Method of applying vegetable polysaccharide as radioprotector and stimulator of colony formation of spleen stem cells in irradiated animals

FIELD: medicine.

SUBSTANCE: invention relates to radiation and experimental biology, biotechnology and medicine, namely to means of radiological protection and to stimulators of colony formation of stem cells. Invention can be used in research and clinical practice for protection of living organisms in case of exposure to ionising radiation, in emergency and military medicine, in case of emergency situations as means for protection against radiation injury. Claimed is application of preparation gamma-plant as radioprotector with colony-stimulating properties for increasing survival and improving state of organisms, subjected to irradiation. Gamma-plant is non-toxic polysaccharide of vegetable origin, registered by Ministry of Health of the Russian Federation and allowed for medical application; Gamma-plant is included into Russian Pharmacopoeia in section FG-9 "non-narcotic analgesics".

EFFECT: invention makes it possible to increase efficiency of colony formation of stem cells in spleen 2,0-2,3 times, increases survival of experimental animals to 95% with coefficient of dose reduction factor DRF, equal 2-2,2.

2 tbl

 

The invention relates to radiation and experimental biology, biotechnology and medicine, in particular to the means of radiotoxic and stimulants colony of stem cells can be used in research and clinical practice for the protection of living organisms when exposed to ionizing radiation, emergency and military medicine, in emergency situations as a means of protection from radiation damage.

The invention consists in the use of plant polysaccharide gamma-plant (Czekanowska L. A. Polysaccharide gamma-plant (γ pl), having anti-infective activity and not having hemaglutinin action. EN 2028303 C1) as a radioprotector and stimulant colonies of stem cells in the spleens of irradiated animals.

Currently there is an acute problem of protection from radiation injuries. To mitigate exposure use radioprotectors. To radioprotectors are substances that are capable for prophylactic use to have a protective effect, resulting in the preservation of life is irradiated to the living body or the weakening of the severity of radiation injury with rollover state capacity and lifetime (Century. And. Legeza, V., Vladimirov. A new classification of preventive radioprotection means. the radiation biology. Radioecology, T. 38, vol.3, page 416, 1998). Basic requirements radioprotectors, are that they should be quite effective and do not cause adverse reactions. Known radioprotectors in full these requirements are not responsible and the selection of biologically active substances with radiomodification activity is limited.

Known radioprotectors - hamatos and tsistamin included in military first aid kit AI-1,2,3 Thu, 4 with FOOD (to reduce the absorbed dose) of 1.6 and 1.15, respectively. (Swerdlow and collaborators 1974); They are radioprotectors short-term actions are considered to be the most effective. The disadvantage is widely used in the practice of the radioprotector of applied Kuna P. Chemical radiocasete. TRANS. with the construction. M.: Medicine, 1989, S. 61-75) is that its effect is observed at doses that are close to toxic. The use of these doses is in laboratory animals acute side effects: low blood pressure, bradycardia, decreased cardiac output with disturbance of blood supply to tissues. Have nausea, vomiting, defecation, skeletal muscle cramps, death through paralysis of the respiratory center.

Other known radioprotectors - biogenic amines have FOOD to 1.3-1.5, also have a number of serious side action is s: cause a spasm of the blood vessels and circulatory changes in the blood supply in radiosensitive organs and tissues (C. A. Kutsenko. Military toxicology, radiobiology and health protection. SPb: LLC "Publishing house" FOLIO", 2004). The use of these drugs is limited due to the difficulty to establish the optimal dose, time and route of administration.

Common deficiencies the most effective of the known radioprotectors should also be noted that their radioprotective properties, mainly manifested when used before exposure. In extreme situations, in particular in case of accidents at nuclear installations, the use of nuclear weapons by the enemy, when to enter the drug should after exposure, the protective effect is significantly reduced (Shashkov B. C. Anashkin O. D. Suvorov, N. N. Manaeva I. A. the Effectiveness of serotonin, maxamine, AET and applied with the re-introduction after gamma irradiation. // Radiobiology, 1971, T. XI, vol.4, S. 621-623).

There is a method of colony-stimulating stem cells in the spleen after exposure to drugs of IL-1a and IL-1b in a dose of 1 μg/animal after 1 or 3 h after radiation exposure [Chigarev N. G. Interleukin-1b as a means early treatment of acute radiation sickness / N. G. Chigarev, A. F. Myasoedov, N. In. Petkevich, etc. // Fundamental and applied problems of biotechnology and medicine: abstracts. Dokl. nauch.-technology. proc. - SPb., 2000. - S. 31]. It is shown that preparations of IL-1 had a positive effect on the blood, which then manifested itself in the increase of the content of formed elements in the peripheral blood, the increase in the number of myelokaryocytes in the bone marrow, increasing the number of colony forming units in the spleen [Legeza Century. And. cytokines as a means early pathogenetic therapy of radiation injuries. Efficacy and mechanism of action / Century And. Legeza, N. G. Chigarev, Y. A. Abdul, I. W. Galeev // Radiat. biology. Radioecology. - 2000. - So 40, No. 4. - S. 420-424]. In the literature a large number of papers indicate that the most promising drug for early treatment of radiation injuries in Russia is recombinant IL-1β (betalain). Preparation betalain, which dosage form of recombinant IL-1β person authorized by order of the Ministry of health of the Russian Federation No. 51 from 18.02.1997, to clinical application as an immunostimulant and as a means of recovery of bone marrow hematopoiesis in cancer patients after intensive courses of chemo - and radiotherapy (registration number 97/51/6). The disadvantage of this method is the use of pharmacological drug has certain contraindications for use. IL-1 is a proinflammatory cytokine secreted by the cells of the body, and its level in the blood has a fairly broad meaning, which cannot be considered in emergency situations. Errors in establishing the optimal dose, time and route of administration lead to severe side effects - immune disorders the regulation, stimulation reaction, inflammation, high cytotoxicity; application causes fever, sweating, headache. A strong increase in the level of IL-1 leads to hypotension, anorexia, destruction of cartilage in joints. IL-1 is involved in regulation of body temperature, increased its production leads to the development of fever. In addition, there are a number of diseases, the pathogenesis of which has increased levels of IL-1 and application of therapy cytokines are contraindicated.

The purpose of this invention is to develop a method that allows the use of polysaccharide of plant origin of gamma-plant for radiation protection, ensuring a higher survival of living organisms before and after exposure to radiation damage, which will give the opportunity not only to expand your Arsenal of radioprotective substances, but also to find the most effective connection with no toxicity.

The problem is solved in that the detected new radioprotective properties of the drug gamma-plant, natural non-toxic polysaccharide (drug Gamma-plant" was registered by the Russian Ministry of health and approved for medical use and industrial production. Registration certificate of Ministry of health of the Russian Federation No. 99/47/12 February 10, 1999 Registration certificate of Ministry of health of the Russian Federation No. 99/47/1 February 10, 1999, In the Case lcars the governmental funds of Russia "Gamma-plant" is listed in the section FG-9 "analgesics" on page 386 edition 2001), tested dose and the way of its introduction, before and after irradiation of animals on a background of reception of stimulation colonies of stem cells in irradiated animals. When this is achieved: high value FOOD 2-2,2, stimulation of growth of colonies of stem cells in 2-2,3 times compared to non-irradiated animals and increase the survival rate of 95%.

Gamma-plant is a glycoprotein containing up to 95% of carbohydrates and not less than 0.5% protein with high molecular weight isolated from plant cells. Dosage form gamma-Planta presented in ampoules in a 0.01% solution for injections. Latitude therapeutic range gamma-Planta more than 1000.

To perform the tasks carried out an impact assessment of gamma-Planta on the survival of irradiated animals - males line F1(CBA×S57B1/6) weighing 20-25 g of the Compound was administered intravenously in doses of 1, 10, 100 γ/animal. Radioprotective activity of the compounds was studied at a dose of 800 rad (LD80/30). Irradiation of animals produced at facilities IGUR. The control were only irradiated animals. Within 30 days were observed for General condition and behavior of animals. Control animals differed low activity, poor appetite, loss of hair, was also observed dyspeptic syndrome. Animals with the introduction of the gamma-Planta showed great is th activity, normal appetite and no loss of hair.

The evaluation of the preventive radioprotective properties of gamma-Planta experimental animals were injected intravenously 1 hour before radiation exposure dose of 800 rad (dose 158 rad/min, the exposure time 5 min 4 sec).

To evaluate therapeutic radioprotective properties of gamma-Planta experimental animals were subjected to radiation at a dose of 800 rad (dose 158 rad/min, the exposure time 5 min 4 sec), after which he entered the gamma-plant intravenously through 1.6 and 24 hours.

The results are shown in table 1.

100
Table 1
The results of the experiment for the evaluation of the radioprotective activity of gamma-Planta in mice by intravenous method of administration in the conditions of acute exposure.
TimeThe dose of the drug, γ/animalThe number of animalsSurvived
Abs.%
1 hour1201365
10201680
100201260
After 1 hour1201680
10201680
100161487,5
After 6 hours1201995
10191785
100191684,2
After 24 hours1201680
10201680
201680
At this dose, the survival rate of mice in the control groups was equal to 20%.

The results indicate that under these conditions the compound gamma-plant has radioprotective activity. Studied doses of 1, 10 and 100 μg/animal have the same activity, which further increases therapeutic latitude. Draws the attention of high therapeutic radioprotective effect of gamma-Planta during the day - this is a very important quality of the connection, which gives the ability to provide emergency assistance in almost any point of the globe. The use of gamma Planta as a radioprotector provides high radioprotective effect, it was hypothesized that this effect is based on the stimulation of growth of colonies of hematopoietic stem cells.

Gamma-plant prepared from a source of sterile solution containing 100 µg of dry polysaccharide in 1 ml of physiological solution. In the experiments used a solution with a concentration of 10 μg/0.2 ml, which was prepared immediately before the experience after dilution of the initial solution of sterile saline. Was administered once.

To assess stimulating action of polysaccharide schemes the-plant on kolonialapologie hematopoietic precursor cells in the spleen before or after irradiation experimental studies were performed on mice-hybrids F 1(CBA×S57In1/6), males aged 3 months, weighing 20-22 g

The evaluation of the preventive effects of gamma Planta experimental animals for 1 hour and 3 hours before irradiation was injected intravenously 10 µg/animal polysaccharide. All animals were subjected to gamma irradiation (source of Co60) at a dose of 6 Gy on the device "Ray" (dose - 52,63 with Gr/min, irradiation time - 11 min 24 sec).

To determine therapeutic effects of gamma Planta animals were first subjected to gamma irradiation (source of Co60) at a dose of 6 Gy on the device "Ray" (dose - 52,63 cGy/min, irradiation time - 11 min 24 sec), and then experimental animals after 1 hour and 24 hours after irradiation was injected intravenously 10 µg/animal polysaccharide.

All experimental groups of animals were compared with control group of animals, in which mice were injected with saline under the scheme the introduction of the polysaccharide. All animals were subjected to gamma irradiation (source of Co60) at a dose of 6 Gy on the device "Ray" (dose - 52,63 cGy/min, irradiation time - 11 min 24 sec).

On the eighth day after exposure, all animals were euthanized by injection of a solution of Nembutal from a rate of 60 mg/kg body weight, were extracted from the spleen, which was weighed and placed for fixation in freshly prepared solution of acetic acid alcohol (a mixture of 3 parts 95% ethyl sleep the TA and 1 part glacial acetic acid). After 2 hours of fixation of the spleen were transferred to 70% ethanol, and were counted formed splenic colonies on the surface of spleens with a diameter exceeding 0.2 mm, the Average number of formed colonies and standard error (M±M) was calculated on the basis of guidelines prepared by the Ministry of health of the USSR assessment in radiobiological experiments survival of CFU-S (colony forming units splenic), 1975

Criterion stimulating action of polysaccharide gamma-plant was the formation of colonies in the spleens of irradiated animals. The number of colonies is proportional to the number of surviving precursor cells.

The results are shown in table 2.

Table 2
The results of the experiment for the evaluation of the radioprotective activity of gamma-Planta (SE) on the test of survival of hematopoietic stem cells that form the splenic colonies (CFU-C), 8 days after irradiation of mice hybrids F1(CBA×C57B1/6)
no groupGroupWeight of spleen (mg M±MThe number of CFU-C in the group, M±M
1. Control, irradiation of 6 Gy31,5±1,50,8±0,1
2.Introduction SE for 3 h to 6 Gr32,1±0,51,6±0,2∗
3.The introduction of a GP for 1 h to 6 Gr33,8±1,81,9±0,4∗
4.Introduction SE after 1 h after 6 Gy34,7±1,41,9±0,3∗
5.The introduction of the GP within 24 h after 6 Gy33,0±1,31,6±0,3∗
∗ is statistically significant (p<0.05) difference with the index of the control group by the number of CFU-S.

The table shows that all the intervals of polysaccharide gamma-plant are stimulants hematopoietic stem cells.

A single injection to mice of the polysaccharide before irradiation increases the number of colonies in 2-2,3 times, with the greatest preventive effect was observed in the group in which gamma-plant was administered one hour before irradiation.

A single injection to mice of the polysaccharide after irradiation also increases the number is about colonies in 2-2,3 times, with the greatest therapeutic effect was observed in the group in which gamma-plant was administered one hour after exposure.

Examples of specific performance:

Example No. 1.

Studies were performed on mice-hybrids F1(CBA×C57B1/6), males aged 3 months, weighing 20-22 g Were used 5 groups of mice containing 12 animals in each group.

1st group (control) animals were injected with saline.

2nd group - mice were administered 10 μg of gamma-Planta in a volume of 0.2 ml for 3 hours before irradiation.

3rd group - mice were administered 10 μg of gamma-Planta in a volume of 0.2 ml for 1 hour before irradiation.

4-I group - mice were administered 10 μg of gamma-Planta in a volume of 0.2 ml 1 hour after irradiation.

5th group - mice were administered 10 μg of gamma-Planta in a volume of 0.2 ml 24 hours after exposure.

Gamma-plant immediately before the experience bred from the original sterile solution containing 100 µg of dry polysaccharide in 1 ml of physiological solution, to a solution with a concentration of 10 μg/0.2 ml sterile saline and administered to mice once intravenously into the tail vein or subcutaneously.

Then the animals are subjected to gamma irradiation (source of Co60) at a dose of 6 Gy on the device "Ray" (dose - 52,63 cGy/min, irradiation time - 11 min 24 sec).

On the eighth day after irradiation anyone alive who Tim was performed euthanasia by injection of a solution of Nembutal from a rate of 60 mg/kg body weight, were extracted from the spleen, which was weighed and placed for fixation in freshly prepared solution of acetic acid alcohol (a mixture of 3 parts 95% ethyl alcohol and 1 part glacial acetic acid). After 2 hours of fixation of the spleen were transferred to 70% ethanol, and were counted formed splenic colonies on the surface of spleens with a diameter exceeding 0.2 mm

The results of the experiment are presented in table 2.

The table shows that the introduction of polysaccharide gamma-plant increases the number of endogenous colonies in the spleen of 2.0-2.3 times.

The observed increase in the number of colonies in the spleens of irradiated animals is associated with an increase in the number of survivors of hematopoietic precursor cells. This indicates the presence of the investigational product's ability to be not only therapeutic, but also radioprotective action on stem cells from ionizing radiation.

Example # 2

Studies were performed on mice-hybrids F1(CBA×C57B1/6), weighing 20-25 g Were used in 13 groups of mice containing 10 animals in each group.

1st group (control) animals were injected with saline.

2nd group - mice was injected intravenously at 1 mg gamma-Planta in a volume of 0.2 ml for 1 hour before irradiation.

3rd group - mice intravenously injected p is 10 µg gamma-Planta in a volume of 0.2 ml for 1 hour before irradiation.

4th group of mice was injected intravenously at 100 µg gamma-Planta in a volume of 0.2 ml for 1 hour before irradiation.

5th group of mice was injected intravenously at 1 mg gamma-Planta in a volume of 0.2 ml after 1 hour after irradiation.

6th group of mice was injected intravenously at 10 mg gamma-Planta in a volume of 0.2 ml after 1 hour after irradiation.

7th band - mice intravenously injected 100 μg gamma-Planta in a volume of 0.2 ml after 1 hour after irradiation.

8th group of mice was injected intravenously at 1 mg gamma-Planta in a volume of 0.2 ml after 6 hours after exposure.

9th group of mice was injected intravenously at 10 mg gamma-Planta in a volume of 0.2 ml after 6 hours after exposure.

10-th group of mice was injected intravenously at 100 µg gamma-Planta in a volume of 0.2 ml after 6 hours after exposure.

the 11th group of mice was injected intravenously at 1 mg gamma-Planta in a volume of 0.2 ml 24 hours after exposure.

the 12th group of mice was injected intravenously at 10 mg gamma-Planta in a volume of 0.2 ml 24 hours after exposure.

13th group of mice was injected intravenously at 100 µg gamma-Planta in a volume of 0.2 ml 24 hours after exposure.

Gamma-plant immediately before the experience bred from the original sterile solution containing 500 μg of dry polysaccharide in 1 ml sterile saline to concentrations of 1 and 10 μg/0.2 ml sterile physio is ogicheskom solution. The original solution was a concentration of 100 µg/0,2 ml

The resulting solutions were injected into mice once intravenously into the tail vein.

Then animals were irradiated at facilities IGUR at a dose of 800 rad (dose 158 rad/min, the exposure time 5 min 4 sec)).

Within 30 days were observed for General condition and behavior of animals, and then evaluated the survival of animals of all groups.

The results are shown in table 1.

The introduction of this substance as a radioprotector for the needs of nuclear power plant workers, rescue workers, medical workers and the military will allow to raise efficiency of their work in case of emergencies or when interacting with radioisotopes, providing more than double the value of FOOD in the absence of negative side effects, including toxic.

The use of the drug gamma-plant as a radioprotector with distinct colony-stimulating properties to increase survival and improve the condition of the organisms exposed.



 

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1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical industry, in particular to a medication, possessing a nephroprotective action. The medication with the nephroprotective action, contains an extract of grass with roots and rhizomes of slender-leaf iris (Iris tenuifolia), stabilising agents and preservatives, taken in a certain ratio, where the extract is obtained by extraction of the grass with roots and rhizomes of slender-leaf iris (Iris tenuifolia) with a 30-70% water solution of polyol.

EFFECT: medication possesses an effective nephroprotective action.

3 cl, 3 dwg, 7 tbl, 14 ex.

FIELD: veterinary science.

SUBSTANCE: preparation of local therapy contains pine resin, chlorophyll carotin paste, birch oil, castor oil in the following proportions: pine resin: chlorophyll carotin paste: birch oil: castor oil - 10:5:5:80.

EFFECT: using the declared preparation enables the higher therapeutic effect as compared to the existing agents for treating animal skin diseases, including dermatitis.

1 ex

FIELD: biotechnology.

SUBSTANCE: invention is a composition having antibacterial, immunostimulating, anti-allergic and anti-inflammatory action, containing bacterial waste products useful for human body, in the form of exometabolites and fermentolysis products, characterised in that it is a culture medium of lactic acid bacteria, containing laxarane in an amount of 5-10 g/ml, caseicyne, isracydine or their mixture and lectins in an amount of 0.05-2.5 mol/l, histamine in an amount of 0.8-2.0 mmol/l and monocarboxylic fatty acid with an unbranched chain, namely, acetic acid, propionic acid, butyric acid and valeric acid - in an amount of 10-20 mg/ml.

EFFECT: expanding the range of agents having complementary antibacterial, immunomodulating, anti-allergic and anti-inflammatory action.

4 cl, 5 ex

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