Method for treating acute radiation disease

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

 

The invention relates to medicine and veterinary medicine, namely, lactoferrin, and can be used for the treatment of acute radiation sickness.

Lactoferrin (LF) is a component of the milk of mammals was discovered over 50 years ago. He is a natural glycoprotein with a molecular weight ˜80 kDa. LF belongs to the class of iron-containing proteins transferrin and exists in two forms: free and bound with iron ions. The LF molecule contains two active centre fixing iron (Fe3+), which are located on the two functional domains of the protein [1, 2]. LF is present in external secretions of mammals, in the saliva, in granules of polymorphonuclear neutrophils. Blood concentration is 1 μg/ml, increasing in acute inflammation to 20 ág/l, the Highest concentration observed LF in milk animals in the early stage of lactation (7-10 g/l) [1, 2, 3]. In an ongoing study of the characteristics of action of LF in the body, which due to its multifunctional [1]. The most important biological function LF is anti-infectious [4, 5, 6]. LF is included in the scheme of complex treatment of candidiasis with antifungal drugs that significantly increases the effectiveness of their therapeutic applications [7, 8, 9]. Found that LF induces in the body's synthesis of interleukin-18 and interferon-γto the which are anti-inflammatory cytokines [10, 11]. LF strengthens the body's immune defenses [11, 12, 13]. LF has been used successfully for the treatment of asthma [11] and treatment of gastric ulcers [14]. Known application of human LF to stimulate myelopoiesis, restore the content of neutrophils, treatment of anemia, inhibition of inflammation, inhibition of growth of solid tumors and activation of NK-cells [15]. LF used in the treatment of cancer of the stomach, intestines, lungs, bladder, uterus, lymphosarcoma (intravenous, intracavitary injection or local application) [16], to prevent the formation of metastases in adenoma and carcinoma of the lung, bladder, esophagus and rectum [17, 18, 19]. It is also used in the form of applications for the treatment of local inflammatory processes after chemoradiation therapy of tumors of the oropharyngeal area and esophagus [20].

The aim of the present invention is to research and prove that it is possible therapeutic use of LF in radiation damage to the body that cause acute radiation sickness. To achieve this goal we studied the effect of LF on the survival and haematopoiesis irradiated Guinea pigs. In the course of the research showed that LF is able to significantly increase the survival of irradiated animals and weaken the postradiation cytopenia.

In our experiments we used LF, received biotechnologica the Kim method of colostrum mammals (methodology TU/TR 9380-001-424118073-04). LF was saturated by 15-20% iron ions (Fe3+) and dissolved in a biologically combined buffer. The homogeneity of the LF was 95% according to the results of chromatography and polyacrylamide gel electrophoresis. Gel-filtration chromatography did not reveal the presence of protein oligomers in LF.

therapeutic effectiveness of LF was investigated in experiments on 27 Guinea pigs weighing about 350, the Randomization of the control and experimental groups was carried out according to the body mass and the number of peripheral blood leukocytes. Blood counts are consistent with normal values. Animals were irradiated γ-quanta137Cs dose of 2.5 Gy at the "energy spectrum" (dose 2,045 rad/sec). Following exposure of animals developed bone marrow form of acute radiation sickness.

Experimental animals after exposure daily subcutaneously injected with a solution of LF in physiological solution in the 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 untreated control animals received physiological solution of sodium chloride in a volume of 0.2 ml.

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

Statistics is the processing of experimental data was performed using standard methods of variation statistics.

When statistical processing of experimental data was calculated simple averages and their standard errors in all stages of the survey. About the statistical significance of the difference in the performance in some time after exposure in animals compared groups was assessed by student's criterion. To estimate the total effect of LF on the content of lymphocytes during all periods of survey has used characters.

Acute radiation sickness is a secondary immunodeficiency type. Its development is accompanied by a decrease in the number of cells in the bone marrow, peripheral blood. For acute radiation sickness depends on the dose of irradiation. The higher it is, the harder the course of the disease. The use of different preventive and curative medicines reduces the severity of acute radiation sickness and increases the probability of survival of an organism.

In our research it was found that untreated animals of the control group survived 53.8% of cases. Therapeutic use of LF increased the survival of irradiated Guinea pigs up to 85.7% (65 mg/kg) and 100% (300 mg/kg) (table 1).

Table 1

Survival of Guinea pigs exposed γ-quanta137Cs dose of 2.5 Gy and treated with LF
№ p/pLF, mg/kg Of them% survival
survivedPalo
1-137553,8
26576185,7
3300770100,0

In addition to monitoring the clinical course of acute radiation sickness experimental groups of animals were examined and determined the content of lymphocytes and neutrophils in the peripheral blood. The results of haematological examinations of animals are presented in table 2. Table 2 shows that the content of lymphocytes in the peripheral blood of all patients LF groups was higher compared to control untreated animals. The most significant effect recorded during the survey in the midst of cytopenia on the 12th day after irradiation.

Note: 1)*a statistically significant excess compared with the control group by student's criterion p<0,05.
Table 2

The content of lymphocytes and neutrophils in the peripheral blood of Guinea pigs irradiated γ-quanta137Cs dose of 2.5 Gy and treated with LF
IndexLF, mg/kgTime after exposure, day.
0 71216182132
Lymphocytes (×109/l M±m)-5,3±0,41,7±0,11,4±0,15,6±0,56,8±1,14,1±0,53,8±0,2
656,9±0,91,9±0,22,5±0,2*5,9±0,88,0±0,54,7±0,35,2±0,4
3005,2±0,42,2±0,42,5±0,3*6,0±0,77,2±0,74,6±0,65,0±0,8
Neutrophils (×109/l M±m)-3,6±0,20,3±0,10,006±0,0050,8±0,22,9±0,55,4±0,93,3±0,8
653,6±0,40,2±0,10,06±0,0042,0±0,3*5,9±0,6*6,2±0,74,0±0,3
3003,2±0,40,2±0,040,1±0,063,5±0,9*4,6±1,04,1±0,75,2±0,7

At this time, the content of lymphocytes in the treated group was 2.5±0.2 and in the untreated control group to 1.4±0,1 (×109/l). The difference between the indices is statistically significant (student test t=4,7; p<0,01).

The content of lymphocytes in the treated groups at all follow-up period was higher than in the control group of Guinea pigs not treated with LF. The criterion marks suggests that this effect LF is statistically significant (p=0.05).

A pronounced stimulating effect on haematopoiesis was observed when determining the number of neutrophils in the peripheral blood. On the 16th day after irradiation after administration of LF in a daily dose of 65 or 300 µg/kg content of neutrophils was equal to 2.0±0,3 (×109/l) and 3.5±0,9 (×109/l) in treated groups and 0.8±0,2 (×109/l) in the control group. Student test was equal to, respectively, t=of 3.3 and 2.9; p<0,01. At 18 days after irradiation in the group treated animals (65 mg/kg) was also observed excess performance over the control group. In the treated group was 5.9±0,6 (×109/l)in the control group and 2.9±0,5 (×109/l). The marked difference between the indices is statistically significant x is the character (student test t=3,8; p<0,01).

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

Example

Therapeutic efficacy LF study in the experience of 20 Guinea pigs (7 treated and 13 control) weighing about 350 g, located on the customary diet of the vivarium. Randomization of animals to groups is according to the body mass and the number of peripheral blood leukocytes.

For modeling of acute radiation exposure are γ-quanta137Cs dose of 2.5 Gy, the installation "energy spectrum" (dose 2,045 rad/sec). Animals irradiated groups 6 goals in special plastic boxes, separated by partitions 6 (3+3) sections. Radiation effects in animals developing bone marrow form of acute radiation sickness.

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

The development of acute radiation sickness naturally accompanied by a decrease in the content of cells in the peripheral blood. Development is the development of acute radiation sickness in control animals accompanied by loss of body weight, the decrease in motor activity, loss of appetite, theresaknott wool, which loses its Shine and becomes contaminated faeces.

In the group treated animals (LF, 300 μg/kg) of the clinical manifestations of acute radiation is observed only reduce body weight. Other symptoms are absent. The animals are active, neat, eat food in the usual quantities.

Most marked reduction of cells in the peripheral blood of Guinea pigs is celebrated on the 12th day of radiation sickness. At this time, the content of lymphocytes in the peripheral blood of immunized LF animals higher compared to control untreated animals. The content of lymphocytes in the treated group is 2.5±0.2 and in the control group to 1.4±0,1 (×109/l). The difference between the indices is statistically significant (student test t=4,9; p<0,01).

The number of neutrophils in the peripheral blood on the 16th day after irradiation is equal to 3.5±0,9 (×109/l) in the treated group and 0.8±0,2 (×109/l) in the control group. Student test t=2,9; p<0,01.

An integrated assessment of the impact of LF on the course of acute radiation disease in Guinea pigs is determining their survival for 30 days after irradiation. The reduction in the severity of clinical symptoms of acute radiation sickness and less severe cytopenia caused increased compared with the control group is (53.8%) of survival of treated animals (100%).

Thus, LF enhances survival of irradiated animals and reduces severity of postradiation cytopenia. This gives grounds to consider it effective in the treatment of acute radiation sickness.

Sources of information

1. Konysheva YEAR, Buneva C. N., Premium GA // Biochemistry, 2001, CH, No. 1, Pp.5-13.

2. Brigens Y. // Scand. J. Haematolog., 1985, V.34, No. 4, P.326-331.

3. Shimazaki, K., Tazume T., Tanaka M. // Journal of Dairy Science, V.81, No. 11, P.2841-2849.

4. Ott E, Dikareva SV, Melnikov O.R. // Matters. nutrition, 1993, No. 1, P.6-13.

5. Arnoid R, Brever M., Gauthier J. // Infect. Immun., 1980, V.28, No. 8, P.893-898.

6. Ajello M., Greco R., Doimaramma G. // Igiene Moderna, 1998, V.110, no. 6, P.671-682.

7. Hanson L., Lonnroth I., Bjersing J. // Nutrition Reviews, 2000, V.58, (2, Part 2), P.931-937.

8. Ikeda M., Nozaki, A. Sgiyama K. // Virus Research, 2000, V.66, No.1, P.51-53.

9. Ikeda M, Sugiyama K., Tonaka So // Biochem. Biophys. Res. Com., 1998, V.245, P.549-553.

10. Debbabi H., M. Dubarry, Rautureau, M. // Journal of Dairy Research, 1998, V.65, No. 2, P.283-293.

11. Lygren Century, Sveir N., Hjeltes Century // Fish and Shellfish Immunology, 1999, V.9, 2, P.95-107.

12. Kakuka J., Kurokura H. // Fish Pathology, 1995, V.30, №2, P.289-290.

13. Miyauchi, N., Kaino A., Shinoda I. // Journal of Dairy Science, 1997, V.80, No. 10, P.2330-2339.

14. Miyauchi, N., Hashimoto, S., Nakajima V. // Cellular Immunology, 1998, V.187, No. 1, P.34-37.

15. Nuioens J., van Berkel N. Useful properties of human laktoferrin and variants thereof. U.S. patent No. 6333311. December 25, 2001.

16. Chissov V.I., Jakubowski R.I., A.V. Boiko, Nemtsov ER, Sergeeva T., Osipova ON. Antibacterial, antioxidant, immunomodulatory and anticarcinogenic drug and a method for its use. RF patent №2165769, 2001

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20. Chissov V.I., Borisov V., Jakubowski R.I., A.V. Boiko, Nemtsov ER, Demidov L.V., Sergeeva T., Teleus T.A. drug for the treatment of local complications of conservative anti-tumor treatment in oropharyngeal zone and a method of treatment of complications of conservative anti-tumor therapy in oropharyngeal area. RF patent №2099065, 1997

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



 

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