RU2551619C1 - Method for preventing and treating acute radiation disease experimentally - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: melanin having water-solubility of at least 80% and an paramagnetic centre concentration of at least 8·1017 spin/g is administered orally into the animals having been exposed to the radiation in a dose high enough to cause a spinal radiation injury; melanin is administered after dissolved in distilled water in the effective concentration. Melanin water is used as drinking water for the mice having been exposed to single and fractionated acute radiation, which is able to cause acute radiation disease. Melanin water is taken from the 1^st to 30^th day following the single radiation, or from the 1st day of the fractionated radiation to the 30th day on completion of the radiation.

EFFECT: higher survival rate, faster recovered haemopoiesis, body weight and orientation and motion activity.

7 tbl, 5 ex

Description

The invention relates to medicine and veterinary medicine and can be used to treat acute radiation sickness.

Melanins - natural pigments, biologically active substances contained in the tissues of microorganisms, plants of animals and humans (give color to hair, eyelashes, skin), in a number of foods (tea, coffee, mushrooms, buckwheat, etc.). They are formed as a result of the polymerization of tyrosine, dioxiphenylalanine or catecholamines on a protein matrix. The chemical structure of natural melanins has not yet been fully established due to their extremely complex high polymer structure.

According to pharmacological studies, water-soluble melanins are non-toxic. A single oral or intraperitoneal administration in the dose range of 100-3000 mg / kg did not cause the death of mice. When administered within 15 days, there were also no fatalities, violations of the general condition, pathological changes in internal organs, no carcinogenic effect was detected [1-4].

и перекисные радикалы органических соединений [2, 3, 13, 14 и др.].The information available in the literature demonstrates the presence of a variety of biological properties in melanins, which are manifested when administered orally or intraperitoneally. In small laboratory animals (mice, rats) it was shown that melanin has antistress [1, 5], anticonvulsant [5] and antimutagenic effects [6, 7]. It has been established that melanin increases physical performance [8], enhances the body’s immune defense [1, 8, 9], causes the production of cytokines such as IL-6, IL-8, tumor necrotizing factor, endothelial growth factor [10, 12]. There is evidence of the successful use of melanin in the complex therapy of cancer of the lungs, intestines, breast, and diabetes [1]. One of the most important properties is pronounced antioxidant activity. Melanins inhibit lipid peroxidation due to their ability to intercept the superoxide anion radical $$O_2^{-}$$

and peroxide radicals of organic compounds [2, 3, 13, 14, etc.].

Separate sources of information have been found in the literature database, which describe the use of melanin as a radioprotector, administered parenterally before exposure to ionizing radiation, which is practically impossible in case of emergency situations. Under experimental conditions, a single or daily intraperitoneal administration of melanin for 4 days before irradiation in doses causing the death of 40-80% of mice increased survival by 35% and 60%, respectively [15, 16]. It was also shown that the intravenous injection of nanomelanin during the radioisotope therapy of melanoma in experiments on mice did not have a protective effect on tumor cells, but at the same time contributed to the reduction of hematopoiesis [17 - prototype, 18].

With exposure of pregnant rats chronic for 20 days at a total dose of ~ 1.25 Gy, not accompanied by the development of acute radiation sickness (ARS), the daily intragastric administration of a suspension of melanin in starch gel (10 mg / kg) eliminated somatic deficiency in the postnatal period rat pup development [8, 9]. There are indications of the ability of melanin to reduce the accumulation of radionuclides in the body [19].

The objective of the work is to study and prove the possibility of optimal therapeutic and prophylactic (administration after irradiation) use of water-soluble melanin after radiation exposure in doses that cause acute radiation sickness (ARS). The development allows to increase the survival rate of irradiated animals, accelerating the restoration of body weight, locomotor activity and blood formation indices disturbed by radiation.

The technical result consists in increasing the survival rate of the irradiated animals, accelerating the restoration of body weight, locomotor activity and hematopoiesis indicators impaired by radiation.

To achieve this goal, the effect of melanin on survival, hematopoiesis, and spontaneous motor activity of irradiated mice was studied.

During the study, it was found that the best effects are achieved when using melanin in dissolved form. Moreover, it is optimal to dissolve it in distilled water at the rate of 12.5 mg per 100 ml, using this water in animals as drinking. We have revealed that it is in this form that melanin is capable of exerting the most adequate radioresistant effect on animals that have already undergone acute radiation.

The experiments used melanin manufactured in accordance with TU 9197-001-6735746-0, with a water solubility of at least 80% and a concentration of paramagnetic centers of at least 8 · 10 ¹⁷ spin / g.

The therapeutic efficacy of melanin was studied on 262 CD-1 outbred mice, females weighing 22-30 g. Only clinically healthy individuals were selected for the experiments. The control and experimental groups were formed by a random choice from animals of equal mass.

Tests of the therapeutic efficacy of melanin were performed with total - acute and fractionated radiation exposure. Short-term irradiation was carried out on an x-ray biological installation RUST-M1 (RUB RUST-M1): voltage 200 kV, beam current 2 × 2.5 mA, aluminum filter 1.5 mm. The dose rate of 0.85 Gy / min ± 10%. Some mice were irradiated with ⁶⁰ Co gamma rays with a power of 1 Gy / min at the ROCUS-M facility. Doses of a single exposure were 5.0; 6.5; 7.0; 7.5 g

Fractional total irradiation of 0.5 Gy or 1 Gy was carried out daily for 5 days, then again after a 2-day break.

Melanin dissolved in distilled water at the rate of 12.5 mg per 100 ml, mice were freely available instead of drinking water from the 1st to the 30th day after a single exposure, and with fractionated exposure - from 1 day from the start of the exposure and on the 30th day after the end of irradiation. At the same time, control untreated animals consumed distilled water.

The experiments were carried out in compliance with the "Rules for the use of experimental animals, regulated by order of the Ministry of Health of the USSR No. 75 of 08/12/1987."

The effect of melanin on the general condition of irradiated animals was evaluated by their 30-day survival and weight dynamics, which was measured 2 times a week, as well as by the level of locomotor activity in the "open field" and the state of hematopoiesis.

Statistical processing of the data obtained in the experiments was carried out using generally accepted methods of variation statistics. Arithmetic mean values and their standard errors were calculated over the entire duration of the examination. The statistical significance of differences in animals of the compared groups was judged by Student, χ ^2, and Wilcoxon-Mann-Whitney criteria.

In the conducted studies, the authors found that melanin, when taken after irradiation, has a therapeutic effect, manifested in an increase in radioresistance. The results obtained in a generalized form and in various series are presented in tables 1 and 2. Irradiation in doses of 6.5; 7.0 and 7.5 Gy by X-ray and gamma rays caused the development of the bone marrow form of ARS, after which the vast majority of mice in the control groups (68 out of 70) died. The survival rate of untreated animals when observed within 30 days after irradiation was 20% (with DM ₈₀ ) and 0% (with DM ₁₀₀ ). The therapeutic use of melanin with drinking water has increased the survival rate up to 40% (with DM ₈₀ ) and 8.3% (with DM ₁₀₀ ). The total indicator was 16.3%, in the control - 2.9%. The difference between the groups is statistically significant (criterion χ ² , p <0.01).

Observation of the clinical condition of the animals also showed that taking melanin after irradiation helps to maintain body weight at a higher level than in the control, and accelerate its growth during the recovery period. Differences on days 18-21 are statistically significant (Table 3).

In addition to monitoring the clinical course of ARS in experimental mice, the state of hematopoiesis was determined on the 8th day after irradiation with X-rays in a sublethal dose of 5 Gy. Table 4 shows that the mass of the thymus, the mass of the spleen, and the number of endogenous colony forming units in the spleen are significantly higher in the treated animals than in the control irradiated mice. The registered differences in the mass of these organs are statistically significant (p <0.01).

In mice that had ARS, the use of melanin had a beneficial effect on the state of locomotor activity and psychophysiological status. When testing in an “open field” after irradiation at a dose of 6.5 Gy, most of the indicators (the number of crossed squares, the number of rising, peeping in minks) in the treated mice were at a significantly higher level than in the control. The total index was 94.6 ± 6.7 and 52.8 ± 19.8 (p <0.05), respectively, with the physiological norm for this animal species in our experiments being 125.0 ± 8.3. (Table 5)

The therapeutic and prophylactic effect of melanin with fractionated total exposure, which caused in mice clinical manifestations of the lesion characteristic of the bone marrow form of ARS, is illustrated in table 6.

The studies found that in the control group, the survival rate was 43.7%. The therapeutic use of melanin increased survival to 100% (p = 0.002). Weight gain after the end of irradiation in treated animals was noted already on the 7th day, in the control - on the 11th day.

30 days after the end of fractionated exposure, the behavioral activity of the treated mice in the “open field” was also higher than that of the control: 141.5 ± 7.6 and 87.7 ± 44, respectively.

At a lower dose per fraction of 0.5 Gy and a similar irradiation pattern in dynamics, the effect of melanin on hematopoiesis was studied (table 7).

It can be seen from the presented data that during the period of the greatest suppression of hematopoiesis, treatment with melanin softened the changes caused by irradiation, helping to maintain at a higher level (on the 7th day) the number of platelets in the peripheral blood (873 ± 10.6 × 10 ⁹ / l, in the control - 715 ± 46.6 × 10 ⁹ / L; p <0.02), myelocaryocytes in the bone marrow (13.7 ± 2.8 × 10 ⁶ , in the control - 8.1 ± 1.17 × 10 ⁶ ; p <0.1), accelerated the restoration of hematopoiesis on the 14th and 21st days.

An analysis of the totality of the results allows us to conclude that melanin has a therapeutic effect when taken orally after radiation exposure. On 2 models of the bone marrow form of ARS, it was shown that the use of melanin increases survival, reduces the negative impact of radiation on the dynamics of mass, hematopoiesis, and motor activity.

Thus, the proposed method is as follows.

After irradiation, animals are orally administered in liquid form to melanin. Moreover, melanin is used with a water solubility of at least 80% and a concentration of paramagnetic centers of at least 8 · 10 ¹⁷ spin / g, when dissolved in distilled water in an effective concentration. Water with melanin is used in mice as drinking water after a single and fractionated acute exposure that can cause acute radiation sickness. Water with melanin is consumed from the 1st to the 30th day after a single exposure, and during fractionated exposure from 1 day from the start of irradiation and 30 days after the end of irradiation.

Example 1. The therapeutic and prophylactic radioprotective efficacy of melanin is studied in an experiment on 32 outbred mice SD-1, females weighing 28-30 g, which are in vivarium on a normal diet.

As a model of radiation damage, fractionated 10-fold total irradiation of 1 Gy is used daily for 5 days, then after a 2-day break, again in the same mode. Irradiation is carried out with ⁶⁰ Co gamma-quanta at the ROCUS-M installation (average dose rate of 1 Gy / min). Animals are irradiated in groups of 10-16 heads in plastic boxes. After the 1st fraction, the animals are divided into two groups of mass equivalent; one of them receives melanin for free drinking, which is dissolved in distilled water (DV) at the rate of 12.5 mg / 100 ml, the other group receives DV throughout observation period (up to 30 days after the end of irradiation).

The development of ARS is accompanied by a decrease in body weight, decreased motor activity, refusal to feed, loss of neatness, more pronounced in control animals.

An integral assessment of the effect of melanin on the course of ARS is the survival rate. By the 30th day, the use of melanin ensured the survival of all animals (16 of 16), while 7 of 16 survived in the control group.

Thus, the therapeutic effect of melanin was manifested in an increase in survival up to 100% compared with 43.7% of survivors in the control group. The difference between the indicators is statistically significant (p = 0.02).

Example 2. The therapeutic and prophylactic efficacy of melanin is studied on outbred mice of CD-1, females weighing 22-30 g, which are in normal vivarium conditions with free access to water. Mice are exposed to a single total irradiation in a dose of 6.5 Gy on an X-ray biological installation RUST-M1 (RUB RUST-M1): voltage 200 kV, beam current 2 × 2.5 mA, aluminum filter 1.5 mm. The dose rate of 0.85 Gy / min ± 10%.

Animals are irradiated in groups in special plastic containers divided into individual cells.

One group of mice immediately after irradiation and then up to 30 days instead of tap water receives melanin, which is dissolved in the AI at the rate of 12.5 mg / 100 ml. At the same time, control animals consume DV. The experimental and control groups are formed by random sampling from animals of equal mass. Observation is carried out for 30 days, after which the number of surviving mice is recorded in the experiment and control.

The development of ARS is accompanied by a decrease in body weight, decreased motor activity, refusal to feed, loss of neatness, more pronounced in control animals. When ingesting melanin, in the group of treated mice out of 20, 8 survived, in the control group - from 10 - 2.

Thus, the therapeutic effect of melanin in acute short-term exposure was manifested by an increase in survival by 20%.

Example 3. The effect of melanin on a change in body weight - an integral indicator that reflects the general condition of the animals, is studied on 30 outbred mice SD-1, females with an initial weight of 22-30 g, which are in vivarium conditions. Distribute the animals by weight into 2 groups.

Irradiation is carried out by x-rays in a dose of 6.5 Gy at the RUST-M1 installation (RUB RUST-M1): voltage 200 kV, beam current 2 × 2.5 mA, aluminum filter 1.5 mm. The dose rate of 0.85 Gy / min ± 10%. Animals are irradiated in groups of 10-15 animals in special plastic containers. After irradiation, one group receives for drinking a solution of melanin in DV at the rate of 12.5 mg / 100 ml, the other - DV throughout the entire post-radiation observation period for 30 days.

In dynamics 2 times a week, changes in body weight are recorded. Melanin helps to accelerate the growth of body weight and its return to its original level. During the period of 18-21 days in treated mice, the weight indices were 18.9 ± 1.09 and 20.9 ± 1.2 g, in the control - 13.5 ± 1.24 g and 15.0 ± 2.9 g. The difference between the indicators is statistically significant (Student's test, p <0.02-0.05).

Example 4. The effect of melanin on the post-radiation restoration of hematopoiesis is studied on 24 outbred mice - female SD-1 with an initial weight of 22-30 g (8 - irradiated treated, 8 - irradiated control and 8 - non-irradiated, biocontrol), under normal vivarium conditions diet.

To simulate acute radiation damage, X-ray radiation at a dose of 5 Gy is used on the RUST-M1 installation (RUB-RUST-M1): voltage 200 kV, beam current 2 × 2.5 mA, aluminum filter 1.5 mm. The dose rate of 0.85 Gy / min ± 10%. Animals are irradiated in groups of 8 animals in plastic containers. After irradiation, 3 mass randomized groups are formed, one of which ad libitum receives DV with melanin (12.5 mg / 100 ml), and the other two (radiation control and biocontrol) - DV.

On the 8th day after irradiation, animals are killed by cervical dislocation and thymus and spleen mass are determined by standard methods, the number of endogenous colonies detected after 2-hour fixation in Buen’s fluid is counted.

Indicators of organ mass in the treated mice were: thymus - 32 ± 2.9 mg; spleen - 38.9 ± 5.3 mg; in the irradiated control, respectively, 22.1 ± 2.0 mg and 25.8 ± 1.6 mg; in biocontrol - 82.6 ± 14.4 mg and 120.8 ± 26.0 mg. The differences between the indices of the irradiated treatment and control groups are statistically significant (according to the Mann-Whitney test, p <0.01).

The level of endogenous colonies in the spleen of treated animals was 7.4 ± 2.1, which is significantly higher than in the control irradiated group 4.0 ± 0.8.

Example 5. The effect of melanin on the approximate research and total locomotor activity is studied on 40 outbred CD-1, female mice weighing 22-30 g, irradiated in a dose of 6.5 Gy with X-rays on the RUST-M1 (RUBUST-M1): voltage 200 kV, beam current 2 × 2.5 mA, aluminum filter 1.5 mm. The dose rate of 0.85 Gy / min ± 10%. The animals randomized by weight are divided into 3 groups, one of which ad libitum with DV receives melanin (12.5 mg / 100 ml) from 1 day, the other two (radiation control and biocontrol - non-irradiated) - DV.

At the end of the convalescence period (32-34 days), the state of spontaneous motor activity in an “open field” is determined in mice that have undergone ARS, as well as in intact (biocontrol). The mouse is placed in the center of a special arena, the floor of which is divided into sectors. Within 3 minutes, the number of racks, crossed squares, approaches to the center, peeps into openings in the floor and the total number of actions are counted.

In mice treated with melanin, the total score was 94.6 ± 6.7 and was significantly higher than in irradiated control animals — 52.8 ± 19.8, with a physiological norm (biocontrol) of 125 ± 8.3. The differences between the irradiated animals in the treated and control groups are statistically significant (Mann-Whitney test, p <0.05). Statistically significant differences were recorded, including by the number of peeps into “minks” (29.5 ± 1.46 and 16.8 ± 5.3) and crossed squares (51.8 ± 6.0 and 27.8 ± 12.4 )

Thus, melanin increases the survival of animals after a single or fractionated radiation exposure in doses that cause the development of the bone marrow form of ARS. The effect is achieved with a course of therapeutic oral administration with drinking water at the rate of 12.5 mg / 100 ml. Therapeutic efficacy is also clinically registered in the form of maintaining at a higher level of body weight, orientative-motor activity and some indicators of blood formation.

Radiological properties with almost complete harmlessness, a simple and convenient method of application allow the use of melanin for solving problems of radiation medicine, in particular for the treatment of bone marrow form of radiation damage.

Sources of literature

1. Water-soluble plant melanin http // factor.net.ua / melanin / vodorasvorimyj-rastitelnyj-melanin.html. 2011.

2. Ostrovsky M.A., Dontsov A.E. Physiological functions of melanin in the body // Human Physiology. 1985.V.11. Number 4. S.670-678.

3. Zherebin Yu.M., Bondarenko N.A., Makan S.Yu. et al. Pharmacological properties of enomelanin pigments // Doklady AN SSSR. 1984. No. 3. series 5. S.64-67.

4. Borschevskaya M.I., Vasilyeva S.M. The development of ideas about the biochemistry and pharmacology of melanin pigments // Questions honey. chemistry. 1999. V. 45. issue 1. S.13-24.

5. Zherebin Yu.M., Sava V.M., Kolesnik A.A., Bogatsky A.V. Studies of the antioxidant properties of enomelanin // DAN SSSR. 1982. 262. No. 1. S.112-115.

6. Alekseeva T.N., Oreshchenko A.V., Kulikova A.V. et al. Effect of plant melanin pigment on the clastogenic effects of chemical mutagens in mice // J. "Experimental and clinical pharmacology." 2001. No.6. S.51-61.

7. Mosset IB // Radiation and heredity: Genetic aspects of radiation protection. Minsk: University Publishing House. 1990.208 p.

8. Izmestieva O.S., Dubovik B.V., Zhavoronkov L.P. Experimental evaluation of the radioprotective effect of melanin on somatic development during irradiation in the antenatal period of ontogenesis // Radiation Biology. Radioecology. 2007.V. 47. No. 6. S.684-689.

9. Izmestyev O.S., Dubovik B.V., Zhavoronkov L.P. Experimental evaluation of the neuroembryoprotective properties of melanin during irradiation in the antenatal period of development // Radiation Biology. Radioecology. 2007.V. 47. No. 6. S.690-695.

10. El-Obeid A., Al-Harbi S., AL-Jomah N., Hassib A. Herbal melanin modulates tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF ) production // Phytomedicine: international journal of phytotherapy and phytopharmacology. 2006./May; 13 (5). P.324-333.

11. El-Obeid A., Hassib A., Ponten F., Westermark B. Effect of herbal melanin on IL-8: a possible role of Toll - like receptor 4 (TLR4) // Biochemical and Biophysical research communications. 2006. Jun. 16; 344 (4); 1200-6.

12. Oberg F., Hassib A., Ahnfelt M. et. al. Herbal melanin activates TLR 4 / NF-kappa In the signaling pathway // Phytomedicine: international journal of phytotherapy and phytopharmacology. 2009. May; 16 (5). P. 477-484.

13. Grossi G.F., Durante M., Gvalanella G. et al. Effects of melanin on high-LET radiation response of human epithelial cells // Radiation and environmental biophysics. 1998. V.37. P.63-67.

14. Zhdanova N.N. On the protective effect of pigment. The peculiarity of the chemical structure of melanins // Microbiology. 1970.T. XXXXIX. issue 4. S.431-434.

15. Kunwar A., Adhihary B., Jayakumar S. et al. Melanin, a promising radioprotector: Mechanisms of actions in a mice model // Toxicol. Appl. Pharmacol 2012. Oct. fifteen; 264 (2): 202-11.

16. Berdyshev G.D. On the protective effect of melanin upon irradiation of mice // Radiobiology. 1964.V.4. issue 4. S.644-645.

17. Schweitzer A.D., Revskaya E., Chu P. et al. Melanin-coated nanoparticles for protection of bone marrow during radiation therapy of cancer // Int. J. Radiat. oncol. Biol. phys. 2010. V.78. No. 5. P.1494-1502.

18. Revskaya E., Iongco A.M., Celler R.S. et al. Radioimmunotherapy of experimental human metastatic melanoma with melanin - binding antibodies and in combination with decarbazine // Clin. Cancer Res. 2009. V.15. Number 7. P.2373-2379.

19. Mosset I.B., Plotnikova S.I., Lyakh P.P. and others. Radioprotector, which reduces the accumulation of radionuclides in the body. // Application for A.S. 12/06/90. No. 1888566 (116992).

Claims (1)

A method for the prevention and treatment of acute radiation sickness in an experiment, including oral administration of melanin in liquid form to animals after irradiation, characterized in that melanin is used with a water solubility of at least 80% and a concentration of paramagnetic centers of at least 8 · 10 ¹⁷ spin / g, in dissolved form in distilled water in an effective concentration, and water with melanin is used in mice as drinking after a single and fractionated acute exposure, which can cause acute radiation sickness, while water with lanine is used from 1 to 30 days after a single exposure, and during fractionated exposure from 1 day from the start of irradiation and 30 days after the end of irradiation.