RU2294755C1 - Method for treating acute radiation disease - Google Patents

Abstract

FIELD: medicine, radiation medicine.

SUBSTANCE: it has been suggested to apply lactoferrin as a curative preparation in case of acute radiation disease. Lactoferrin should be applied in the experiment at reproducing madullary form of acute radiation disease. Subcutaneous injection of lactoferrin in physiological solution at the dosage of 300 mcg/kg is accompanied with statistically significant increase of lymphocytes and neutrophils in peripheral blood that provides 100%-survival rate of irradiated animals in experimental group.

EFFECT: higher efficiency of therapy.

1 ex, 2 tbl

Description

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

Lactoferrin (LF) - a component of mammalian milk was discovered more than 50 years ago. It is a natural glycoprotein with a molecular weight of ~ 80 kDa. LF belongs to the class of iron-containing transferrin proteins and exists in two forms: free and associated with iron ions. The LF molecule contains two active centers fixing iron (Fe ³⁺ ), which are located on two functional domains of the protein [1, 2]. LF is present in the outer secrets of mammals, in saliva, in granules of polymorphic nuclear neutrophils. In the blood, its concentration is 1 μg / ml, increasing in acute inflammation to 20 μg / L. The highest concentration of LF is observed in the milk of animals at an early stage of lactation (up to 7-10 g / liter) [1, 2, 3]. Currently, the study of the features of the action of LF in the body is ongoing, which is explained by its multifunctionality [1]. The most important biological function of LF is anti-infection [4, 5, 6]. LF is part of the complex treatment of candidiasis with antifungal drugs, which significantly increases the effectiveness of their therapeutic use [7, 8, 9]. It was found that LF induces in the body the synthesis of interleukin-18 and interferon-γ, which are anti-inflammatory cytokines [10, 11]. LF enhances the body's immune defense [11, 12, 13]. LF is successfully used to treat bronchial asthma [11] and treat gastric ulcers [14]. The use of human LF is known to stimulate myelopoiesis, restore neutrophil content, treat anemia, suppress inflammation, suppress the growth of solid tumors and activate NK killers [15]. LF is used in the treatment of cancer of the stomach, intestines, lungs, bladder, uterus, lymphosarcoma (intravenous, intracavitary injections or local applications) [16], to prevent the formation of metastases in adenoma and carcinoma of the lungs, bladder, esophagus and rectum [17 , 18, 19]. It is also used in the form of applications for the treatment of local inflammatory processes after chemoradiotherapy of tumors of the oropharyngeal zone and esophagus [20].

The aim of the present invention is the study and evidence of the possibility of therapeutic use of LF in radiation damage to the body that causes acute radiation sickness. To achieve this goal, the effect of LF on the survival and hematopoiesis of irradiated guinea pigs was studied. In the course of studies, it was found that LF is able to significantly increase the survival of irradiated animals and weaken post-radiation cytopenia.

In the experiments, LF obtained by the biotechnological method from colostrum of mammals was used (method TU / TR 9380-001-424118073-04). LF was saturated with 15–20% iron ions (Fe ³⁺ ) and dissolved in a biologically combined buffer. LF homogeneity was 95% judging by the results of chromatography and polyacrylamide gel electrophoresis. Gel filtration chromatography did not reveal the presence of protein oligomers in LF.

The therapeutic efficacy of LF was studied in experiments on 27 guinea pigs weighing about 350 g. The control and experimental groups were randomized according to body weight and the number of peripheral blood leukocytes. Blood counts corresponded to normal values. The animals were irradiated with ¹³⁷ Cs γ-rays at a dose of 2.5 Gy on the IGUR facility (dose rate 2.045 rad / s). As a result of irradiation in animals, a bone marrow form of acute radiation sickness developed.

After irradiation, the experimental animals were injected subcutaneously daily with a solution of LF in physiological saline in an amount of 65 or 300 μg / kg, in a volume of 0.2 ml on the 1st - 14th day of radiation sickness. At the same time, the control untreated animals received a physiological solution of sodium chloride in a volume of 0.2 ml.

The effect of LF on the status of irradiated animals was evaluated by their survival for 30 days after irradiation, as well as by the number of lymphocytes and neutrophils in the peripheral blood. The examination was carried out 3-4 days before and on the 7th, 12th, 16th, 18th, 21st and 32nd days after irradiation.

Statistical processing of the obtained experimental data was carried out using standard methods of variation statistics.

In the statistical processing of experimental data, the arithmetic mean values and their standard errors were calculated for all periods of the survey. The statistical significance of the difference in indicators at certain times after irradiation in animals of the compared groups was judged by Student's criterion. To evaluate the total effect of LF on the content of lymphocytes at all stages of the examination, the sign criterion was used.

Acute radiation sickness is a secondary type of immunodeficiency. Its development is accompanied by a decrease in the number of cells in the bone marrow, in peripheral blood. The course of acute radiation sickness depends on the dose. The higher it is, the more severe the course of the disease. The use of various therapeutic and prophylactic agents reduces the severity of acute radiation sickness and increases the likelihood of survival of the body.

In our studies, it was found that untreated animals of the control group survived in 53.8% of cases. The therapeutic use of LF increased the survival of irradiated guinea pigs to 85.7% (65 μg / kg) and 100% (300 μg / kg) (Table 1).

Table 1
Survival of guinea pigs irradiated with γ-quanta of ¹³⁷ Cs at a dose of 2.5 Gy and treated with LF No. p / p LF, mcg / kg The number of pigs in the group Of them % survival survived fell one - 13 7 5 53.8 2 65 7 6 one 85.7 3 300 7 7 0 100.0

In addition to monitoring the clinical course of acute radiation sickness in the experimental groups of animals, we examined and determined the content of lymphocytes and neutrophils in the peripheral blood. The results of hematological examination of animals are presented in table 2. From table 2 it is seen that the content of lymphocytes in the peripheral blood of all treated LF groups was higher compared to control untreated animals. The most significant effect was recorded during examination in the midst of cytopenia on the 12th day after irradiation.

table 2
The content of lymphocytes and neutrophils in the peripheral blood of guinea pigs irradiated with γ-rays of ¹³⁷ Cs at a dose of 2.5 Gy and treated LF Indicator LF, mcg / kg Time after irradiation, days 0 7 12 16 eighteen 21 32 Lymphocytes (× 10 ⁹ / L, M ± m) - 5.3 ± 0.4 1.7 ± 0.1 1.4 ± 0.1 5.6 ± 0.5 6.8 ± 1.1 4.1 ± 0.5 3.8 ± 0.2 65 6.9 ± 0.9 1.9 ± 0.2 2.5 ± 0.2 ^* 5.9 ± 0.8 8.0 ± 0.5 4.7 ± 0.3 5.2 ± 0.4 300 5.2 ± 0.4 2.2 ± 0.4 2.5 ± 0.3 ^* 6.0 ± 0.7 7.2 ± 0.7 4.6 ± 0.6 5.0 ± 0.8 Neutrophils (× 10 ⁹ / L M ± m) - 3.6 ± 0.2 0.3 ± 0.1 0.006 ± 0.005 0.8 ± 0.2 2.9 ± 0.5 5.4 ± 0.9 3.3 ± 0.8 65 3.6 ± 0.4 0.2 ± 0.1 0.06 ± 0.004 2.0 ± 0.3 ^* 5.9 ± 0.6 ^* 6.2 ± 0.7 4.0 ± 0.3 300 3.2 ± 0.4 0.2 ± 0.04 0.1 ± 0.06 3.5 ± 0.9 ^* 4.6 ± 1.0 4.1 ± 0.7 5.2 ± 0.7 Note: 1) ^* statistically significant excess of indicators compared with the control group by student criterion p <0.05.

At this time, the lymphocyte content in the treated group was 2.5 ± 0.2, and in the untreated control group, 1.4 ± 0.1 (× 10 ⁹ / L). The difference between the indicators is statistically significant (Student's test t = 4.7; p <0.01).

The content of lymphocytes in the treated groups at all observation periods was higher than in the control group of guinea pigs not receiving LF. The sign criterion indicates that this LF effect is statistically significant (p = 0.05).

A pronounced stimulating effect on hematopoiesis was noted in determining the number of neutrophils in peripheral blood. On the 16th day after irradiation after the administration of LF in a daily dose of 65 or 300 μg / kg, the neutrophil content was 2.0 ± 0.3 (× 10 ⁹ / L) and 3.5 ± 0.9 (× 10 ⁹ / l) in the treated groups and 0.8 ± 0.2 (× 10 ⁹ / l) in the control group. Student's criterion was equal to t = 3.3 and 2.9, respectively; p <0.01. On the 18th day after irradiation in the group of treated animals (65 μg / kg), an increase was also observed over the control group. In the treated group - 5.9 ± 0.6 (× 10 ⁹ / L), in the control group - 2.9 ± 0.5 (× 10 ⁹ / L). The marked difference between the indicators is statistically significant (Student's t test = 3.8; p <0.01).

An analysis of the obtained data allows us to conclude that the treatment of acute radiation sickness of LF increases the survival of animals and reduces the severity of post-radiation cytopenia judging by the content of lymphocytes and neutrophils in the peripheral blood.

Example

The therapeutic efficacy of LF is studied in an experiment on 20 guinea pigs (7 treated and 13 control) weighing about 350 g on the usual diet of vivarium. The animals are randomized into groups according to body weight and the number of peripheral blood leukocytes.

To simulate acute radiation exposure, ¹³⁷ Cs γ-quanta in a dose of 2.5 Gy are used, the IGUR installation (dose rate 2.045 rad / s). Animals are irradiated in groups of 6 animals in special plastic boxes divided by partitions into 6 (3 + 3) sections. As a result of radiation exposure in animals, a bone marrow form of acute radiation sickness develops.

After irradiation, the experimental animals are daily injected subcutaneously with a solution of LF in physiological saline in an amount of 300 μg / kg, in a volume of 0.2 ml on the 1st - 14th day of radiation sickness. At the same time, control untreated animals receive a physiological solution of sodium chloride in a volume of 0.2 ml.

The development of acute radiation sickness is naturally accompanied by a decrease in the content of cells in peripheral blood. The development of acute radiation sickness in control animals is accompanied by a decrease in body weight, decreased motor activity, refusal to feed, ruffled wool, which loses its luster and is contaminated with feces.

In the group of treated animals (LF, 300 μg / kg) of the clinical manifestations of acute radiation, only a decrease in body weight is noted. No other symptoms. Animals are active, neat, eat food in normal quantities.

The most pronounced decrease in cells in the peripheral blood of guinea pigs is observed on the 12th day of radiation sickness. At this time, the content of lymphocytes in the peripheral blood of treated LF animals is higher compared to control untreated animals. The content of lymphocytes in the treated group is 2.5 ± 0.2, and in the control group, 1.4 ± 0.1 (× 10 ⁹ / L). The difference between the indicators is statistically significant (Student's criterion t = 4.9; p <0.01).

The number of neutrophils in the peripheral blood on the 16th day after irradiation is 3.5 ± 0.9 (× 10 ⁹ / L) in the treated group and 0.8 ± 0.2 (× 10 ⁹ / L) in the control group. Student's criterion t = 2.9; p <0.01.

An integral assessment of the effect of LF on the course of acute radiation sickness in guinea pigs is to determine their survival for 30 days after irradiation. A decrease in the severity of the clinical symptoms of acute radiation sickness and less pronounced cytopenia led to an increased survival of the treated animals (100%) compared with the control group (53.8%).

Thus, LF increases the survival of irradiated animals and reduces the severity of post-radiation cytopenia. This gives grounds to consider it an effective treatment for acute radiation sickness.

Information sources

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Claims (1)

The use of lactoferrin as a therapeutic agent in acute radiation sickness.