RU2245143C2 - Amino acid preparation eliciting antitumor effect and method for its preparing - Google Patents

Abstract

FIELD: medicine, oncology, amino acids.

SUBSTANCE: invention relates, in particular, to the development of an antitumor preparation based on natural substances. Invention relates to an amino acid preparation comprising at least one modified essential amino acid obtained by treatment of amino acid by ultraviolet radiation (UV) at wavelength 250-350 nm for 12-80 h at temperature 15-30^oC or with ozone at temperature 15-25^oC. The modified amino acid has no toxicity for health cells. Also, invention relates to a method for preparing such preparation. Invention provides the development of an antitumor preparation based on modified amino acids and expanded assortment of antitumor preparations being without cytotoxicity for normal cells.

EFFECT: valuable medicinal antitumor properties of preparation.

8 cl, 4 tbl, 2 dwg, 4 ex

Description

The invention relates to medicine, in particular to the creation of an antitumor drug based on natural substances.

Currently, the search for anticancer chemotherapeutic agents is directed not only towards new synthetic substances, but to a large extent natural, more physiological and environmentally friendly agents. In particular, researchers often turn to the metabolites of plant and animal cells and tissues, the vital products of microorganisms, vitamins, amino acids and their derivatives.

Known antitumor agent, which is p-di- (2-chloroethyl) -D, L-phenylalanine hydrochloride and pharmaceutically acceptable excipients (see RF Patent No. 2144821, 2000). Tests were conducted in laboratory animals with experimental lymphosarcoma, bone reticulosarcoma, testicular and ovarian cancer. The purpose of the invention is to increase the life expectancy of animals, increasing the bioavailability of the drug. However, the presented preparation containing chlorine ions is quite toxic to healthy mammalian tissues.

As another analog of our development, we can cite a composition for the prevention of cancer with AIDS. The composition comprises mainly three components: at least one amino acid, at least one vitamin, and one substance selected from the group: adenine, a monosaccharide derivative, malic acid (US Pat. RF No. 2138257, 1999). The role of amino acids in such a composition is not clear enough, and the drug is more likely to support the life of a weakened organism, for example, in AIDS.

Closest to the claimed invention is a composition consisting of ten natural amino acids: valine, leucine, isoleucine, phenylalanine, tryptophan, lysine, methionine, histidine, serine, monosodium glutamate, and three trace elements: manganese ions, cobalt, copper (US Pat. RF No. 2125874, 1999). The composition has an antitumor and antihypoxic effect. The study was conducted on laboratory animals, while the composition of amino acids and trace elements is compared in its action with classical antitumor drugs, for example cyclophosphamide, as well as copper glycinate-serinate.

The role of amino acids as antitumor agents in this drug, in our opinion, is not clear, most likely the cytotoxic effect is ensured by the action of divalent copper ions, which, as follows from some reports, have a cytotoxic effect against tumor cells (see, for example, Treschalin EM and other Bulletin of the Academy of Medical Sciences of the USSR, 1986, No. 5, pp. 51-56), and amino acids only ensure the penetration of metal ions toxic to the tumor into the cell.

We have created a fundamentally new antitumor drug based on essential amino acids that do not have essentially additional chemical agents in their composition.

In our opinion, in addition to the usual classification of amino acids for irreplaceable amino compounds, a distinction should be made between the latter and those capable of participating in anabolic (plastic) processes in the cell and not capable of such participation as “aplastic”. In the process of the metabolic cycle of nitrogen in biological tissue, in our opinion, amino acids “wear out” as they are reused in the processes of anabolism, their fine electronic structure changes, which capture sensitive enzymes involved in protein synthesis. In normal tissue, such amino acids are catabolized to ammonia and carbon dioxide, the latter subsequently enter the soil, and through the work of nitrogen-fixing microorganisms and some other processes, they continue the nitrogen cycle with the formation of new “plastic” native amino acids, including irreplaceable into the body with food. Tumor cells, we believe, do not “recognize” “aplastic” amino acids and continue to incorporate them into their metabolic cycle.

Information about the violation of nitrogen metabolism in the body with progressive tumors indirectly confirms our hypothesis. Nitrogen balance, as you know, means the difference between the total amount of nitrogen received in the human body and the total amount of nitrogen excreted. If more nitrogen is supplied than excreted, it is said that this individual has a positive nitrogen balance: the latter is positive in both a healthy growing child and a healthy pregnant woman. An adult is normally in a state of nitrogen balance: nitrogen consumption is balanced by its excretion in the composition of feces and urine. In a state of negative nitrogen balance, the amount of nitrogen released exceeds the amount of nitrogen consumed by the body. Such a condition is observed, for example, with progressive forms of cancer (see, for example, A. White and others. Fundamentals of biochemistry, vol. 2. M .: Mir, 1981, pp. 902-904).

According to our data, less than 1% of “aplastic” phenylalanine (ACE) is contained in the blood of a healthy person compared to “plastic”, while in the urine ACE figures increase by 3-5 times, i.e. in a normal body, ACE is largely eliminated from the body.

An object of the present invention is to provide a preparation based on artificially created “aplastic” ones, i.e. “Modified” with the help of our development, amino acids and the method of obtaining such a drug.

The problem is solved in that an amino acid preparation having an antitumor effect is developed, while it contains at least one modified essential amino acid obtained by treatment with ultraviolet radiation (UV) at a wavelength of 250-350 nm and / or ozone, while modified amino acid is non-toxic to healthy cells.

In particular, the amino acid preparation contains UV-irradiated phenylalanine with an emission band in the region of 410 nm.

An amino acid preparation processed as described above may contain one or more individually modified amino acids selected from the group: phenylalanine, tryptophan, lysine, leucine.

An amino acid preparation processed as described above may contain a mixture of four modified amino acids: phenylalanine, tryptophan, lysine, leucine.

An amino acid preparation modified as described above is characterized in that each amino acid is treated with UV for 12-80 hours at a temperature of 15-30 ° C.

An amino acid preparation is characterized in that it may additionally contain one or more pharmaceutically acceptable additives: microelements, and / or B vitamins, and / or vitamin C, and / or essential and / or essential native amino acids.

For the preparation, individually processed various irreplaceable amino acids in various numerical and / or qualitative combinations can be used individually individually in the aforementioned manner.

The following substances can be used as pharmaceutical additives: trace elements, such as Fe, Cu, Mg, Mn, Se, Te, Ti, Co, Mo, etc., B vitamins, such as thiamine, riboflavin, pyridoxine, etc., vitamin C. In addition, it may contain native amino acids, such as asparagine, glutamine, glycine, valine, etc.

Another object of the invention is a method for producing a preparation.

The method is as follows.

At least one essential amino acid (in dry form) is treated with UV radiation at a wavelength of 250-350 nm and at a temperature of 15-30 ° C for 12-80 hours, or with ozone at a temperature of 15-25 ° C to obtain a modified amino acids.

Modified amino acids are mixed in various combinations, while amino acids are taken in equal proportions.

The study of the modified preparation was carried out by the method of fluorescence analysis of irradiated phenylalanine (see figure 1). The spectrum was recorded for 72 hours on 11 samples taken at the following time intervals (T) of exposure:

Sample No. 1 2 3 4 5 6 7 8 nine 10 eleven T, hour 0 2 4 8 24 28 32 48 52 56 72

The spectra presented for each exposure time are the result of averaging over 10 measurements (5 samples were taken from each tube, measurements were repeated 2 times).

It should be noted that not one fluorescence band appears, but two.

On spectra No. 2 and No. 3 (Fig. 1), the emission band in the region of 392 nm (25500 cm ^-1 ) is clearly visible. The same (extremely weak) band can also be seen in the spectrum of the initial amino acid before exposure. (An insignificant amount of a fluorescent substance is also formed during natural irradiation of a sample prior to fluorescence analysis).

With an increase in the exposure time in the spectra, the intensity of a certain “main” component, which is formed under the influence of UV radiation with an emission band in the region of 410 nm (24380 cm ^-1 ), rapidly increases. The appearance of this “main” component gives the studied amino acid “modification”. However, the first band is also present in the spectra in the form of a small “shoulder” to the left of the maximum emission of the main band (the third band at 330 nm can be ignored - this is the Raman scattering peak of the OH group of water).

The maximum spectrum intensity was observed after 72 hours, after the start of UV irradiation (wavelength 250-350 nm). Further UV irradiation (105, 120 hours) did not increase the formation of the “main component” and did not enhance the cytotoxic effect on tumor cells.

The exposure time for other amino acids was as follows.

Tryptophan 12-18 hours

Lysine 60-72 hours

Leucine 46-56 h.

With an increase in the UV irradiation time, the destruction of the studied amino acid was recorded (apparently due to oxidation of the amino group), which can be determined by a decrease in the reaction with ninhydrin.

Figure 2 presents samples of three-dimensional fluorescence spectra of irradiated phenylalanine. The original sample (sample 1), irradiated for 36 hours (sample 2) and irradiated for 72 hours (sample 3). From the presented figure one can see an increase in the peak of the “main component” in the region of 410 nm.

The biological activity of the drug was tested on the following amino acids: phenylalanine, tryptophan, leucine, lysine. Phenylalanine was exposed to UV for 72 hours at 18 ° C to give “modified” phenylalanine (MF), after which water for injection was added.

The invention is confirmed by the following examples:

EXAMPLE 1. Tests were performed on a culture of H-9 cells (passivated human lymphocytic leukemia cells). Peripheral blood mononuclear cells of healthy donors were used as a control. The experiment was carried out according to the following scheme:

- N-9 cells were planted in a 24-well culture plate at a concentration of 250 × 10 ³ / ml, 1.0 ml per 1 well. Each experiment was set up in 5 repetitions.

- The MF preparation was diluted in water for injection and added to the experimental wells in the amount of 500, 250 and 100 μg / ml.

- An appropriate amount of culture medium was added to the control wells.

- The composition of the culture medium: RPMI-1640 with 10% ETS + glutamine 146 mg per 500 ml + gentamicin 20 mg per 500 ml.

- Cells were incubated 24 and 48 hours in a CO ₂ incubator at 37 ° C.

- After incubation, the number and% of dead cells with the addition of trepan blue were counted.

The result of the study is shown in table 1. From the data presented in the table it is seen that MF has a cytotoxic effect on the viability of H-9 tumor cells at doses of 100, 250 and 500 μg / ml, and the table also shows that increasing the dose of the drug does not lead to cytotoxic effect.

Table 1 The concentration of the drug MF, number of experiments After 24 hours of incubation After 48 h of incubation The number of cells in the well in million / ml % dead cells % deviation from control The number of cells in the well in million / ml % dead cells % deviation from control 1. Control, n = 5 0.22 ± 0.12 8.3 ± 2.4 - 0.25 ± 0.11 8.1 ± 2.3 - 2.100 μg / ml, n = 10 0.19 ± 0.11 33.3 ± 4.2 301 ± 27.5 0.19 ± 0.10 35.5 ± 4.3 338 ± 18.7 3.250 mcg / ml, n = 10 0.17 ± 0.11 52.5 ± 7.3 532 ± 38.6 0.16 ± 0.09 52.7 ± 8.3 550 ± 45.7 4. 300 μg / ml, n = 10 0.18 ± 0.10 58.3 ± 7.4 557 ± 20.1 0.18 ± 0.11 52.5 ± 7.8 536 ± 29.4

The MF preparation in the studied concentrations exerts a cytotoxic effect on the viability of H-9 leukemia cells. A greater severity of the effect is observed at doses of 250 and 500 μg / ml.

EXAMPLE 2. In the control series of experiments, mononuclear cells (lymphocytes) of the peripheral blood of a healthy person were used, which were placed in a 24-well culture plate and the experiment was carried out as described in Example 1 with the participation of MFs (see table 2).

table 2 The concentration of the drug MF, number of experiments After 24 hours of incubation After 48 h of incubation The number of cells in the well in million / ml % dead cells % deviation from control The number of cells in the well in million / ml % dead cells % deviation from control 1. Control, n = 10 0.24 ± 0.1 6.0 ± 1.4 - 0.25 ± 0.12 6.1 ± 1.3 - 2.100 μg / ml, n = 10 0.24 ± 0.10 4.9 ± 1.2 18.3 ± 5.3 0.24 ± 0.11 5.5 ± 1.3 9.8 ± 2.6 3.250 mcg / ml, n = 10 0.23 ± 0.12 4.7 ± 1.4 21.7 ± 5.6 0.24 ± 0.10 5.7 ± 1.3 6.6 ± 1.7 4. 500 μg / ml, n = 10 0.24 ± 0.11 4.7 ± 1.6 21.7 ± 4.2 0.23 ± 0.11 6.2 ± 1.9 1.6 ± 0.3

The MF preparation in doses of 100-500 μg / ml did not have a cytotoxic effect on the lymphocytes of a healthy donor.

EXAMPLE 3. In the experiments, the cytotoxic effect of “modified” tryptophan (MT) was evaluated in a suspension culture of human leukemia cells H-9. The results are shown in table 3.

Table 3 The concentration of the drug MT, number of experiments After 24 hours of incubation After 48 h of incubation The number of cells in the well in million / ml % dead cells % deviation from control The number of cells in the well in million / ml % dead cells % deviation from control 1. Control, n = 10 0.23 ± 0.08 7.5 ± 0.6 - 0.24 ± 0.08 9.3 ± 0.7 - 2.10 μg / ml, n = 10 0.21 ± 0.09 16.2 ± 0.9 116 ± 10.8 0.23 ± 0.08 27.3 ± 2.8 193 ± 18.9 3. 50 μg / ml, n = 10 0.21 ± 0.08 18.4 ± 1.3 145 ± 11.9 0.22 ± 0.07 25.2 ± 2.6 171 ± 17.1 4.100 μg / ml, n = 10 0.19 ± 0.06 20.1 ± 1.4 168 ± 12.3 0.20 ± 0.05 31.3 ± 3.6 236 ± 21.5 5.250 μg / ml, n = 10 0.18 ± 0.07 31, b ± 2.1 321 ± 23.5 0.19 ± 0.06 43.3 ± 4.3 366 ± 33.7 6.500 mcg / ml, n = 10 0.17 ± 0.08 33.7 ± 3.1 349 ± 31.2 0.19 ± 0.07 49.0 ± 4.9 427 ± 39.3

The MT preparation in the studied concentrations has a cytotoxic effect on the viability of H-9 cells. A linear increase in cytotoxicity is observed depending on the concentration.

EXAMPLE 4. Studies were conducted on a mixture of “modified” amino acids (MCA): phenylalanine, leucine and lysine. The experimental conditions are similar to those described above, related to human leukemia cells N-9.

The results are shown in table 4.

Table 4 MSA concentration, number of experiments After 24 hours of incubation After 48 h of incubation The number of cells in the well in million / ml % dead cells % deviation from control The number of cells in the well in million / ml % dead cells % deviation from control 1. Control, n = 10 0.22 ± 0.08 7.6 ± 0.5 - 0.24 ± 0.08 9.2 ± 0.7 - 2.400 μg / ml, n = 10 0.17 ± 0.08 58.5 ± 5.1 680 ± 58.1 0.18 ± 0.06 67.5 ± 6.1 626 ± 59.9 3.1000 μg / ml, n = 10 0.17 ± 0.07 82.3 ± 6.3 997 ± 82.1 0.18 ± 0.07 95.5 ± 8.1 927 ± 89.7

A mixture of the above “modified” amino acids in the studied concentrations has a strong cytotoxic effect on tumor cells.

The present invention opens up new ways to search for antitumor drugs of natural origin, physiological, not possessing cytotoxicity in relation to normal cells, allowing to fight against such serious diseases as cancer.

Claims (8)

1. An amino acid preparation having an antitumor effect, characterized in that it contains at least one modified essential amino acid obtained by treatment with ultraviolet radiation (UV) at a wavelength of 250-350 nm for 12-80 hours at a temperature of 15-30 ° C or ozone at a temperature of 15-25 ° C, while the modified amino acid is not toxic to healthy cells. 2. The amino acid preparation according to claim 1, characterized in that it contains phenylalanine with an emission band in the region of 410 nm UV-irradiated for 12-18 hours. 3. The amino acid preparation according to claim 1, characterized in that it contains one or more modified amino acids selected from the group of phenylalanine obtained by exposure for 36-72 hours, tryptophan obtained by exposure for 12-18 hours, lysine obtained when exposed for 60-72 hours, leucine obtained by exposure for 46-56 hours 4. Amino acid preparation according to claim 1 and 3, characterized in that it contains a mixture of four modified amino acids: phenylalanine, tryptophan, lysine, leucine. 5. Amino acid preparation according to claim 1, characterized in that each amino acid is treated with UV radiation for 12-80 hours at a temperature of 15-30 ° C. 6. The amino acid preparation according to claim 1, characterized in that it can additionally contain one or more pharmaceutically acceptable additives: microelements, and / or B vitamins, and / or vitamin C, and / or interchangeable and / or irreplaceable native amino acids. 7. A method of obtaining an amino acid preparation with an antitumor effect, characterized in that at least one irreplaceable amino acid is treated with UV radiation at a wavelength of 250-350 nm for 12-80 hours and at a temperature of 15-30 ° C or ozone at a temperature of 15-25 ° C to obtain modified amino acids that are not toxic to healthy cells. 8. The method according to claim 6, characterized in that the modified amino acids are mixed in various combinations, while the amino acids are taken in equal proportions.

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