RU2414223C1 - Medication possessing immunostimulating and hemostimulating action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed is application of immobilised oligonucleotides as immunostimulating and hemostimulating medication for peroral introduction. Immobilised on polyethylene oxide oligonucleotides of DNA of salmon soft roes demonstrate immuno- and hemostimulating activity in conditions in vivo in case of peroral introduction in experimental animals and in vitro, which is manifested in stimulation of phagocytic reactions in vivo and in vitro, increase of IFN-γ in vitro, increase of cytotoxic activity of natural killers, increase of spontaneous proliferative activity of lymphoid cells of experimental animals and index of B-lymphocyte stimulation, increase of nitrogen oxide production, as well as in enchancement of hemopoiesis regeneration in early terms after cytostatic impact.

EFFECT: immobilised by physical method oligonucleotides are protected from destruction in gastrointestinal tract, which facilitates their permeability through intestine wall and increases bioavailability.

11 tbl

Description

The invention relates to medicine, specifically to pharmacology, and relates to agents that affect the immune and hematopoietic systems.

To maintain immunity in acute inflammatory diseases (for example, pneumonia), the load on the bone marrow only increases hundreds of times. And the most intense, long and costly moment in maintaining the number and activity of immunocompetent cells is the synthesis of nucleic acids: DNA and RNA. Deficiency of oligonucleotides - nucleic acid fragments - leads to a chronic course of various diseases, a decrease in immunity and a decrease in the body's ability to self-regulate and restore damaged structures, which significantly reduces the quality of human life. When conducting radiation or cytostatic therapy, first of all, the functions of rapidly renewing cell structures, including the bone marrow, are affected. It is in the bone marrow that the formation of key cells responsible for the immunity and protection of the internal environment — lymphocytes, neutrophils, macrophages — takes place. Therefore, the suppression of bone marrow functions, the depletion of its reserves affects the whole body. The therapeutic use of stimulants of the immune and hematopoietic systems is largely limited due to their side effects [1, 2]. In addition, a number of drugs is a protein and therefore they are used exclusively parenterally (in the form of injections), which also has its negative consequences.

The closest analogue to the proposed tool for the achieved result is the sodium salt of deoxyribonucleic acid (deoxinate) obtained from sturgeon milk and partially depolymerized by ultrasound to a molecular weight of 2.7-4.0 × 10 ⁵ Daltons. According to the literature, deoxinate has a therapeutic effect in acute radiation sickness of the II-III severity and in hypo- and aplastic conditions of the blood system caused by radiation or polychemotherapy [3]. It is assumed that due to the ingress of deoxinate into the blood-forming tissue damaged by cytostatics or ionizing radiation, the synthesis of nucleic acids increases, which leads to an increase in the rate of division of hematopoietic progenitor cells and, as a result, accelerated restoration of the cellularity of hematopoietic cells.

The use of the drug is limited due to the development of complications characteristic of the parenteral route of administration. When using deoxinate, soreness at the injection site is noted, body temperature may increase in patients, allergic and anaphylactic reactions are possible [3, 4].

The objective of the invention is to expand the arsenal of immuno-and hemostimulating agents.

The problem is solved by the use of immobilized oligonucleotides as an immunostimulating and hemostimulating agent for oral administration.

The preparation of immobilized oligonucleotides is short DNA fragments, highly purified by proteolytic enzymes, molecular weight in the range of 500-700 kDa obtained from raw materials - "Salmon milk extract - low molecular weight deoxyribonucleic acid." The oligonucleotides are immobilized by irradiating a 10% aqueous solution of polyethylene oxide (PEO) with a molecular mass of 1.5 kDa with a stream of accelerated electrons in a dose of 1.5 Mrad and introducing salmon fish milk DNA into the irradiated solution of oligonucleotides to a final concentration of 10 mg in 1 ml, the mixture is mixed 10 minutes to obtain an opalescent solution. Pre-irradiated polyethylene oxide forms micelles containing condensed DNA, which protects the DNA molecule from destruction in the gastrointestinal tract and facilitates the permeability of the micelle through the intestinal wall. The increased bioavailability of such a drug suggests the possibility of its use by ingestion.

New in the present invention is that the oligonucleotides (im-OGN) immobilized on PEO are first proposed for use as an immuno-and hemostimulating agent for oral administration.

Therefore, the drug that we offer for oral administration may be the drug of choice if the patient has contraindications for the use of other drugs and does not have adequate analogues. The benefits of this drug are obvious. Firstly, the use of nanotechnology of radiation synthesis and gamma radiation to immobilize drug molecules on carriers of low molecular weight prevents their degradation in the gastrointestinal section of the digestive tract and creates conditions for transport through the intestinal mucosa cells into the bloodstream. Secondly, the use of a unique technology allows the creation of water-soluble conjugates of polymers and protein pharmacological preparations with sizes of 20-100 nm, which have increased bioavailability during enteral administration and retained pharmacological activity and therapeutic efficacy [5].

The use of oligonucleotides immobilized on PEO became possible due to the discovery of immunostimulating properties in them, namely, stimulation of phagocytic reactions in vivo and in vitro, increased production of interferon-gamma (IFN-γ) in vitro, increased cytotoxic activity of natural killer cells (EC), increased spontaneous proliferative the activity of lymphoid cells in experimental animals and the stimulation index of B-lymphocytes, increased production of nitric oxide, as well as hemostimulating properties with the introduction of per os.

The use of oligonucleotides immobilized on PEO for oral administration as an immuno-and hemostimulating agent is not described in the literature. This property does not explicitly follow for a person skilled in the art. Immobilized oligonucleotides can be used in patients with immune deficiency associated with acute and chronic infectious diseases, with hypo- and aplastic states of the blood system during radiation therapy and treatment with cytostatics.

Thus, this technical solution meets the criteria of the invention: "novelty", "inventive step", "industrial applicability".

The immunostimulating properties of oligonucleotides immobilized on PEO were discovered through experimental studies.

To establish the immunostimulating activity of oligonucleotides immobilized on PEO, we studied their effect on neutrophil phagocytosis in vivo and in vitro, IFN-γ production in vitro, EC activity, proliferative activity of lymphoid cells with the oral route of administration of the studied drug, and production of nitric oxide by peritoneal animal macrophages when im-OGN is added in vitro.

The study of the immunotropic activity of oligonucleotides immobilized on PEO was carried out according to the “Guidelines for the study of the immunotropic activity of pharmacological substances” [6].

The study used 175 male CBA / CaLac mice and 22 BALB / C male mice weighing 18-20 g at the age of 1.5-2 months, obtained from the nursery of the Scientific Research Institute of Pharmacology SB RAMS. BALB / C mice were used as a source of peritoneal macrophages. The animals were conventional of the 1st category (certificate of the State Research Center for Biomedical Technologies of the Russian Academy of Medical Sciences No. 188-05). Keeping, feeding, caring for animals and removing them from the experiment was carried out in accordance with the requirements of the "Rules for the Use of Experimental Animals" (Appendix to the order of the Ministry of Health of the USSR dated 08.08.1977, No. 755).

Immobilized oligonucleotides were administered to mice orally for 10 or 14 days once a day at a dose of 10 mg / kg, 100 mg / kg and 250 mg / kg in a volume of 0.2 ml of sterile physiological saline, control animals received equivalent volume of solvent (saline). Intact animals of the corresponding sex and age were used as the background.

In in vitro experiments, im-OGN was added to the culture of neutrophils and mononuclear cells of healthy donors (in the amount of 40 people aged 24-38 years) at doses of 0.07 μg / ml, 0.7 μg / ml and 7 μg / ml, and into the culture of peritoneal macrophages of mice in doses varying by an order of magnitude, from 0.00034 μg / ml to 33.775 μg / ml.

Cytostatic myelosuppression was modeled by a single, intraperitoneal administration to animals of a solution of 5-fluorouracil fluoropyrimidine antimetabolite in 1/3 of the maximum tolerated dose (MTD) (76 mg / kg).

Animals were killed by decapitation under ether anesthesia, or an overdose of chloroform.

The phagocytic activity of peritoneal neutrophils was evaluated 24 hours after the end of the 14-day course of administration of immobilized oligonucleotides by the ability of phagocytes to absorb daily Staph culture. aureus strain 209. On the smears of the contents of the peritoneal cavity, the percentage of neutrophils that absorbed microbes (phagocytic index — PI) and the average number of staphylococci absorbed by one cell (phagocytic number — PS) were taken into account [6].

To assess the effect of the drug on in vitro phagocytosis, blood was drawn from healthy donors from a cubital vein into a test tube with heparin at a rate of 25 PIECES per 1 ml of test blood and mixed with 0.8 ml of a 3% gelatin solution prepared on medium 199. A cell suspension was prepared containing 2 million neutrophils in 1 ml. In parallel, a suspension of Staph was prepared. aureus strain 209 at a concentration of 20 million / ml. When setting up the reaction, 90 μl of a cell suspension and 90 μl of staphylococcus (ratio 1:10) were mixed in 96-well round-bottom plates and the plates were incubated at 37 ° C for 30 min. To study the effect of im-OGN on phagocytosis, 20 μl of the studied drug was added to the mixture at concentrations of 0.07 μg / ml, 0.7 μg / ml and 7 μg / ml. After incubation, the plates were centrifuged for 10 min at 1000 rpm, smears were prepared from a precipitate on a glass slide, fixed with ethanol for 30 min, and azure II-eosin was stained for 30-40 min. On smears, the percentage of neutrophils that absorbed microbes (phagocytic index - PH) and the average number of staphylococci absorbed by one cell (phagocytic number - PS) were taken into account [6].

Evaluation of the effect of the drug on the production of IFN-γ was performed on a culture of peripheral blood mononuclear cells (MNCs) of healthy donors [6]. For this, the test drug was added to mononuclear cultures at concentrations of 0.07 μg / ml, 0.7 μg / ml and 7 μg / ml in a volume of 50 μl. IFN-γ in the daily supernatants of mononuclear cultures was determined using the enzyme-linked immunosorbent assay using the appropriate kit manufactured by Vector-Best CJSC (Novosibirsk).

A study of the effect of 14-day course administration of im-OGN on the functional activity of EC was determined in the cytotoxic reaction by their ability to lyse K-562 myeloblastoid line cells using the colorimetric method [7].

To assess the effect of a 14-day course administration of im-OGN on the proliferative activity of T and B lymphocytes of experimental animals, the blast transformation reaction was used, which to a certain extent is an integral method for determining their functional activity [6]. This method is based on the ability of some lectins to induce polyclonal activation and proliferation of lymphocytes, which was evaluated colorimetrically [8]. Phytohemagglutinin (PHA) was used to activate T-lymphocytes; to activate B-lymphocytes - lipopolysaccharide (LPS). Splenocytes from experimental animals were used as a source of lymphoid cells.

To determine the effect of the studied drug on the production of nitric oxide by peritoneal macrophages of intact mice, im-OGN was added to the macrophage culture in doses varying by an order of magnitude from 0.00034 μg / ml to 33.775 μg / ml. Macrophages were cultured in flat-bottomed 96-well plates at a concentration of 2 × 10 ⁵ cells per well for 48 h, after which they were collected from supernatant wells and the amount of nitrite was measured therein using a Grace reagent using a Titertek Multis-can MCC spectrophotometer (Titertek) , Finland) at a wavelength of 540 nm [9].

As the ligand of TLR-9, the CpG oligonucleotide ODN1826 (InvivoGen, USA) was used. The inhibitor oligonucleotide ODN2088 (InvivoGen, USA) was used as a TLR-9 blocker.

The hemostatic properties of im-OGN were studied using a model of cytostatic myelosuppression caused by the introduction of 5-fluorouracil (5-FU). The preparation of immobilized oligonucleotides was administered to mice per os daily from 1 to 10 days of the study at a dose of 250 mg / kg.

On the 3rd, 5th, 7th, 9th, 11th, and 13th day of the experiment in mice, using the ABACUS automatic hematology analyzer (Diatron, Austria), the state of the peripheral unit of the erythron system (hemoglobin content, number of red blood cells, hematocrit, and average corpuscular hemoglobin concentration) was evaluated in veterinary mode. . Further, by the generally accepted methods, the content of reticulocytes and various forms of leukocytes in peripheral blood was studied [10]. After killing by the craniocervical dislocation method under ether anesthesia, the total number of myelocaryocytes and the absolute content of the cellular elements of individual hematopoietic sprouts in the bone marrow were evaluated [11].

Statistical processing of the results was carried out using the statistical software package Statistica for Windows (version 5.0) with a preliminary assessment of the normality of the distribution and the use of Student t-test, when the variational series deviated from the normal distribution, the non-parametric Wilcoxon-Mann-Whitney statistic method was used.

Results confirming the immunostimulating properties of oligonucleotides immobilized on PEO by the oral route of administration.

Example 1. The study of the phagocytic activity of peritoneal neutrophils was evaluated 24 hours after the end of the 14-day course of administration of immobilized oligonucleotides at a dose of 10 mg / kg (group 3), at a dose of 100 mg / kg (group 4) or solvent (group 2) according to ability phagocytes absorb the daily culture of Staph. aureus strain 209. The course administration of im-OGN at a dose of 10 mg / kg led to a significant increase in the relative number of phagocytic neutrophils in experimental animals as compared to the control group and the background, as well as to the group of mice that received the studied drug at a dose of 100 mg / kg (table 1). Moreover, the number of absorbed bacteria (PS) in the group of animals using im-OGN at a dose of 10 mg / kg was statistically significantly higher relative to the values of the control group. The course introduction of im-OGN at a dose of 100 mg / kg did not lead to significant changes in the phagocytic index and the phagocytic number of neutrophils in experimental animals compared

Table 1
Assessment of the effect of the course administration of immobilized oligonucleotides on the phagocytic activity of peritoneal neutrophils of CBA / CaLac mice

Research indicators Groups of experimental animals Group 1
Background
(n = 7) Group 2
The control
(n = 7) Group 3
im-ogn
(10 mg / kg)
(n = 7) Group 4
im-ogn
(100 mg / kg)
(n = 7) FI,% 12.0 ± 0.36 14.0 ± 0.73 * 24.14 ± 2.06 *
_2-3 P <0.001 16.57 ± 1.41 *
_3-4 P <0.05 Ff 2.97 ± 0.14 2.60 ± 0.16 3.69 ± 0.31
_2-3 P <0.01 2.96 ± 0.15 Note: * - differences between the experimental groups and the background, between the control group and the background are significant (p <0.05), n is the number of animals in the group.

With the control group, only with respect to the background was a statistically significant increase in the relative number of phagocytic neutrophils observed.

Example 2. Evaluation of the effect of immobilized oligonucleotides on phagocytosis of neutrophils in vitro. When im-OGN was added to healthy blood donors of peripheral blood neutrophils at various concentrations (0.07 μg / ml, 0.7 μg / ml and 7 μg / ml), a significant increase in the phagocytic number was observed relative to the control group when a dose of immobilized neutrophils was introduced into the culture oligonucleotides 0.7 μg / ml (table 2). It should also be noted that when im-OGN was added to neutrophils in various doses, the phagocytic activity indices exceeded the control values, although not statistically insignificantly, while with increasing concentration of im-OGN added, the phagocytic index decreased, and the phagocytic number increased.

table 2
Evaluation of the effect of immobilized oligonucleotides at doses of 0.07 μg / ml, 0.7 μg / ml and 7 μg / ml when added in vitro to the donor peripheral blood phagocytosis

Research indicators Experimental groups Group 1 Control
(n = 10) Group 2
im-ogn
(0.07 mcg / ml)
(n = 10) Group 3
im-ogn
(0.7 mcg / ml)
(n = 10) Group 4
im-OGN (7 μg / ml)
(n = 10) FI,% 26.90 ± 2.65 35.50 ± 3.53 33.80 ± 3.34 32.10 ± 3.06 Ff 3.86 ± 0.36 4.73 ± 0.41 4.91 ± 0.26
_1-3 P <0.05 5.09 ± 0.50 Note: n is the number of donors in the group.

Example 3. Evaluation of the effect of immobilized oligonucleotides on the synthesis of IFN-γ by peripheral blood mononuclear cells. When im-OGN was added, a statistically significant increase in the production of IFN-γ was observed at a dose of 0.7 μg / ml of the introduced drug (Table 3). The spontaneous production of the studied cytokine in this group was significantly higher than in comparison with

Table 3
Assessment of the effect of immobilized oligonucleotides at doses of 0.07 μg / ml, 0.7 μg / ml and 7 μg / ml when added in vitro to mononuclear culture on IFN-γ synthesis

Experimental groups The concentration of cytokine (PG / ml) Group 1 Control (no drug added) (n = 10) 9.64 ± 1.17 Group 2 im-OGN (0.07 μg / ml) (n = 10) 10.31 ± 1.14 Group 3 im-OGN (0.7 μg / ml) (n = 10) 21.29 ± 5.21
_1-3 P <0.05
_2-3 P <0.05 Group 4 im-OGN (7 μg / ml) (n = 10) 9.52 ± 1.46 Note: n is the number of donors in the group.

control, and with the group in which im-OGN was added at a dose of 0.07 μg / ml.

Example 4. The study of the effect of immobilized oligonucleotides on the functional activity of natural killer mice. After a 14-day course administration of im-OGN at a dose of 10 mg / kg, the functional activity of the natural killer cells of mice increased both in comparison with the background and the control group for all studied ratios of targets to effectors, but statistically insignificant (Table 4). Moreover, the use of im-OGN in a dose of 100 mg / kg significantly enhanced

Table 4
Evaluation of the effect of course administration of immobilized oligonucleotides at doses of 10 mg / kg and 100 mg / kg on the natural killer activity (ESA) of CBA / CaLac mice

Research indicators Experimental groups Group 1
Background (n = 7) Group 2 Control (n = 7) Group 3
im-ogn
(10 mg / kg) (n = 7) Group 4
im-ogn
(100 mg / kg) (n = 7) ESA (%) target: effector ratio 1:10 35.70 ± 2.66 32.67 ± 1.44 38.90 ± 3.32 38.86 ± 2.43
_2-4 p <0.05 ESA (%) target: effector ratio 1:25 35.24 ± 2.26 34.20 ± 1.23 39.61 ± 2.54 39.36 ± 1.55
_2-4 p <0.05 ESA (%) target: effector ratio 1:50 37.49 ± 1.50 37.60 ± 1.42 42.68 ± 2.78 49.32 ± 4.47
_1-4 p <0.05
_2-4 p <0.05 Note: n is the number of animals in the group.

the cytotoxic activity of the EC of experimental animals compared with the control for all studied ratios of targets to effectors, and at a ratio of targets to effectors of 1:50, the functional activity of natural killers increased statistically significantly compared to the background.

Example 5. The study of the effect of immobilized oligonucleotides on the proliferative activity of lymphocytes. After a 14-day course administration of im-OGN at a dose of 10 mg / kg, a significant increase in the spontaneous proliferative activity of lymphoid cells of experimental animals was observed both in comparison with the background and the control group (Table 5). In this case, the stimulation index (IS) of T-lymphocytes is statistically

Table 5
Evaluation of the effect of a course of administration of immobilized oligonucleotides at doses of 10 mg / kg and 100 mg / kg on the proliferative activity of lymphocytes of CBA / CaLac mice

Research indicators Groups of experimental animals Group 1
Background (n = 7) Group 2 Control (n = 7) Group 3
im-ogn
(10 mg / kg) (n = 7) Group 4
im-ogn
(100 mg / kg) (n = 7) Spontaneous proliferation (unit density) 0.196 ± 0.016 0.217 ± 0.005 0.238 ± 0.007
_1-3 p <0.05
_2-3 p <0.05 0.206 ± 0.006
_3-4 p <0.01 IS (FGA) 1.11 ± 0.08 1.06 ± 0.02 1.12 ± 0.03 1.23 ± 0.16 IP (LPS) 1.94 ± 0.22 1.53 ± 0.15 2.09 ± 0.07
_2-3 p <0.01 1.99 ± 0.14
_2-4 p <0.05 Note: n is the number of animals in the group.

did not significantly change both in the group using im-OGN at a dose of 10 mg / kg, and at a dose of 100 mg / kg. However, the stimulation index of B-lymphocytes in both experimental groups with course administration of im-OGN at a dose of 10 mg / kg and 100 mg / kg was significantly higher than the corresponding indicator of the control group.

Example 6. The study of the effect of immobilized oligonucleotides on the production of nitric oxide in a culture of peritoneal macrophages in vitro. The studied drug (Table 6) dose-dependently stimulated the production of nitric oxide at concentrations of 3.3775 and 33.775 μg / ml.

Table 6
The effect of immobilized oligonucleotides on the production of nitric oxide by peritoneal macrophages

Concentration
im-OGN (μg / ml) NO (nitrites, μm) control (no drug added) 19.0 ± 1.0 0,00034 21.4 ± 1.9 0,00338 19.9 ± 1.3 0,03378 19.7 ± 1.5 0.33775 18.9 ± 1.7 3,37750 27.0 ± 2.5 * 33.7750 49.5 ± 3.2 * Note: * - significant differences with control (p <0.05).

The commercial ligand TLR-9 (CpG oligonucleotide ODN1826) and the blocker TLR-9 (inhibitory oligonucleotide ODN2088) were used to clarify the role of the TLR-9 receptor in stimulation of nitric oxide by the preparation. The results of the study are presented in table 7. The stimulating effect of the CpG oligonucleotide was dose-dependently canceled when the TLR-9 receptor was blocked. The effect of the study drug was also canceled by the inhibitory oligonucleotide ODN2088.

Thus, the studied drug has a stimulating effect against macrophages. This action is mediated by the macrophage receptor TLR-9.

Table 7
The role of TLR-9 in stimulating nitric oxide production by peritoneal macrophages with immobilized oligonucleotides

Cultivation conditions Nitric oxide production in the presence of: control-1 2.5 μM ligand TLR-9 3.3775 μg / ml im-OGN 33.775 μg / ml im-OGN - control-2 0 14.3 ± 0.5 * 6.7 ± 1.0 * 10.4 ± 0.6 * + TLR-9 blocker 1.25 μM 0 7.8 ± 0.6 * # 0 # 4.0 ± 0.5 * # + TLR-9 2.5 μM blocker 0 0 # 0 # 0 # Notes: * - significant differences with control-1 (p <0.05); # - significant differences with control-2 (p <0.05).

Example 7. The study of the effect of immobilized oligonucleotides on the restoration of hematopoiesis, suppressed by the introduction of 5-fluorouracil. The introduction of im-OGN contributed to a significant increase in the absolute number of leukocytes in the peripheral blood during the post-cytostatic restoration of hematopoiesis (9th, 11th, 13th day) (Table 8). Leukocytosis found in the experiment was a consequence of the accumulation of the number of lymphocytes and neutrophils at the indicated times. The course administration of the studied drug at a dose of 250 mg / kg increased the content of immature (on the 3rd and 5th day) and mature (on the 7th day) forms of neutrophilic granulocytes in comparison with the same parameters of the cytostatic control (Table 9). The course administration of im-OGN contributed to an increase in the number of reticulocytes (on the 13th day) and platelets (on the 9th and 13th day) in the peripheral blood after application of 5-FU (Table 10, 11).

Table 10
The effect of the preparation of immobilized oligonucleotides at a dose of 250 mg / kg on the dynamics of the content of reticulocytes in the peripheral blood of CBA / CaLac mice treated with 5-fluorouracil (‰)

Study time, days Experimental groups 5-fluorouracil Immobilized Oligonucleotides Intact control 30.17 ± 1.92 30.17 ± 1.92 3 16.29 ± 1.38 * 13.57 ± 1.19 * 5 15.5 ± 3.06 * 14.78 ± 3.10 * 7 11.78 ± 1.25 * 14.20 ± 1.40 * 9 17.56 ± 1.23 * 18.16 ± 1.36 * eleven 22.85 ± 2.73 * 18.65 ± 2.07 *
P <0.02 13 17.93 ± 5.58 * 31.59 ± 3.45
P <0.02

Thus, the use of the proposed method for the physical immobilization of oligonucleotides of DNA of salmon milk on polyethylene oxide, weighing 1.5 kDa, allows you to get a drug for oral administration, which exhibits significant immunostimulating activity, which is expressed in stimulation of phagocytic reactions in vivo and in vitro, increasing the production of IFN -γ in vitro, increased cytotoxic activity of natural killers, increased spontaneous proliferative activity of lymphoid cells in experimental animals and index timulyatsii B-lymphocytes, enhancing nitric oxide production, and hemostimulating properties when administered per os.

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Claims (1)

The use of immobilized oligonucleotides, which are DNA fragments with a molecular weight of 500-700 kDa, highly purified by proteolytic enzymes, obtained from an extract of salmon milk immobilized on polyethylene oxide by irradiating a 10% aqueous solution of polyethylene oxide with a molecular weight of 1.5 kDa with a stream of accelerated electrons in a dose of 1 , 5 Mrad and introducing salmon fish milk DNA into an irradiated solution of oligonucleotides to a final concentration of 10 mg in 1 ml, as an immunostimulating and hemostimulating medium for oral administration.