Amino acid preparation eliciting antitumor effect and method for its preparing

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 15-30oC or with ozone at temperature 15-25oC. 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.

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

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

Known antitumor agent, representing p-di-(2-chloroethyl)-D,L-phenylalanine hydrochloride and pharmaceutically acceptable auxiliary substances (see Pat of the Russian Federation No. 2144821, 2000). Tests conducted on laboratory animals in experimental lymphosarcoma, bone retikulosarkome, cancer of the testicles and ovaries. The purpose of the invention to increase the life expectancy of animals, increase the bioavailability of the drug. However, the presented product containing chlorine ions is sufficiently toxic to healthy tissues of the mammal.

As another analogue of our development can lead to a composition for the prophylaxis of oncologic diseases in AIDS. The composition includes mainly three components: at least one amino acid, at least odenville and one substance, selected from the group of adenine-derived monosaccharides, malic acid (U.S. Pat. Of the Russian Federation No. 2138257, 1999). The role of amino acids in such a composition is not clear, and the drug is rather suitable for sustaining the weakened organism, for example, in AIDS.

Closest to the claimed invention appears to be a composition consisting of ten natural amino acids: valine, leucine, isoleucine, phenylalanine, tryptophan, lysine, methionine, histidine, serine, glutamate, sodium, and three trace elements: manganese ions, cobalt, copper (U.S. Pat. Of the Russian Federation No. 2125874, 1999). The composition has anti-tumor and antihypoxic effect. The study was conducted on laboratory animals, and the composition of amino acids and trace elements compared in their effect with classical anticancer drugs such as cyclophosphamide and glycinato-Serenata copper.

The role of amino acids as antitumor agents in this drug, in our opinion, is not clear, most likely the cytotoxic effect is due to the action of ions of bivalent copper, which, according to some messages that have a cytotoxic effect against tumor cells (see e.g., Tremaine EM and other Bulletin of the Academy of medical Sciences of the USSR, 1986, No. 5, p.51-56), and amino acids only provide penetration toxic to tumor ions IU allow in the cell.

We have created a fundamentally new anticancer drug based on the essential amino acids that do not have substantive additional chemical agents in its composition.

In our opinion, in addition to the usual classification of amino acids, the essential amino compounds enter the delimitation last able to participate in anabolic (plastic) processes in the cell and is not capable of such participation, “aplastic”. In the metabolic process of the nitrogen cycle in biological tissue, in our opinion, the amino acids “wear out” as they re-use in the processes of anabolism, their slim electronic structure changes that capture sensitive enzymes involved in protein synthesis. In normal tissues such amino acids catabolized to ammonia and carbon dioxide, the latter further into the soil, and through the work of the nitrogen-fixing microorganisms and some other processes continue the cycle of nitrogen with the formation including the new “plastic” native amino acids, including essential that enter the body with food. Tumor cells, as we believe, not “recognize” “aplastic” amino acids and continue to include them in your metabolic cycle.

Information about the metabolic nitrogen in the body when progressives the x tumors indirectly serve as confirmation of 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 produced nitrogen. If nitrogen is supplied more than excreted, they say that this individual has a positive nitrogen balance: the last positive how to have healthy growing baby, and healthy pregnant women. Adult is usually in a state of nitrogen balance: nitrogen consumption is balanced by his selection in the composition of faeces and urine. In a state of negative nitrogen balance the amount of nitrogen exceeds the amount of nitrogen consumed by the body. This condition is observed, for example, in progressive forms of cancer (see e.g., Await and other Fundamentals of biochemistry, volume 2. M.: Mir, 1981, s-904).

According to our data, in the blood of a healthy person contains less than 1% “plastidnogo” phenylalanine (ACE) in comparison with “plastic”, whereas in urine figures ACE increase 3-5 times, i.e. in the normal body ACE largely excreted from the body.

The technical task of the present invention is the creation of a product based on artificially created “plastycznych”, i.e. the “modified” with the help of our development, amino acids and method of producing such a product.

The problem is solved t is m, what amino acid formula designed with antitumor activity, 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, with the modified amino acid is non-toxic to healthy cells.

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

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

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

Amino acid formula, modified as described above, characterized by the fact that each amino acid is treated with UV for 12-80 hours at a temperature of 15-30°C.

Amino acid formula is characterized by the fact that optionally may contain one or more pharmaceutically acceptable additives: minerals and/or vitamins and/or vitamin C, and/or interchangeable and/or irreplaceable native amino acids.

For the drug can be used in a variety of the above about the time separately processed essential amino acids in either numerical and/or qualitative combination.

As pharmaceutical additives can be used the following substances: minerals, such as Fe, si, Mg, Mn, Se, Te, Ti, Co, Mo and others, the b vitamins, such as thiamine, Riboflavin, pyridoxine, etc., vitamin C. in Addition, may contain native amino acids such as asparagine, glutamine, glycine, valine and other

Another object of the invention is a method of obtaining the drug.

The method is as follows.

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

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

The study modified the drug was carried out by the method of fluorescence analysis of irradiated phenylalanine (see figure 1). The range was cleared within 72 hours of 11 samples taken through the following time periods (T) of exposure:

4
No. sample1234567891011
T, h02824283248525672

Shows spectra for each exposure time is the result of averaging 10 measurements from each tube was taken on 5 samples, the measurements were repeated 2 times).

It is necessary to pay attention that there is not one band fluorescence and two.

The spectra 2 and 3 (figure 1)is clearly visible band emission in the area of 392 nm (25500 cm-1). This (extremely weak) stripe can be seen in the spectrum of the original amino acid before exposure. (A small amount of fluorescent substance is formed and during the natural exposure of the sample to the fluorescence analysis).

With increasing exposure time in the spectra of rapidly increasing the intensity of a “main” component, formed under the action of UV-irradiation with band emission in the region of 410 nm (24380 cm-1). The emergence of this “main” component and attaches the amino acid “modifitsirovannoi”. However, the first band is also present in the spectra in the form of a small “shoulder” to the left of the maximum of emission of the main strip (the third band in the region of 330 nm can not be taken into consideration is the peak of the Raman scattering of the Oh-group of water).

The maximum intensity of the spectrum nabludalsia 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 didn't increase the cytotoxic effect on tumor cells.

The exposure time for other amino acids were identified following.

Tryptophan 12-18 hours

Lysine 60-72 hours

Leucine 46-56 PM

With increasing time of UV-irradiation recorded the destruction of the studied amino acids (probably due to oxidation of amino groups), which can be determined by reduction 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 figure one can see the increase of the peak “main component” in the field of 410 nm.

The biological activity of the drug was tested on the following amino acids: phenylalanine, tryptophan, leucine, lysine. Phenylalanine was subjected to UV exposure for 72 hours at a temperature of 18°With obtaining “modified” phenylalanine (MT), after which was added water for injection.

The invention is confirmed by the following examples:

EXAMPLE 1. The tests were carried out on cell cultures N-9 (passiruete cell leukemia person). As control was used mononuclear cells peripheral is why the blood of healthy donors. The experiment was carried out according to the following scheme:

Cells N-9 were planted in 24-well plate for culturing at a concentration of 250×103/ml at 1.0 ml per 1 well. Each version of the experiment was put on 5 repetitions.

- Preparation of MOF was dissolved in water for injection was added to sample wells in the amount of 500, 250 and 100 mg/ml

In the control wells were added an appropriate amount of medium for culturing.

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

Cells were incubated for 24 and 48 hours in CO2incubator at 37°C.

After incubation considered the number and percent of dead cells with the addition of Trypanosoma blue.

The result of the study are shown in table 1. From the data presented in the table shows that the MOF has a cytotoxic effect on the viability of tumor cells N-9 in doses of 100, 250 and 500 μg/ml, the table also shows that increasing the dose does not lead to increased cytotoxic effect.

Table 1
The concentration of the drug MF, number of experimentsAfter 24 h incubationAfter 48 h incubation
 The number of cells per well in a million/ml% dead cells % deviation from controlThe number of cells per well in a million/ml% dead cells% deviation from control
1. Control, n=50,22±0,128,3±2,4-0,25±0,118,1±2,3-
2. 100 µg/ml, n=100,19±0,1133,3±4,2301±27,50,19±0,1035,5±4,3338±18,7
3. 250 µg/ml, n=100,17±0,1152,5±7,3532±38,60,16±0,0952,7±8,3550±45,7
4. 300 µg/ml, n=100,18±0,1058,3±7,4557±20,10,18±0,1152,5±7,8536±29,4

The preparation of MOF in the investigated concentration has a cytotoxic effect on the cell viability of leukemia N-9. The most pronounced effect was observed at doses of 250 and 500 mg/ml

EXAMPLE 2. In the control series of experiments used the mononuclear cells (lymphocytes) in the peripheral blood of a healthy person, which were placed in 24-well plate for culturing and have experience as described in example 1 with the participation of the MOF (see table 2).

After 24 h incubation
Table 2
The concentration of the drug MF, number of experimentsAfter 48 h incubation
 The number of cells per well in a million/ml% dead cells% deviation from controlThe number of cells per well in a million/ml% dead cells% deviation from control
1. Control, n=100,24±0,16,0±1,4-0,25±0,126,1±1,3-
2. 100 µg/ml, n=100,24±0,104,9±1,218,3±5,30,24±0,115,5±1,39,8±2,6
3. 250 µg/ml, n=100,23±0,124,7±1,421,7±5,60,24±0,105,7±1,36,6±1,7
4. 500 µg/ml, n=100,24±0,11of 4.7±1.621,7±4,20,23±0,116,2±1,91,6±0,3

The drug MT at doses of 100-500 μg/ml had no cytotoxic effect on lymphocytes of a healthy donor.

EXAMPLE 3. In experiments assessed the cytotoxic effect “modified” tryptophan (MT) in suspension culture of human leukemia cell N-9. The results are shown in table 3.

Table 3
oncentrate drug MT, number of experimentsAfter 24 h incubationAfter 48 h incubation
 The number of cells per well in a million/ml% dead cells% deviation from controlThe number of cells per well in a million/ml% dead cells% deviation from control
1. Control, n=100,23±0,087,5±0,6-0,24±0,089,3±0,7-
2. 10 µg/ml, n=100,21±0,0916,2±0,9116±10,80,23±0,0827,3±2,8193±18,9
3. 50 µg/ml, n=100,21±0,0818,4±1,3145±11,90,22±0,0725,2±2,6171±17,1
4. 100 µg/ml, n=100,19±0,0620,1±1,4168±12,30,20±0,0531,3±3,6236±21,5
5. 250 µg/ml, n=100,18±0,0731,b±2,1321±23,50,19±0,0643,3±4,3366±33,7
6. 500 µg/ml, n=100,17±0,0833,7±3,1349±31,20,19±0,0749,0±4,9427±39,3

The drug methadone in the investigated concentrations has qi is otoxicity effect on cell viability N-9. There is a linear increase in cytotoxicity, depending on the concentration.

EXAMPLE 4. Studies on a mixture of “modified” amino acids (ACI): phenylalanine, leucine and lysine. The conditions of the experiment similar to the above-described related to human leukemia cell N-9.

The results are shown in table 4.

Table 4
The concentration of the ISA, the number of experimentsAfter 24 h incubationAfter 48 h incubation
 The number of cells per well in a million/ml% dead cells% deviation from controlThe number of cells per well in a million/ml% dead cells% deviation from control
1. Control, n=100,22±0,087,6±0,5-0,24±0,089,2±0,7-
2. 400 µg/ml, n=100,17±0,0858,5±5,1680±58,10,18±0,0667,5±6,1626±59,9
Being 3.1000 µg/ml, n=100,17±0,0782,3±6,3997±82,10,18±0,0795,5±8,1927±89,7

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

The present invention opens up new ways to search for anticancer drugs of natural origin, physiological, non-cytotoxicity against normal cells, allowing to deal with such serious diseases as cancer.

1. Amino acid formula with antitumor activity, 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°or ozone at a temperature of 15-25°With modified amino acid is not toxic to healthy cells.

2. Amino acid preparation according to claim 1, characterized in that the UV-irradiated for 12-18 h phenylalanine with band emission in the region of 410 nm.

3. Amino acid preparation according to claim 1, characterized in that it contains one or more modified amino acids selected from the group of the phenylalanine obtained when the exposure within 36-72 hours, tryptophan, obtained by exposure for 12-18 h, lysine, obtained by exposure for 60-72 h, leucine, obtained by exposure for 46-56 PM

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, wherein each amino acid is treated with UV radiation during 12-80 hours at a temperature of 15-30°C.

6. Amino acid preparation according to claim 1, characterized in that optionally may contain one or more pharmaceutically acceptable additives: minerals and/or vitamins and/or vitamin C, and/or interchangeable and/or irreplaceable native amino acids.

7. The method of obtaining amino acid drug with anti-tumor activity, characterized in that at least one essential 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°or ozone at a temperature of 15-25°obtaining modified amino acids, non-toxic against normal cells.

8. The method according to claim 6, wherein the modified amino acids are mixed in various combinations, while amino acids are taken in equal proportions.



 

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