. L-malic and L-ascorbic acid as activators sautereau and stabilize amino acids in their acid form and thus cause a change in mn the OST or even reverse the development of malignant tumors.

Known [10] a solution of amino acids for intravenous infusion containing specific amino acids except L-isoleucine, are able to exert an inhibitory effect on the increase of tumors. Possible, but not proven action of this drug may be based on limiting amino acid, such as isoleucine.

The purpose of this invention is to provide pharmaceutical compositions comprising natural substances for elimination of disadvantages, such as toxicity, low specificity and restricted limits of known compositions and methods tumor therapy.

The invention is based on the recognition of passive protective systems [hereinafter called passive antitumor defense system (HRLA)] against tumor cells in various organisms. This system is able to destroy the emerging and already existing tumor cells. Agents involved in HRLA, are endogenous and exogenous natural substances present in the circulatory system, namely amino acids, vitamins, nucleic bases, carbohydrates and products of cellular metabolism. It was found that if a joint application is to that all cells and penetrate into them), they synergistically enhance the effects of each other and are therefore able to destroy tumor cells.

Further, the invention is based on the fact that it is found that due to the synergies will exist significant qualitative differences between normal and tumor cells in their behavior toward participants in, which is why the two types of cells become visible. While the classical immune system can recognize and selectively destroy tumor cells by virtue of their external differences from normal cells, the recently discovered mechanism of the passive antitumor defense can do the same due to internal differences.

Further, the invention is based on the fact that it is found that depending on the applied dose and method of application can be achieved prophylactic or antitumor activity for a variety of tumor cells. In the case of different types of tumors that differ from normal cells in varying degrees, on the basis of synergism as a criterion can be defined qualitative and quantitative composition of the most effective mixtures.

Based on the foregoing, the invention provides a pharmaceutical composition is resultvalue in the circulatory system: at least one amino acid, at least one vitamin and at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, malic acid, salewoman acid, ATP and/or their pharmaceutically acceptable salts, provided that if the composition contains in addition to the amino acids(t) only malic acid and vitamin, the vitamin can be any, except ascorbic acid. The compositions can also contain carriers, solvents and/or other auxiliary reagents commonly used in pharmacology.

The composition according to the invention may include L-methionine, L-tryptophan, L-tyrosine, L-phenylalanine, L-arginine, L-histidine, L-benzoylglycine and/or its salt as amino acids and d-Biotin, pyridoxine, Riboflavin, Riboflavin-5'-phosphate, L-ascorbic acid, lipoic acid, Orotava acid and/or salt as a vitamin.

A preferred composition according to the invention include L-tryptophan, L-ascorbic acid, at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-glucosamine and/or their pharmaceutically acceptable salts.

Another preferred composition according to the invention includes L-Argi acid, ATP and/or their pharmaceutically acceptable salts.

Another preferred composition according to the invention includes 30-44% by weight of L-arginine, 27-35% by weight of Riboflavin-5'-phosphate, 38-62% by weight of malic acid and/or their pharmaceutically acceptable salts.

The following preferred composition according to the invention contains 0,002-70% by weight of at least one component selected from the group consisting of L-methionine, L-tryptophan, L-tyrosine, L-phenylalanine, L-arginine, L-histidine, N-benzoylglycine and/or their salts as amino acids, of 0.0004-80% by weight of at least one component selected from the group consisting of d-Biotin, pyridoxine, Riboflavin, Riboflavin-5'-phosphate, L-ascorbic acid, lipoic acid, orotovoy acid and/or their salts as vitamin and 0.003 to 80% by weight of at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, malic acid, salewoman acid, ATP and/or their pharmaceutically acceptable salts.

Another preferred composition according to the invention includes a 0.9-25% by weight of L-methionine, 0.8 to 19% by weight of L-tryptophan, 1,1-48% by weight of L-arginine, 0,9-46% m is the mass of 2-deoxy-D-ribose, 0.7 to 68% by weight of malic acid, 0.6 to 40% by weight of D-mannose and/or their pharmaceutically acceptable salts.

Very effective composition according to the invention includes from 0.005 to 34% by weight of L-methionine, 0,002-25% by weight of L-tryptophan, 0.02 to 23% by weight of L-tyrosine, 0.04 to 30% by weight of L-phenylalanine, 0.04 to 50% by weight of L-arginine, 0,03-34% by weight L-histidine, 0.05 to 22% by weight of N-benzoylglycine, 0.01 to 60% by weight of d-Biotin, 0.01 to 20% by weight of pyridoxine, 0,0004-45% by weight of Riboflavin, 0,0005-45% by weight of Riboflavin-5'-phosphate, 0.003 to 70% by weight of L-ascorbic acid, 00004-15% by weight lipoic acid, from 0.01 to 17% by weight orotovoy acid, 0.001 to 10% by weight of adenine, 0.01 to 63% by weight of 2-deoxy-D-ribose, 0.08 to 42% by weight of D-mannose, 0.05 to 20% by weight of D-glucosamine, 0.01 to 80% by weight of malic acid, 0.02 to 60% mass salewoman acid, 0.001 to 10% by weight of ATP and/or their pharmaceutically acceptable salts.

The next subject of invention is a method of obtaining the above composition, which comprises mixing the active compounds present in the circulatory system, among which at least one amino acid, at least one vitamin and at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, abomasitis composition. The active composition may also be added carriers, solvents and other auxiliary reagents widely used in pharmacology, in an amount necessary to Supplement the weight of the composition to 100%.

The preferred implementation of the method according to the invention for obtaining a pharmaceutical composition involves mixing of L-tryptophan, L-ascorbic acid, at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-glucosamine and/or their pharmaceutically acceptable salts.

Another preferred implementation of the method according to the invention to obtain the pharmaceutical composition includes mixing L-arginine, Riboflavin-5'-phosphate, at least one component selected from the group consisting of D-mannose, malic acid, ATP and/or their pharmaceutically acceptable salts.

The following preferred embodiment of the method according to the invention to obtain the pharmaceutical composition includes mixing 30-44% by weight of L-arginine, 27-35% by weight of Riboflavin-5'-phosphate, 38-62% by weight of malic acid and/or their pharmaceutically acceptable salts.

The preferred embodiment of the method according to image the component selected from the group consisting of L-methionine, L-tryptophan, L-tyrosine, L-phenylalanine, L-arginine, L-histidine, L-benzoylglycine and/or their salts as amino acids, of 0.0004-80% by weight of at least one component selected from the group consisting of d-Biotin, pyridoxine, Riboflavin, Riboflavin-5'-phosphate, L-ascorbic acid, lipoic acid, orotovoy acid and/or their salts as vitamin and 0.003-80% by weight of at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, malic acid, salewoman acid, ATP and/or their pharmaceutically acceptable salts.

Another preferred embodiment of the method according to the invention to obtain the pharmaceutical composition includes mixing 0.9 to 25% by weight of L-methionine, 0.8 to 19% by weight of L-tryptophan, 1,1-48% by weight of L-arginine, 0.9 to 46% by weight of d-Biotin, 1.2 to 16% by weight of pyridoxine, 0.03 to 42% by weight of Riboflavin-5'-phosphate, 0.05 to 18% by weight of D-glucosamine, 0.5 to 60% by weight of 2-deoxy-D-riboy, 0.7 to 68% by weight of malic acid, 0.6 to 40% by weight of D-mannose and/or their pharmaceutically acceptable salts.

The most preferred implementation of the method according to the invention for obtaining the pharmaceutical is in, 0.04 to 30% by weight of L-phenylalanine, 0.04 to 50% by weight of L-arginine, 0,03-34% by weight L-histidine, 0.05 to 22% by weight of N-benzoylglycine, 0.01 to 60% by weight of d-Biotin, 0.01 to 20% by weight of pyridoxine, 0,0004-45% by weight of Riboflavin, 0,0005-45% by weight of Riboflavin-5'-phosphate, 0.003 to 70% by weight of L-ascorbic acid, 0,004-15% by weight lipoic acid, 0.01 to 17% by weight orotovoy acid, 0.001 to 10% by weight of adenine, 0.01 to 63% by weight of 2-deoxy-D-ribose, 0.08 to 42% by weight of D-mannose, 0.05 to 20% by weight of D-glucosamine, 0.01 to 80% by weight of malic acid, 0.02 to 60% by weight salewoman acid, 0.001 to 10% by weight of ATP and/or their pharmaceutically acceptable salts.

For therapeutic applications, the compositions according to the invention is formed in a suitable manner in the pharmaceutical compositions so that after mixing them with non-toxic, inert solid or liquid carriers, solvents, binding agents and/or other additives commonly used in the pharmaceutical industry for enteral or parenteral administration, they became one of the conventional dosage forms. Carriers, solvents and binding agents that meet the above requirements, are, for example, water, gelatin, lactose, sucrose, starch, pectin, Starinov and polyethylene glycol. Pharmaceutical additives and auxiliary components are, for example, preservative agents, such as methyl-4-hydroxybenzoate, various natural or synthetic emulsifiers, dispersing and wetting agents, coloring and aromatic reagents, buffer substances, as well as reagents that stimulate disintegration or dissolution, and other substances that enhance the desired effect.

A conventional dosage form is a composition for oral administration obtained by applying the above-mentioned pharmaceutical additives; these compounds may be a solid form, for example, tablets, capsules, powders, coated tablets, pills or granules, or liquid forms such as syrups, solutions, emulsions or suspensions; in addition, the compositions for rectal administration, such as suppositories, as well as formulations for parenteral administration, for example, solutions or infusions for injection.

The preferred daily dose of the composition according to the invention depends on several factors such as the nature of curable diseases, the patient's condition, treatment, etc. Preferred daily dose of 30-3000 mg/kg body weight. Accordingly, it is acceptable to enter the hedgehog is La infusion, containing 10-200 g/l of active compound.

Further subject of the invention is a method for the prevention and treatment of cancer. This method includes the introduction of a therapeutically effective amount of the composition according to the invention to a patient undergoing treatment.

The invention will be presented in more detail in the following tables, drawings, and examples. Cells used in the following experiments, were obtained from the American type culture collection" (Rockville, MD, USA).

Table 1 presents kill tumor cells action and synergistic joint action of the components of the compositions of examples 1-20, including four and five active components, respectively, Sp2/0-Ag14 myeloma cells (ATSC CRL 1581).

Table 2 shows, on the basis of examples 21-37, strengthening actions, comprising five active components, Sp2/0-Ag14 cells by adding additional components.

In Fig. 1 presents the action and synergistic joint action of the components of the compositions, including four and five active components, respectively, according to examples 1-20.

In Fig. 2 presents a comparison of the actions in vitro compositions include, according to the examples 107-111, on the Sp2/0-Ag14 cells are murine myeloma in comparison with the corresponding control mixture.

In Fig. 3 presents the effect of the compounds, including twenty-one active ingredient according to example 107, Sp2/0-Ag14 cells are murine myeloma as a function of time in comparison with untreated cells and corresponding control mixture.

In Fig. 4 presents the effect in vitro of compounds, including twenty-one active component according to examples 107-111, on K-562 cells of the human erythromelas ADS CCL 243), compared with those of the control mixtures.

In Fig. 5 presents the effect in vitro of compounds, including twenty-one active component according to examples 107-111, HeLa cells human epitheloid carcinoma of the cervix (ATSS CCL 2), compared with those of the control mixtures.

In Fig. 6 presents the effect of the compounds, including twenty-one active component according to examples 107-111, ner-2 cells, a human epidermoid carcinoma of the larynx (ATSS CCL 23) in comparison with the corresponding control mixtures.

In Fig. 7 presents the effect in vitro of compounds, including twenty-one active component according to examples 107-111, normal VP> In Fig. 8 presents the effect in vivo of compounds, including twenty-one active ingredient according to example 112, the tumor developed from Sp2/0-Ag14 cells, mouse myeloma, introduced intraperitoneally BALB/c mice, in comparison with an appropriate control group.

In Fig. 9 shows the in vivo effect of the compounds, including twenty-one active ingredient according to example 112, solid tumor, developed under the skin of HeLa cells human epitheloid carcinoma of the cervix, introduced subcutaneously BALB/c (nu/nu) mice, in comparison with an appropriate control group.

For experiments we used the following environment: in the case of Sp2/0-Ag14 and K-562 cells in RPMI 1640 medium (Sigma Chemie GmbH, D-8024 Deisenhofen, Germany, product number: R 6504), in the case of ner-2, HeLa and Vero cells minimum support MEM medium (Sigma Chemie GmbH, product number: M 4655).

Examples 1-37

In the apparatus, equipped with a stirrer, make active reagents in the same quantities listed in tables 1 and 2, then to the resulting powder mixture is added a quantity of sodium bicarbonate, also listed in tables 1 and 2 are needed to neutralize the acid components of the type. During continuous stirring to the mixture add the appropriate environment that is a of Fig. 1.

Examples 38-67

Carry out also as examples 1-37, with the difference that to neutralize the components of the acid type, apply the appropriate amount of potassium bicarbonate instead of sodium bicarbonate. The effect obtained by this method of solution in any case is not significantly different from the action of solutions of examples 1-37 presented in tables 1 and 2 and in Fig. 1, respectively.

Examples 68-97

Carry out also as examples 1-37, with the difference that to neutralize the components of the acid type, apply the appropriate amount of calcium carbonate instead of sodium bicarbonate. The effect obtained by this method of solution in any case is not significantly different from the action of solutions of examples 1-37 presented in tables 1 and 2 and in Fig. 1, respectively.

Examples 98-101

Carry out also as examples 23, 24, 27 and 28, with the difference that instead of L-arginine, L-histidine, L-methionine and L-tyrosine, respectively, used in examples 23, 24, 27 and 28, respectively, are used 0,053% by weight of L-arginine hydrochloride, 0,052% by weight of L-histidine hydrochloride, 0,009% by weight of L-methionine hydrochloride and 0.025% by weight tyrosine hydrochloride, respectively. In the team by way of solutions does not differ significantly from the action of solutions of examples 23, 24, 27 and 28, are presented in table 2.

Example 102

In the apparatus, equipped with a stirrer, contribute to 0.01% by weight of L-tryptophan, 0,034% by weight of 2-deoxy-D-ribose, 0,003% by weight of adenine, 0,065% by weight of malic acid, to 0.007 mass% L-ascorbic acid and 0,091% by weight of sodium bicarbonate. Then, in order to Supplement the weight of the composition to 100%, during continuous stirring to this mixture add the appropriate environment. The effect thus obtained solution is shown in Fig. 2 for 100% of the composition.

Example 103

Perform well as as an example 102, with the difference that they are only 80% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture, add the appropriate environment. The effect thus obtained solution is shown in Fig. 2 for 80% of the composition.

Example 104

Perform well as as an example 102, with the difference that they are only 60% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture, add the appropriate environment. The effect thus obtained solution is shown in Fig. 2 for 60% of the composition.

Example 105

Perform well as as an example 102, with the ex is to this mixture add the appropriate environment. The effect thus obtained solution is shown in Fig. 2 for 40% of the composition.

Example 106

The method according to example 102, with the difference that contribute only 20% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture adds the appropriate environment. The effect thus obtained solution is shown in Fig. 2 to 20% of the composition.

Example 107

In the apparatus, equipped with a stirrer, contribute to 0.011% by weight of L-methionine, 0.01 percent by weight L-tryptophan, being 0.036% by weight of L-tyrosine, 0,041% by weight of L-phenylalanine, 0,044% by weight of L-arginine, 0,039% by weight L-histidine 0,089% by weight of N-benzoylglycine, to 0.007 mass% L-ascorbic acid, a 0.012% by weight of d-Biotin, 0,010% by weight of pyridoxine, of 0.0004% by weight of Riboflavin, 0,0006% by weight of Riboflavin-5'-phosphate, 0,0006% by weight lipoic acid, is 0.017% by weight orotovoy acid, 0,003% by weight of adenine, 0,034% by weight of 2-deoxy-D-ribose, 0,090% by weight of D-mannose, 0,053% by weight of D-glucosamine, 0,065% by weight of malic acid, 0,040% by weight salewoman acid 0,0015% by weight of adenosine triphosphate and 0.087% by weight of sodium bicarbonate. Then, in order to Supplement the weight of the composition to 100%, during continuous stirring to this mixture is added with the.

Example 108

Perform well as as an example 107, with the difference that they are only 80% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture, add the appropriate environment. The effect thus obtained solution is shown in Fig. 2-7 for 80% of the composition.

Example 109

Perform well as as an example 107, with the difference that they are only 60% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture adds the appropriate environment. The effect thus obtained solution is shown in Fig. 2-7 for 60% of the composition.

Example 110

Perform well as as an example 107, with the difference that contribute only 40% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture, add the appropriate environment. The effect thus obtained solution is shown in Fig. 2-7 for 40% of the composition.

Example 111

Perform well as as an example 107, with the difference that contribute only 20% of the amount of each component. Then, in order to Supplement the weight of the composition to 100%, to this mixture, add the appropriate environment. The action obtained in this way p is make 1,47% by weight of L-methionine, 1,01% by weight of L-tryptophan, being 0.036% by weight of L-tyrosine, 1,63% by weight of L-phenylalanine, 1,71% by weight of L-arginine, 1,53% by weight of L-histidine to 0.18% by weight of N-benzoylglycine, 1,94% by weight of L-ascorbic acid, 1,21% by weight of d-Biotin, 2,02% by weight of pyridoxine, 0,038% by weight of Riboflavin, 0.05% by weight Riboflavin-5'-phosphate, 0.02 mass% lipoic acid, and 0.09% mass orotovoy acid, 0,068% by weight of adenine, 1,32% by weight of 2-deoxy-D-ribose), to 1.8% by weight of D-mannose, and 1.1% by weight of D-glucosamine, 1,32% by weight of malic acid, 0,040% by weight salewoman acid, of 0.11% by weight of adenosine triphosphate, 1,34% by weight of sodium bicarbonate and 0.04% by weight of potassium bicarbonate. Then with continuous stirring to this mixture add 83,168% by weight of a buffer containing 0.02 mass% KH2PO4and 0.3% by weight of Na2HPO4. The effect thus obtained solution is used for treatment of animals, is shown in Fig. 8 and 9.

Example 113

In the mixer is made of 20% by weight of L-tryptophan, 62% by weight of L-ascorbic acid and 18% by weight of D-glucosamine. The resulting full of mixing the powder mixture used for the preventive experiments.

Example 114

The method according to example 113, Orlich">

Example 115

The method according to example 113, with the difference that applies to 23% by weight of L-tryptophan, and 0.2% by weight of pyridoxine, 17,8% by weight of N-benzoylglycine and 59% by weight salewoman acid.

Example 116

The method according to example 113, with the difference that apply 19% by weight of L-tyrosine, 61 mass% L-ascorbic acid, 0.2% by weight of adenine and 19.8% by weight of L-histidine.

Example 117

The method according to example 113, with the difference that applies to 31% by weight of L-methionine, 53% by weight of d-Biotin, 0.2 percent by weight of adenine and 15.8% by weight orotovoy acid.

Example 118

The method according to example 113, with the difference that applies to 27% by weight of L-phenylalanine, 33% by weight of Riboflavin, 0.2 percent by weight of adenosine triphosphate and 39.8% by weight of D-mannose.

Example 119

The method according to example 113, with the difference that apply to 32% by weight L-histidine, 9% by weight lipoic acid and 59% by weight of 2-deoxy-D-ribose.

Example 120

The method according to example 113, with the difference that apply 12% by weight of L-arginine, 11% by weight of pyridoxine and 77% by weight malic acid.

Example 121

In the mixer contribute 10% by weight of L-methionine, 3% by weight of L-tryptophan, 0.02 mass% L-tyrosine, 10.9% of L-fenilalanina, of 0.1% by weight of d-Biotin, 0.2% by weight of pyridoxine, 0.05% by weight of Riboflavin, 0.35 percent by weight Riboflavin-5'-phosphate, 0.1% by weight lipoic acid, 0.6% by weight orotovoy acid, and 0.3% by weight of adenine, 0.9 percent by weight of 2-deoxy-D-ribose, 11.5% by weight of D-mannose, 7% by weight of D-glucosamine, 8% by weight of malic acid, and 0.4% by weight salewoman acid and 0.7% by weight of adenosine triphosphate. The resulting full mixing powdery mixture of uses for the preventive experiments.

The effect of the compositions of examples 1-37 and synergistic joint action of the active ingredients (in comparison with the action of the individual components) was studied on Sp2/0-Ag14 cells of murine myeloma.

In our experiments we use the latest methods described in the scientific literature. In the case of Sp2/0-Ag14 and K-562 lines from among the collected cells in the logarithmic growth phase and resuspendable them in 96-well tablet used for cell culture to a final concentration of 4 104Sp2/0-Ag14 cells and 2 104K-562 cells, respectively, in 250 µl of the medium per well containing the analyte of interest in certain concentrations. In the case of HeLa, ner-2 and Vero cells cultivated cells were collected from 75% of the energy drain Kolb d is. Aliquots (100 ál) was transferred into a 96-well microplate and incubated for 24 hours. Then the medium was removed and replaced it with a 250 ál of fresh medium containing the compounds in certain concentrations. All types of cells were left for proliferation within 48 hours. Then microscopically counting the number of viable Sp2/0-Ag14 and K-562 cells using the method of exclusion dye tripan blue. The survival of HeLa, ner-2 and Vero cells was determined by measuring the activity of endogenous alkaline phosphatase cells. The results were processed using a t-test t-test Data after the mean values in tables and symbols in the drawings denote standard error of the mean" (SOS).

In the first column of table 1 presents the numbers of examples in the second column presents the number of control experiments and in the following six columns presents the number of components used in the experiments and expressed in mass%. Control mix was of the same quantities of chemically similar, but pharmacologically ineffective substances (0,026% by weight of L-serine, 0,033% by weight of L-asparagine, 0,029% by weight of L-valine, 0,018% by weight of L-alanine, 0,006% by weight of glycine, 0,059% by weight of trimethylglycine and the Assos sodium salt of folic acid, 0,001% by weight geekalicious salts of D-Pantothenic acid, 0,012% by weight of uracil and 0,0008% by weight of octanoic acid as vitamins; 0,003% by weight gipoksantina, 0,038% by weight of D(-)ribose, 0,090% by weight of glucose, to 0.055% by weight of N-acetylglucosamine, of 0.081% by weight of disodium salt of succinic acid, 0,080% by weight of disodium salt of fumaric acid and 0,0015% by weight trinational salt GTP-independent) as the active composition. In the last two columns presents the effect of the compounds, including four and five active ingredients, respectively, and the effects of the individual components (control experiments), namely the number of cells after 48 hours of incubation and the number of cells, expressed as a percentage of the amount of untreated cells, respectively (the number of untreated cells is 100%). If, for example, L-tryptophan 2-4 initial control experiments show the effect of 0.002; 0,006; and 0.01% by weight of pure L-tryptophan, respectively, on the number of cells. In the case of examples 2-4 presents the effect of L-tryptophan used in the same quantities, but made jointly with four other active ingredients.

From the data presented in the last two columns tab is wholesae cell activities: in fact, when using these amounts of L-tryptophan cell proliferation even slightly increased. At the same time from the data presented in discussions of columns, it is seen that each substance has exacerbated the effects of the other four active ingredients in proportion to its number of synergistic manner. For example, to 0.01% by weight of L-tryptophan was increased to 73.1% action four other active ingredients to 92.3%. The same number (0,01% by mass) of the L-tryptophan without other four components even slightly increased cell proliferation. This clearly proves synergistic effect.

The difference between the actions of compounds consisting of four active ingredient (examples 1, 5, 9, 13 and 17) and compositions, including the fifth active ingredient (examples 4, 8, 12, 16 and 20) in the maximum used here amounts (for example, in the case of L-tryptophan to 0.01 mass%) was significant in all cases (p < 0.01), and thus, all matter was greatly enhanced the effects of the other four components.

The synergistic effect of the compositions of examples 1-20, including five active ingredients, shown in Fig. 1, where the vertical axis shows the number of Sp2/0-Ag14 cells, expressed in prey axis shows expressed in % by mass, the amount of L-tryptophan, 2-deoxy-D-ribose, adenine, malic acid and L-ascorbic acid. White columns show the number of cells, expressed as a percentage of the quantities of untreated cells after 48 hours incubation with the use of these substances separately, and the black columns show the results obtained by their joint application with four other active components. Varying degrees of differences (in the case of L-tryptophan even the opposite) between white and black columns with increasing quantities of individual substances prove synergistic effect, and the black columns show the powerful destroys the tumor cells to the action of the compounds.

On the basis of examples 21-37 table 2 shows how to intensify the action, comprising five active components, in the case of cells Sp2/0-Ag14 adding other components. Table 2 presents the number of substances, expressed in % by weight and multiplied by 1000, so the numbers in the table should be read, multiplying them by 10-3; for example, in example 21 in the case of L-tryptophan number 6 means 6 of 10-3that is 0,006% by weight. Conditions of the experiments were the same as before.

Similarly to table 1, from dannyt in its pure form has no reducing cell proliferation effect, moreover, under the action of L-phenylalanine (Control 22), L-arginine (Control 23), L-histidine (Control 24) and Riboflavin (Control 29) the number of cells compared to the number of untreated cells (Control 21) slightly increased. It is also seen that the use of these substances in the same quantities they synergistically enhance the effect of the composition of example 21. Without reinforcing substances all 5 active ingredients in example 21, in all cases was significantly less than in a joint application with other compounds.

It was also conducted comparing the effect of the compositions of examples 107-111, which included twenty-one active ingredient, and the effect of the compositions of examples 102 to 106, including five active ingredients, contained the same number of total active ingredients. To facilitate comparison with earlier results were also used cells Sp2/0-Ag14 and conditions of the experiments were the same as before. The results are presented in Fig. 2. The vertical axis shows changes in the number of cells, expressed as a percentage of the amount of untreated cells. The horizontal axis shows the change, expressed in % of the composition (changes in quantities of active Aosta from example 103 contains each component in the amount of 80%. In cases of compositions, including twenty-one active ingredient, 100% passed the composition of example 107.

In order to exclude the possibility that the measured effect is the result of osmotic effect or nonspecific overdose of nutrients or amino acid imbalance, also prepared a control mixture. The control mixture contained a substance that was found in previous experiments, were ineffective in HRLA. The composition of the control mixtures was given in accordance with table 1. The results obtained in experiments with mixtures of examples 102 to 106 and 107-111 and control mixtures, shown in Fig.2, where the line with icons unpainted circles shows the change in the number of cells after 48 hours incubation in the case of the control mixtures, the line with icons filled circles shows the change in the case of compositions, including five active ingredients of examples 102 to 106, and the line with icons unfilled triangles shows the change in the case of compositions, including twenty-one active ingredient from examples 107-111. In experiments conducted with 100% different compositions of examples 102 and 107 and is shown in Fig.2, separate the notches (as is evident from the results of earlier experiments, presented in tables 1 and 2). It is evident from Fig. 2 shows that in case of joint application of these components, they have significant antitumor effect. This fact proves once again the synergistic nature of the joint action of the active ingredients. It is evident from Fig. 2 shows that the control of the mixture does not have any significant effect on the number of cells. Thus, on the basis of these experiments it is possible to exclude the possibility that the measured effects are the result of an osmotic effect or nonspecific overdose of nutrients or amino acid imbalance.

In order to find out whether the counterions any role in the action of the compounds, experiments were carried out, in which instead of Na+ions used K+ions (examples 38-67) or Ca++ions (examples 68-97). No significant difference was detected between the actions of compounds with different counterions, in fact, the results were almost identical.

Studied the effect of 100% composition of example 107 Sp2/0-Ag14 cells as a function of time and compared it with 100% control mixture and untreated cells. Experimental conditions were the same as in the 8 hours, but after 6, 12, 24 and 36 hours. The results of these kinetic experiments is shown in Fig. 3. The vertical axis shows the number of cells/ml On the x-axis shows the time in hours. Line with icons unpainted circles shows the change in the number of cells in the case of untreated cells, in line with icons filled circles shows the change in the case of the control mixture, and the line with icons shaded triangles shows the change in the case of the compositions of example 107, which included twenty-one active ingredient, as a function of time. It is evident from Fig. 3 shows that the control mixture does not exert any significant effect on the number of cells, as it increases exponentially in time, and the line shape corresponds to the shape of the line showing the proliferation of untreated cells, whereas the composition according to the invention is toxic to tumor cells, causing a reduction in their number over time. Thus, the composition according to the invention not only inhibits cell proliferation, but also has cytotoxic activity. The quantity of active ingredients in the above composition, comprising twenty-one substance, were selected so that Thu is even more true for 80% of the energy, 60%-tions, etc. compounds, where the number of active reagents for 20%, 40% etc less). Because in vitro experiments used the same compositions, the results in all cases show a synergistic joint action of active components.

The above results prove that the action is called a synergistic effect of the substances selected in accordance with the provisions constituting the basis of the invention. Further, these results also prove that the positions are correct. Because of various cancer cells differ from normal cells in varying degrees, the most effective compositions according to the invention against cancer cells also differ from each other. This means that against each tumor can be selected the most appropriate structure to use as a criterion synergistic destroys tumor cells actions. However, to prevent, or in cases when the tumor type may not be installed either too much time has passed, it is preferable to apply the basic compounds, including twenty-one active ingredient. Therefore, in the following experiments, it was investigated how General is the action of these compounds and what their performance is, on K562 cells human erythroleukemia and comparison with the action of the control mixture, mentioned earlier. The results of the experiments shown in Fig. 4. Similarly to Fig.2, the vertical axis shows the change in the number of cells, expressed as percent of the untreated cells. The horizontal axis shows the change, expressed in % of the composition (changes in the quantities of active ingredients). Line with icons unpainted circles shows the action of the control mixtures, and the line with icons filled circles shows the effect of compositions containing twenty-one active ingredient. It is evident from Fig. 4 shows that the compositions according to the invention have significant destroying tumor cells effect also in the case of K-562 cells of the human erythroleukemia.

Then it was investigated the effect of the compositions of examples 107-111, containing twenty-one active ingredient, in HeLa cells human epitheloid carcinoma of the cervix and ner-2 cell epidermoid carcinoma of the larynx and comparison with the action of the control mixture. This experiment was necessary to check whether the previous results are General and independent of perterborough steps p and by measuring the activity of endogenous alkaline phosphatase cells. Namely, after the incubation period was removed by washing neslepties (presumably damaged) cells. Then to each well was added 150 mg of substrate for alkaline phosphatase (4-nitrophenylphosphate, Sigma tablets No. 111-112), dissolved in 150 μl of fresh 10% diethanolamine buffer (pH 9,8). The plates were incubated at 30oC as long as the value of the absorption in the case of untreated cells did not reach values of about 1. The reaction was stopped by adding to each well 50 µl of 3 M NaOH. The absorption was measured at 405 nm using a plate reader Dynatech for enzyme-linked immunosorbent assay (ELISA). Background values subtracted from each result. The results are presented in Fig. 5 (in the case of HeLa cells) and Fig. 6 (in the case of ner-2 cells). Similarly to Fig. 2 and 4, on both drawings, the vertical axis shows the change in the number of cells, expressed as percent of the untreated cells. The change in the number of cells in these cases was determined by measuring the extinction. The horizontal axis shows the change, expressed in % of the composition (changes in the quantities of active ingredients). As shown in Fig. 5, and in Fig. 6 lines with icons unpainted circles shows the action of the control mixtures, while the lines with the effective ingredient. In the drawings it is seen that the compositions are effective against both cell lines. This result is even more significant given that the ner-2 known as "resistant cell line, resistant to the effects of the environment [11].

According to theoretical basis of the invention, the compounds selectively operate only on cancer cells. To further prove this statement, it was also investigated the effect of the compositions of examples 107-111, which included twenty-one active ingredient, and the control mixtures on normal cell line Vero kidney cells of African green monkeys. The results of the experiment shown in Fig. 7. The vertical axis shows the change in the number of cells, expressed as percent of the untreated cells. The horizontal axis shows the change, expressed in % of the composition (changes in the quantities of active ingredients). Line with icons unpainted circles shows the action of the control mixtures, and the line with icons filled circles shows the effect of the compositions of examples 107-111, which included twenty-one active ingredient. It is evident from Fig. 7 shows that the compositions according to the invention does not have a cytotoxic effect on normal Vero cell line as there is in Fig. 3 for the case of the control mixtures. Vero is a rapidly proliferating cell line [12], and this experiment proves that unlike drugs, the composition according to the invention is not toxic to all rapidly proliferating cells, it selectively acts only on tumor cells. Based on the above results, the compounds according to the invention can be characterized as anti-tumor and nezatocheny drugs.

To prove the in vivo efficacy of the composition according to the invention were performed pharmacological experiments. In the course of these experiments always applied the methods and conditions described in the scientific literature. The experiments were conducted with Sp2/0-Ag14 cells. For the experiments we used female BALB/c mice aged 5-6 weeks. The experiments were conducted with two groups consisting of 10 mice each. The mice were injected intraperitoneally 5 10 Sp2/0-Ag14 cells suspended in 200 µl of incomplete RPMI 1640 medium. The day of injection was marked as 0 is the first day of the experiment. The number of cells needed for the experiments was determined by test ofwholesale. Handling of animals was started 24 hours after injection of the cells (it was the first day of the experiment) and prodoljalo phosphate saline) in each treatment, while the experimental group was intraperitoneally injected with the same volume of the composition from example 112, which included twenty-one active ingredient.

The most effective way of treatment was continuous infusion, sustained concentration of the components at a fairly high level. Because of the practical difficulties, used approximately continuous, periodic processing, for example, animals were treated six times a day. The experimental results shown in Fig 8. The horizontal axis shows the number of days that have elapsed since the beginning of the experiment. The vertical axis shows the percentage of survival, namely, the number of mice surviving on these days. So, 10 mice up to 100%, and 8 mice constitute 80%, respectively. White columns indicate the survival of treated mice, black columns survival of untreated mice. It is evident from Fig. 8 shows that, for example, for 16 days, none of the untreated mice did not survive, and all treated mice were still alive. All untreated mice died to 16th days processed only to the 25th day. You can see that due to processing can be achieved a significant increase in life expectancy. Average bruiceclienia of these average duration of survival (150%) much more than 125%, taken as a criterion of effectiveness. According to literature data, on the basis of this value, the composition according to the invention can be considered as a drug with potent antitumor effect against this cell line.

In order to prove that the increase in survival time is the result of a toxic effect of the composition of example 112 on tumor cells, but not in a strong strengthening effect, was delivered the following experiment. 10-10 mice treated in the same way. After a 10-day processing period from the abdominal cavities of both groups of mice cells were removed, and then counted. The difference between the average numbers of cells (5,55 105and 5,95 107respectively) is significant (p < 0.001) and proves in vivo efficacy of the composition according to the invention.

In order to further prove the effectiveness of the composition according to the invention and to exclude the possibility that the results are only consequences of local actions (cells were in the abdominal cavity and were treated intraperitoneally) was conducted another experiment on mice with congenital immunodeficiency (thymus deficient) (nude), injected subcutaneously human solid tumors. For e and subcutaneously in the hind limb of mice. Processing began with the composition of example 112, when the size of tumors reached the average volume of 50 mm3. The treatment plan, time of treatment and solutions (example 112), used for processing were the same as in the previous pharmacological experiments. The tumor size [length (L) width (W) height (In)] was measured twice a week using finger concircular (Mitutoyo. Inc., Tokyo, Japan). Calculate the volume of tumors (calculated according to the equation DSW/2), and the relative volumes of the tumors Vt/Vo[13], where Vtis the actual tumor volume, and Vois the tumor volume at the beginning of processing. The results are summarized in Fig. 9. The vertical axis shows the average values of Vt/Voand on the horizontal axis - the day after the start of treatment. Line with icons unpainted circles shows the increase in the relative volume of the tumor after the start of treatment in case of control mice, and the line with icons filled circles shows the same in the case of treated animals. It is evident from Fig. 9 you can see that the treatment significantly slowed tumor growth. For example, in the case of the control mice tumor volume was increased over 16.5 days prior to the nine-fold volume, while ka is UB>t/Vovalues of the treated and control groups. The values of T/C % was in all cases below 42%, which is acceptable as a criterion of effectiveness [14]. The experiment shows again that the compositions according to the invention possess significant antitumor effect. These results are promising, in view of the fact that due to technical difficulties, the researchers were far from the optimum process to the maximum tolerated dose, and that in the clinic such processing can be performed much more efficiently (more processing time, permanent effect, for example, by infusion, and so on).

Change in body weight during treatment is a good and widely used indicator to characterize the toxicity of the treatment; therefore, during the above experiments measured the weight of the animals. According to literature data, in cases where the average weight of a body decreases by 10-15%, the drug is considered toxic [13]. In our experiment, the average body weight was -5,9 4.1% in the case of the control group and -6,6 3.9% in the case of the treated group. Thus, there are no significant differences between the two groups, which means that the composition according to the invention has no t to the invention, 2-2 groups of BALB/c mice (10 mice per group) were processed so that the processed food for animals was mixed in a 1:1 ratio with the compositions of examples 113-121 and the animals were fed ad libitum. Later, 5 days after the start of treatment in each group of animals were injected with 1 to 104Sp2/0-Ag14 cells intraperitoneally. Processing was carried out for 100 days. Mice that are fed a 1-1 mixture of the compositions of examples 113-121 and normal food, showed no evidence intraperitoneal tumor growth after 100 days after injection of cells. At the same time the survival of control animals in the different experimental groups ranged from 21 to 26 days after injection Sp2/0-Ag14 cells.

Depending on the dose and method of processing the compositions according to the invention can be applied to cancer prevention, to suppress the development of tumors in the case of AIDS and organ transplants to prevent the formation of metastases and for the auxiliary, combined and direct therapy of tumor patients.

The main advantages of the compositions according to the invention are the following:

a) the Use of adequate doses of the compositions can be used for cancer prevention, for the suppression of the Oh, auxiliary or combination of processing and treatment of cancer.

b) They are not toxic. The experimental studies of toxicity, based on the measurement of body weight loss in animals, proved that the compounds are not toxic. According to literature data, the toxicity of the individual components of the compositions is very low and even this low toxicity decreases when the joint application of these substances. In addition, primates carry these substances are better than small animals used to determine the toxicity characteristics. Considering all these facts, it can be stated that the compounds should be considered non-toxic.

C) They do not have or have only minor side effects. There have been numerous observations of adverse effects of its components, as almost all of them (except 2-deoxy-D-ribose) was used in different types of therapy (cancer treatment - only ascorbic acid) or as food additives. According gained in this field experience, it is obvious that the compounds have little or no side effects.

d) They are selective. Selectivity prove special expetec and experimental animals or normal cells, respectively. Selectivity is enhanced due to the fact that there is much more normal cells than cancer cells, and the last cells accumulate components of the compositions, while in the case of normal cells, their absorption is regulated.

d) They can have a wider application, their action is common. The effect of the compounds was investigated in the case of cell lines are mouse myeloma (Sp2/0-Ag14), human erythroleukemia (K-562), human epitheloid cervical carcinoma (HeLa) and human epidermoid carcinoma of the larynx (ner-2). These cells represent a wide range, as they include both human tumors and tumors of animals and of human tumors presents leukemia and two very different solid tumors. The results obtained from experiments with these cell lines, confirming the above statement that the compositions have the General effect and can have a wider application.

e) They can be easily obtained. The components of the compositions are substances with a low molecular weight, readily available, and so obtaining them is not difficult.

W) They are soluble in water and therefore they are easy to dose.

al Medicine, 12th ed., In one example is international Edition, McGraw-Hill, Inc., New York, Vol. 2, pp. 1587-1599 (1991).

3. Scientific American Medicine, Scientific American, Inc., New York, Vol. 2, 12(V), pp. 1-14 (1984).

4. Pharmac. Ther., 49, 43-54 (1991).

5. Proc. Roy. Soc. Med., 63, 1063-1066 (1970).

6. Chest, 99, 557-561 (1991).

7. Eur. J. Cancer, 27, 936-939 (1991).

8. J. R. Soc. Med., 84, 321 (1991).

9. PCT No. WO 86/025555.

10. Published patent application Japan N 62-135 421.

11. American Type Culture Collection Catalogue of Cell Lines and Hybridomas, 5th ed., pp. 15-16 (1985).

12. American Type Culture Collection Catalogue of Cell Lines and Hybridomas, 5th ed., pp. 45-46 (1985).

13. Eur. J. Cancer. Clin. Oncol., 21, 1253-1260 (1988).

14. Europ. J. Cancer, 17, 129-142 (1981).

1. Pharmaceutical composition for the prevention and treatment of cancer, characterized in that it includes at least three active compounds present in the circulatory system: at least one amino acid, at least one vitamin and at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, malic acid, salewoman acid, ATP and/or their pharmaceutically acceptable salts, considering the fact that if the composition contains, in addition to amino acids, only malic acid and vitamin, the vitamin may not be the t also media the thinners and/or other auxiliary reagents widely used in pharmacology.

3. The composition according to p. 1, wherein the amino acid is L-methionine, L-tryptophan, L-tyrosine, L-phenylalanine, L-arginine, L-histidine, N-benzoylglycine and/or their salts.

4. The composition according to p. 1, wherein the vitamin is d-Biotin, pyridoxine, Riboflavin, Riboflavin-5'-phosphate, L-ascorbic acid, lipoic acid, orotovoy acid and/or their salts.

5. Composition under item 1, characterized in that it includes L-tryptophan, L-ascorbic acid and at least one component selected from the group consisting of adenine, 2-deoxyribose, D-glucosamine and/or their pharmaceutically acceptable salts.

6. Composition under item 1, characterized in that it includes L-arginine, Riboflavin-5'-phosphate and at least one component selected from the group consisting of D-mannose, malic acid, ATP and/or their pharmaceutically acceptable salts .

7. Composition under item 1, characterized in that it includes 0,002 - 70 wt.% at least one component selected from the group consisting of L-methionine, L-tryptophan, L-tyrosine, L-phenylalanine, L-arginine, L-histidine, N-benzoyl the dust, consisting of d-Biotin, pyridoxine, Riboflavin, Riboflavin-5'-phosphate, L-ascorbic acid, lipoic acid, orotovoy acid and/or their salts, as vitamin and 0.003 to 80 wt.% at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine, malic acid, salewoman acid, ATP and/or their pharmaceutically acceptable salts.

8. Composition under item 1, characterized in that it includes a 0.9 - 25 wt.% L-methionine, 0.8 to 19 wt.% L-tryptophan, 1,1 - 48 wt.% L-arginine, 0,9 - 46 wt. % d-Biotin, 1,2 - 16 wt.% pyrodoxine, 0,03 -0,42 wt.% Riboflavin-5'-phosphate, 0.05 to 18 wt.% D-glucosamine, 0.5 to 60 wt.% 2-deoxy-D-ribose, 0.7 to 68 wt.% malic acid, 0.6 to 40 wt.% D-mannose and/or their pharmaceutically acceptable salts.

9. Composition under item 1, characterized in that it comprises from 0.005 to 34 wt.% L-methionine, 0,002 - 25 wt.% L-tryptophan, 0,02 - 23 wt.% L-tyrosine, 0.04 to 30 wt.% L-phenylalanine, 0.04 to 50 wt.% L-arginine, 0,03 - 34 wt.% L-histidine, 0.05 to 22 wt.% N-benzoylglycine, 0.01 to 60 wt.% d-Biotin, 0.01 to 20 wt.% pyridoxine, 0,0004 - 45 wt.% Riboflavin, 0,0005 - 45 wt.% Riboflavin-5'-phosphate, 0.003 to 70 wt.% L-ascorbic acid, 0,0004 - 15 wt. % lipoic acid, from 0.01 to 17 wt.% orotovoy kislota, of 0.001 - 10 wt.% adenine, 0.01 to 63 wt.% 2-kusnoy acid, of 0.001 - 10 wt.% ATP and/or their pharmaceutically acceptable salts.

 

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