RU2734243C1 - Method for treating radiation injuries of an organism - Google Patents

Abstract

FIELD: veterinary medicine; medicine.SUBSTANCE: invention relates to use of preparations for preventing and treating radiation injuries, achieved by inhibiting lipid peroxidation products (LP) induced by ionizing radiation. Method of treating radiation injuries of an organism involves administering into a body a biological preparation of plant origin—composition obtained by mixing purified turpentine and highly purified refined sunflower oil, single intramuscularly in doses 0.25–0.5 ml/kg of live weight for first 12 days before and during first 1–4 days after lethal exposure.EFFECT: method of treating radiation injuries of an organism is disclosed.1 cl, 2 tbl, 6 ex

Description

The invention relates to veterinary medicine and medicine, in particular, to the use of drugs intended for the prevention and treatment of radiation damage, is achieved by inhibiting the formation of lipid pyraxidation products (LPO) induced by ionizing radiation.

A known method of treating radiation damage to the body using extracts from plant (tincture of calendula, pollen, polyextract from rowan berries) products with antiradiation effect (see article L.M. Ovsyannikov et al. - Basic principles of correction of damage to oxidative homeostasis in individuals, exposed to the factors of accidents at the Chernobyl NPP // Problems of radiation protection: Mater, conf. M .: RAS, 1998. - P. 116-117).

A known method of treatment (rehabilitation) of exposed persons using a basic complex of biologically active food additives containing infusions of aspen bark, chaga mushroom, viburnum or mountain ash fruit juice and vitamin herbal tea (application for invention RU No. 2004109896, class A23L 1/30, published date . 10.10.05).

The disadvantage of these methods is that, firstly, they involve the oral use of aqueous or alcoholic extracts of apiphytoproducts, and secondly, the radioprotective effect of these drugs is manifested only with prolonged (for a year or for at least 21 days) continuous use of drugs in the form of food additives to the main diet. In case of need for emergency radioprophylactic or radiotherapeutic use, these methods do not provide emergency protection.

A known method for the prevention and treatment of radiation injuries to the body by a single subcutaneous injection of antiradiation serum in doses of 100-125 mg / kg body weight for young and 200-250 mg / kg for adults during the first 10 days after irradiation for therapeutic purposes (Patent RU No. 2169372, IPC A61K 35/28, June 27, 2001). The disadvantage of this method is the complex technology for preparing the drug (double irradiation of large animals using a powerful radiation-hazardous gamma device and, most importantly, the use of high therapeutic doses of an expensive drug - antiradiation horse serum, the consumption of which for large animals is 200-400 ml of the drug per head ).

A known method for the treatment and prevention of radiation injuries with prolonged use of biologically active substances from tree greenery, spruce and pine, which, when fed at a rate of 0.2 ml / kg of body weight, ensured the survival rate of 60% of lethally irradiated animals (Kurilo A.I. Veterinary and hygienic bases of application biologically active substances of plant origin for the prevention of radiation injuries: Abstract of the thesis ... Doctor of Veterinary Sciences - St. Petersburg, 1977-46 p).

The closest in technical essence is the use of a triterpenoid of plant origin - miliacin, to inhibit toxic radicals, the intraperitoneal administration of which at a dose of 2 mg / kg had a stress-protective effect by inhibiting stress-induced toxic radicals - products of lipid peroxidation of lipids (LPO) (see article T.V. Panfilova, AA Shtil, BA Frolova - Meliacin triterpenoid reduces stress-induced LPO // Bull Experimental Biology and Medicine. - 2006. - T. (4). - No. 6. - P. 633- 635).

The disadvantage of this method is the low efficiency of treatment for radiation injuries, the complexity of obtaining the drug and the duration of its use (30 days).

Herbal preparations - pine terpenoids have the most important mechanism - a protective effect against stress damage by blocking the formation of toxic radicals against the background of exposure to stressors (ionizing radiation, pathogenic agents, chemical toxicants, etc.). One of the most important terpenes used in medicine and veterinary medicine is turpentine oil - turpentine, which has a wide range of biological effects (anti-inflammatory, analgesic, antibacterial, activation of granulation, central nervous system, immuno- and hematopoiesis) (I.E. Mozgov - Pharmacology: Guide for veterinarians. - M .: Selkhozgiz. - 1954. - p. 207-209).

The technical result, which the invention is aimed at, is to increase the effectiveness of the body's protection against radiation damage, to simplify and reduce the cost of the treatment method and to shorten the treatment period.

To achieve the named technical result in the proposed method, including the introduction into the body of a biological preparation of plant origin, a composition obtained by mixing purified turpentine with highly purified refined sunflower oil in a ratio of 70:30, respectively, is used as a biological preparation, which is administered to animals once intramuscularly at a dose of 1 , 0-2.0 ml for small animals and 5.0-8.0 ml for large animals within 1-12 days before irradiation and during the first 1-4 days after irradiation.

Use of a composition obtained by mixing 70 parts of purified turpentine (GOST 1571-82) and 30 parts of highly refined refined sunflower oil "Solpro" (TU 9141-006-70316851-2012) produced by JSC "Aktarskiy MEZ" (Russia) and its intramuscular injection once in doses of 1.0-2.0 ml for small and 5.0-8.0 ml for adult animals within 1-12 days before irradiation and during the first 1-4 days after irradiation, it allows to increase the effectiveness of treatment, to create a radioprotective, radiotherapy the dose of the drug, to simplify the technology of obtaining a biological drug and use instead of oral and long-term (within 30 days) administration of biologically active substances from woody greenery or pine for a single intramuscular parenteral administration.

The choice of the optimal route of drug administration and the reduction of the demarcation effect of turpentine in combination with highly purified vegetable oil provides an urgent and effective action of the composition due to the direct action on the biomembranes of radiation-affected cells and, by stabilizing their function and increasing their resistance to lipid peroxidation products and toxic decay products, the target whose attacks are cells highly sensitive to radiation - lymphocytes, which undergo radiation apoptosis. Thus, the drug used, urgently reaching target cells affected by ionizing radiation - lymphocytes, protects them from the attacking agent - lipid radiotoxins (lipid peroxidation products - LPO) by stabilizing membranes and blocking the metabolic (biochemical and pathochemical) effects of toxic agents.

A method for treating radiation injuries is illustrated by the following examples.

Example 1. Study of the possibility of a parenteral method of using purified turpentine.

White mice were used as a biological model for these studies, and purified pure turpentine was used as a test preparation. The drug was administered to white mice subcutaneously, intramuscularly, and intraperitoneally at doses of 0.1; 0.2; 0.3; 0.4 and 0.5 ml, respectively. The animals were clinically observed, registering the number of deaths and the timing of the death of animals after drug administration.

It has been established that the most acceptable way of using turpentine is its subcutaneous administration. When administered intraperitoneally, the drug has a toxic effect, and when administered intramuscularly, it causes tissue necrosis at the injection site and the death of animals at all tested doses (0.1-0.5 ml) of application.

However, despite the absence of a toxic effect upon subcutaneous administration of purified (native) turpentine, its use undiluted is undesirable, since with this method a diffuse edema is formed at the injection site in the form of a small roll without clear boundaries.

The foregoing implies the search for a diluent (solvent) to accelerate the resorption of the drug and its targeted delivery to target cells affected by ionizing radiation.

Example 2. Selection of the optimal solvent (depositing agent) of turpentine.

Considering that turpentine does not dissolve in water, but easily dissolves in alcohol, chloroform, mixes in any ratio with ether, chloroform, gasoline and vegetable oils, we stopped our choice on vegetable oils, since the other components listed above are highly toxic to the body.

Sunflower oils were used as potential solvents (diluents): unrefined, refined and high oleic refined sunflower oil. Using various oils, turpentine emulsions were prepared in various component ratios (refined turpentine: oil): 70:30; 50:50; 30:70; 20:80.

Prepared by the above method, 70, 50, 30 and 20% emulsions of turpentine and sunflower oil were injected subcutaneously, intraperitoneally and intramuscularly to white mice, 3 animals for each variant of the experiment (12 variants with different ratios of components (4) and 3 types of oils used).

It was found that of all the tested emulsions based on sunflower oils of various degrees of purification, the most acceptable were emulsions (compositions) obtained by mixing highly purified refined sunflower oil "Solpro", which, unlike emulsions based on unrefined and refined oils, when administered intramuscularly did not cause the formation of edema, quickly resolved at the injection site without the formation of edema and a roller with extended boundaries. Intraperitoneal administration had a toxic effect on animals, which was accompanied by the death of 70% of white mice. When administered subcutaneously, edema was formed in the form of a small roll with clear boundaries, which indicates poor resorption and long-term deposition of the drug at the injection site.

Therefore, the optimal solvent (diluent) for purified turpentine is highly purified high oleic sunflower oil "Solpro" (TU 9141-006-70316851-2012) produced by JSC "Atkarskiy MEZ" (Russia), and the method of parenteral administration is intramuscular.

Example 3. Study of the radioprotective effectiveness of an emulsion (composition) based on purified turpentine and sunflower oil.

To assess the radioprotective effectiveness of purified turpentine and sunflower oil, various ratios of components were prepared: 70:30 (70% emulsion), 50:50 (50%), 30:70 (30%) and 20:80 (20 % emulsion). The experiments were carried out on 45 white mice, divided into 9 groups of 5 animals each. The prepared compositions with the indicated ratios of the components were administered intramuscularly to white mice once at a dose of 0.1 ml per mouse 24 hours before (1st, 2nd, 3rd and 4th groups) and 24 hours after (5th , 6th, 7th, 8th groups) of irradiation in a lethal dose (8.0 Gy, LD _100/30 ) 9th group served as a control of exposure.

The animals were monitored for 30 days, before and after irradiation, test preparations were administered, taking into account the number of dead and surviving animals in each group.

The results of studying the radioprotective effectiveness of the tested compositions are presented in Table 1.

It can be seen from the data in the table that the tested compositions have radioprotective activity both for prophylactic (administration of the drug 24 hours before irradiation) and therapeutic (administration of the drug 24 hours after irradiation), providing 80% and 60% survival of lethally irradiated animals respectively. At the same time, the optimal ratio of components in both variants of the use of the drug is 7 parts of purified turpentine and 3 parts of sunflower oil (OS: PM = 70: 30). Any changes in this ratio of components leads to a decrease in the radioprotective effectiveness of the components (groups 2, 3, 4 and 6, 7, 8, respectively).

Example 4. Determination of the optimal therapeutic and prophylactic dose of a radioprotective drug for parenteral (intramuscular) administration.

To determine the optimal therapeutic and prophylactic dose of a radioprotective preparation of a composition based on purified turpentine and sunflower oil, various dilutions of the preparation were prepared, containing 0.1 ml of 0.1; 0.01; 0.05; 0.025 and 0.0012 ml of the initial (70% + 30%) solution - emulsions, which were once intramuscularly injected into 5 white mice (live weight - 20 g) for each dilution (dose) 24 hours before and after 24 hours after lethal exposure (7.7 Gy).

It was found that the maximum radioprotective effect (60-80% protection in therapeutic and prophylactic use, respectively) was manifested when doses of 0.1; 0.01 and 0.005 ml. The introduction of the drug in a dose of 0.0025 ml leads to a decrease in the radioprotective effect (20% survival). Therefore, the optimal therapeutic and prophylactic doses of the drug are 0.01-0.005 ml per mouse, which is 0.5-0.25 ml per 1 kg of body weight.

With the practical use of the composite preparation, the effective therapeutic and prophylactic dose for animals with live weight of 100-500 kg is 5.0-8.0 ml, 50-100 kg - 1.0-2.0 ml.

Example 5. Study of stress-protective antioxidant action of a composition based on purified turpentine and sunflower oil.

To assess the antioxidant activity of the test drug, experiments were carried out on 30 white rats, divided into three groups of 10 animals each. Animals of the 1st and 2nd groups were subjected to radiation stress by irradiating them with a gamma device "Puma" at a dose of 9.0 Gy. 24 h after lethal irradiation, the animals of the 2nd group were injected intramuscularly once with a 70% emulsion of purified turpentine and sunflower oil in a dose of 0.5 ml. The animals of the 3rd group were not irradiated or treated - they served as biological control. Blood samples were taken from all animals 3.7 and 14 days after irradiation and treatment, and the concentration of stress-induced lipid peroxidation products (LPO) was determined in plasma. The content of lipid hydroperoxides (LPO) was determined in blood serum according to M.S. Goncharenko and AM Latinova (Method for the determination of malondialdehyde in the blood serum of animals. - // Laboratory. - 1985. - No. 1. - P. 60-61). The LPO content was expressed in the equivalent amount of malondialdehyde (MDA) using the molar ecotinction coefficient equal to 1.56 to 10 ⁵ (mol / l) cm ^-1 .

The absorption spectrum of the colored product with thiobarbituric acid (TBA) was recorded on a double-beam spectrophotometer "SF-6".

The results of the studies are presented in table 2.

As can be seen from the table, radiogenic stress in white rats is accompanied by a sharp increase in the intensity of lipid peroxidation - after 3 days, the concentration of MDA in the blood of irradiated rats increased by 1.21 times (P <0.05) and gradually increasing by 14 days reached its maximum value ( 10.13 ± 0.57 μmol / L versus 4.65 ± 0.09 in the control).

The use of a composition obtained by mixing 70% purified turpentine and 30% highly refined refined sunflower oil 24 h after lethal irradiation led to a prolonged decrease in the intensity of lipid peroxidation (LPO) compared with only irradiated animals, which caused an increase in the survival rate of lethally irradiated animals while using the test drug.

Thus, the use of purified turpentine in a mixture with highly purified refined sunflower oil has a radioprotective effect on the body, based on the inhibition of the formation of toxic products of lipid peroxidation (LPO - radiotoxins), the target of attack of which are immunocompetent cells - lymphocytes, the devastating apoptosis of which leads to radiation death of the animal. Inhibition of lipid peroxidation formation under the influence of a mixture of terpenes (turpentine and sunflower oil) against the background of lethal radiation enhances the body's resistance to the destructive effect of ionizing radiation.

Example 6. Determination of the optimal timing of the use of the composition before (for prophylactic purposes) and after (for therapeutic purposes) lethal irradiation of animals.

For this purpose, experiments were carried out on 80 white mice, divided into 16 groups. The antiradiation efficacy of a composition based on purified turpentine and sunflower oil was tested before and after lethal training of animals. Forty white mice of the first 8 groups were injected intramuscularly with 0.1 ml of 70% emulsion of the drug for prophylactic purposes for 1, 2, 4, 6, 8, 10, 12 and 14 days (5 white mice for each period) before lethal exposure. Forty white mice of the second subgroup (8 groups of 5 animals in each) 1, 2, 4, 6, 8, 10, 12 and 14 days after lethal irradiation were injected with the drug in similar doses and in the same way as the first subgroup of animals.

White mice of all groups were irradiated with a lethal dose of 8.0 Gy (LD _100/30 ) on the Puma gamma device and were monitored for 30 days after irradiation, taking into account the number of dead and surviving animals in each group.

The results of the experiments showed that a sufficiently high prophylactic (70-80% survival rate) and therapeutic (60% survival rate) effect is achieved within the first 1-12 days before and during the first 1-4 days after lethal exposure.

Claims (1)

A method for the treatment of radiation injuries to the body, providing for the introduction into the body of a biological preparation of plant origin, characterized in that the biological preparation uses a composition obtained by mixing 70 parts of purified turpentine and 30 parts of highly purified refined sunflower oil, which is administered to animals once intramuscularly during the first 1 -12 days before irradiation and during the first 1-4 days after irradiation at a dose of 0.25-0.5 ml / kg of live weight.