RU2320354C2 - Method for obtaining hemopoiesis-affecting immunostimulating thymic polypeptides - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: the present innovation deals with obtaining immunostimulating preparations out of raw material of animal origin indicated to enhance hemopoiesis. Raw material should be homogenized, tissue particles should be deposited due to centrifuging, supernatant obtained should be heated at 80°C for 20 min, the residue should be separated due to centrifuging, supernatant should be filtered, chilled acetone should be added to the filtrate at the ratio of 1:5, precipitated residue should be washed with acetone to be dried, the obtained powder should be dissolved in 0.01 M sodium-phosphate buffer solution pH 7.0 at 1:10 ratio, to be centrifuged. It is necessary to achieve supernatant pH to be 4.0 to be mixed then by adding crystalline ammonium sulfate up to concentration of 23-28.6%, carry out ion-exchange chromatography upon carboxymethyl sephadex S-25, eluate polypeptides with the help of linear gradient of concentration and pH sodium-acetate buffer from 0.01 M, pH 4.0-5.0 up to 2 M, pH 7.0, lyophilize the obtained polypeptides to fulfill desalinization upon a column with sephadex G-25. The innovation enables to isolate hemopoiesis-stimulating polypeptides of high biological activity and apply them for obtaining medicinal preparations to treat anemia.

EFFECT: higher efficiency.

7 ex, 20 tbl

Description

The invention relates to medicine, in particular to a method for producing hematopoietic stimulating polypeptides from the thymus and can be used in the pharmaceutical industry for the production of medicines for the treatment of anemia.

Various methods are known for the preparation of biologically active polypeptides from mammalian thymi, affecting mainly the T-cell immunity.

A known method for producing polypeptides (Goldstein AL, Guha A., Zatz MM, Hardy MA & Wheit A. Purification and Biological Activity of Thymosin, a Hormone of the Thymus Gland // Proc. Nat. Acad. Sci. USA Vol.69, No .7, pp. 1800-1803, July 1972.), which consists in the extraction of calf thymi with a 0.15 M sodium chloride solution, separation of the cake, heating of the supernatant to 80 ° C, separation of the heat-labile proteins and precipitation of the desired product with acetone.

The resulting product, designated as thymosin fraction-3, was dissolved in 0.1 M sodium phosphate buffer, pH 7.2, and subjected to fractional precipitation with ammonium sulfate at 25% and 50% saturation.

The protein precipitate obtained at 50% salt saturation was dissolved in a buffer and reprecipitated with ammonium sulfate at 50% saturation. The precipitate is dissolved in phosphate buffer, the solution is passed through a millipore filter with a pore diameter of 0.45 μm, dialyzed against distilled water and lyophilized. The powder was dissolved in 0.01 M sodium phosphate buffer, pH 7.2, and subjected to gel filtration through a Sephadex G-150 column. The yield of the active fraction, designated as “thymosin fraction-5”, was 150 mg per 1 kg of calf thymus.

Fraction-5 was further purified by column chromatography on Ecteol-cellulose balanced with 0.01 M sodium phosphate buffer, pH 7.2 in a gradient of sodium chloride concentration from 0 to 1 M. Active fraction (thymosin fraction-6, 60 mg) dialyzed against 0.01 M sodium phosphate buffer, pH 6.5 passed through a column of hydroxylapatite in the same buffer. Sorbed peptides elute using a stepwise gradient of sodium phosphate concentration, pH 6.5: 0.01 M → 0.05 M → 0.1 M → 0.15 M → 0.2 M in the form of three peaks.

The yield of the second peak containing the active fraction (thymosin fraction-7) is 20 mg with 1 kg of raw material. Fraction-7 was manifested by polyacrylamide gel electrophoresis at pH 8.6 and 3.1 as a single band of peptides with a molecular weight of about 13,000 Da. The biological activity of fraction-7 was manifested in a dose of 0.12 μg in the test of spontaneous outlets.

The disadvantage of this method is the large number of purification steps, low yield of thymosin fraction-5 and further purification products of this fraction. Peptides affecting hematopoiesis were apparently lost during the purification process.

A known method of producing thymosin (AS USSR 975017, MKI A61K 35/56. Published. 11/23/82, Bull. No. 43 - 3 C.), which is based on the same sequence of techniques as in the previous method.

But to simplify the technology and increase the yield, the stages of reprecipitation of ammonium sulfate at 50% saturation, passing through a millipore filter, dialysis against water are omitted. The method is as follows.

The thymus of the Caspian seal is crushed, homogenized and extracted with a 0.15 M sodium chloride solution, the cake is separated and the supernatant is heated to 80 ° C to separate thermolabile proteins. Raw material is precipitated from the solution with acetone, which is then dissolved in 0.01 M sodium phosphate buffer, pH 7.0, and a saturated solution of ammonium sulfate is added to a concentration of 25% saturation. The precipitate was separated and discarded, the solution was acidified with a 10% solution of acetic acid to pH 4.0, and a 50% solution of ammonium sulfate was added. The precipitate was dissolved in 0.01 M Tris-HCl buffer, pH 8.0, and passed through a Sephadex G-50 chromatographic column. The obtained thymosin fraction 5 with a molecular weight of 12,200 Da was lyophilized. The yield of fraction-5 is 250 mg with 1 kg of thymus.

The drug at a dose of 15 μg per mouse provided 100% survival of animals infected with Salmonella Nephimurium. Thymosin fraction-5, isolated according to the method proposed by Goldstein, showed similar activity, however, animal survival was only 60% at a dose of 15 μg per mouse.

The disadvantage of the above method is the low yield of Fraction-5, although it is 2 times higher than in the base method, and low biological activity. The described method did not allow to detect fractions that affect hematopoiesis.

There is also a method of producing thymosin from mammalian thymi (A.S. 1255136, MKI A61K 35/26, 35/00, published. 07.09.86, Bull. 33 - 2 S.), in which by selecting the mode of homogenization of mammalian thymus tissue and To simplify the cleaning process of acetone powder, it was possible to increase the yield of thymosin fraction-5 to 1.4 g from 1 kg of thymus, which is 5-6 times higher than the yield of the previous analogue.

For this, the thymus of mammals (calves, sheep or seals) is crushed, homogenized in a 0.15 M sodium chloride solution, tissue particles are separated, heated to 80 ° C, thermolabile proteins are separated, the solution is treated with chilled acetone. The precipitate was dried with acetone, the resulting powder was dissolved in 0.01 M sodium phosphate buffer, pH 7.0, and a saturated solution of ammonium sulfate was added to 25% saturation (3 parts of a solution and 1 part of a saturated solution of ammonium sulfate). The precipitated precipitate is separated and discarded, and a 10% solution of acetic acid is added to the supernatant to pH 4.0-4.5, then crystalline ammonium sulfate is calculated as 14.6 g of salt for every 100 ml of solution,

The wet cake was dissolved in 0.01 M Tris-HCl buffer pH 8.0 and gel filtered on a Sephadex G-25 column equilibrated with a 0.05 M ammonium bicarbonate solution, pH 8.0. The yield of thymosin fraction-5 is 1.2-1.4 g per 1 kg of thymus, depending on the type of animal.

Biological activity was manifested at a dose of 10 ^-2 and 10 ^-4 μg per mouse and restored the number of nucleated thymus cells in old animals to the level of nucleated cells of mature animals 48 hours after injection. This method allowed to obtain the maximum yield of fraction-5 in comparison with the previous methods.

The disadvantage of this method is the insufficient degree of purification of fraction-5, which, as you know, consists of about 30 different peptides with a molecular weight of 1000-1500 Da.

The closest in technical essence and the achieved result is a method of obtaining an immunoactive substance from the thymus glands of mammals (A.S. 1392680, MKI A61K 35/26, 37/24. From 24.03.86; published. 07.09.86.).

According to this method, the thymus gland of mammals (calves, sheep, seals) is crushed and homogenized in a 0.15 m sodium chloride solution and stirred for 1 hour at a temperature of 8-10 ° C. The cake is separated by centrifugation, and the supernatant is heated to 80 ° C to precipitate thermolabile proteins. They are separated by centrifugation, the resulting supernatant is filtered through an asbestos-cellulose plate and mixed with 5 volumes of chilled acetone. After 12-16 hours, the supernatant solution is siphoned. The precipitate is dried on a Buchner funnel, washing with acetone. The raw yield is 18 g with 1 kg of raw material.

The raw material (thymosin fraction-3) was dissolved in 0.01 M sodium phosphate buffer with a pH of 7.0, centrifuged and a saturated solution of ammonium sulfate was added to the supernatant at the rate of 3 volumes of supernatant per 1 volume of saturated salt solution, in which pH 7 had previously been adjusted , 0 with an aqueous solution of ammonia. The precipitate was separated by centrifugation, the pH was adjusted in the supernatant to 4.0-4.05 with a 10% solution of acetic acid. Crystalline ammonium sulfate is added to the resulting solution at the rate of 14.6 g of ammonium sulfate for every 100 ml of solution. The resulting precipitate was separated by centrifugation.

The wet cake was dissolved in 0.01 M Tris-HCl buffer, pH 8.0, the insoluble matter was separated by centrifugation and discarded. The solution was applied to a Sephadex G-25 column and eluted with a 0.05 M ammonium bicarbonate solution, pH 8.0. The fractions corresponding to the first protein peak are combined and subjected to ion exchange chromatography on a column with carboxymethyl cellulose in 0.025 M sodium acetate buffer with a pH of 3.4-4.2. After passing the solution and the solution, after washing with the initial buffer, they are combined and mixed with 10 volumes of acetone. The precipitate was collected and dried with acetone.

The resulting precipitate was subjected to gel chromatography on a Sephadex G-50 column in a 0.05 M solution of ammonium bicarbonate, pH 8.0.

The material of the second protein peak is combined and lyophilized. An immunoactive non-pyrogenic substance (thymosin fraction-7) is obtained with a yield of 100 mg per 1 kg of thymus.

Biological activity was studied in the test of restoration of the number of nucleus-containing cells (NSC) of the thymus with age-related immunodeficiency. The biological activity was manifested in a dose of 0.5 × 10 ^-4 μg per 1 kg of animal weight (i.e., 10 ^-6 μg per mouse) and led to the restoration of the thymic UCS of old mice to the level of mature animal CSCs on the second day after the injection of the substance.

The method allowed to obtain the maximum known amount of thymosin fraction-7 with high biological activity. The product is pyrogen-free, stable during storage.

Based on it, the drug Timoptin for Injection was created, which is considered the "gold standard" among immunomodulators.

The disadvantage of the prototype is that during the cleaning process, peptides active against hematopoiesis were also lost.

The technical problem to which the invention is directed is the creation of such a method for producing immunostimulating thymic polypeptides, with which it is possible to isolate polypeptides with higher biological activity that stimulate hematopoiesis, and use them to obtain drugs for the treatment of anemia.

To solve the problem in a method for producing polypeptides from the thymus, including the homogenization of raw materials, extraction with sodium chloride solution, precipitation of tissue particles by centrifugation, removal of thermolabile proteins from the supernatant by heating, precipitation of the target product with acetone, purification by salt fractionation with ammonium sulfate, gel and ion exchange chromatography , according to the invention for the isolation of peptides that enhance hematopoiesis, salt fractionation is carried out by a single addition to the solution crystalline ammonium sulfate to a concentration of 23-28.6% at pH 4.0, ion-exchange chromatography is performed on carboxymethyl Sephadex C-25, polypeptides are eluted using a linear concentration gradient and a pH of sodium acetate buffer from 0.01 M, pH 4.0-5.0 to 2 M, pH 7.0, lyophilize the obtained polypeptides and desalinate on a column with Sephadex G-25.

The proposed method is as follows.

The thymi of young cattle, sheep and seals are minced in a meat grinder and homogenized in a 0.15 M sodium chloride solution. The homogenate is mixed and after the extraction of the cake is separated by centrifugation, and the supernatant is heated to 80 ° C. The resulting suspension is cooled, the precipitate of thermolabile proteins is separated by centrifugation, and the supernatant is filtered through an asbestos cellulose plate. The clear solution is mixed with chilled acetone in a ratio of 1: 5. The precipitate formed is separated on a Buchner funnel, washed with acetone and dried in air. The resulting powder was dissolved with stirring in 0.01 M sodium phosphate buffer, pH 7.0 in a ratio of 1:10, the insoluble part was separated by centrifugation, and acetic acid was added to the supernatant to lower the pH to 4.0. Crystalline ammonium sulfate is added to the resulting suspension to a concentration of 23% -28%. The precipitate was separated by centrifugation and dissolved in 0.01 M Tris-HCl buffer, pH 8.0. The insoluble part is separated by centrifugation, and the solution is applied to a Sephadex G-25 column and eluted with a 0.05 M ammonium bicarbonate solution, pH 8.0, measuring the optical density of the eluate fractions at 276 nm. Fractions corresponding to the first protein peak in the densitogram are combined and lyophilized. The resulting powder is dissolved in 0.01 M sodium acetate buffer, pH 4.0-5.0, the insoluble part is separated by centrifugation, and the clear solution is introduced into the column with an ion exchanger - carboxymethylsephadex C-25, balanced with 0.01 M sodium acetate buffer, pH 4.0-5.0. Collect the passing solution and elute the peptides using a linear concentration gradient and a pH of sodium acetate buffer from 0.01 M, pH 4.0-5.0 to 2 M, pH 7.0. The optical density of the eluate fractions was measured at 276 nm. Then the fractions corresponding to the second protein peak on the densitogram are combined and lyophilized. The powder is dissolved in a 0.05 M solution of ammonium bicarbonate, pH 8.0, and desalted on a Sephadex G-25 column.

The following are specific examples of the method.

Example 1. 1 kg of slightly thawed thymus calves are crushed in a meat grinder and homogenized in small portions, adding a 0.15 M sodium chloride solution. Each batch is homogenized for 3 minutes. A total of 3 liters of a 0.15 M sodium chloride solution was taken. All portions are combined and stirred for 1 hour on a laboratory stirrer. The mixture is centrifuged for 30 minutes at 3000 rpm, the cake is discarded, and the supernatant is transferred to a round bottom flask of heat-resistant glass and heated in a boiling water bath to 80 ° C for 20 minutes. The suspension is cooled to room temperature and the thermolabile proteins are separated by centrifugation (30 minutes, 3000 rpm, 6-8 ° C) and discarded.

The supernatant is filtered through an asbestos-cellulose plate and the resulting clear solution is poured into 5 volumes of acetone, cooled to -10 ° C, with stirring. The mixture is left in the refrigerator until (t = 6-8 ° C) overnight, for 16-18 hours to form a precipitate. The next morning, the liquid is siphoned, the precipitate is transferred to a Buchner funnel and washed with several portions of chilled acetone. Next, the precipitate is dried in air to remove traces of acetone. The raw yield is 12 g.

Salt fractionation. 12 g of crude are dissolved in 120 ml of 0.01 M sodium phosphate buffer, pH 7.0 on a magnetic stirrer for 30 minutes. The insoluble portion was separated by centrifugation (30 minutes, 3000 rpm, 6-8 ° C.), and glacial acetic acid was added dropwise to the supernatant to lower the pH to 4.0. To the resulting suspension was added crystalline ammonium sulfate at the rate of 35 g for every 100 ml of solution (a 26% salt solution was obtained). Stirred on a magnetic stirrer for 30 minutes and left overnight in the refrigerator. Then the precipitate was separated by centrifugation (20 min, 3000 rpm, 6-8 ° C) and subjected to further purification using column chromatography.

Gel filtration on Sephadex G-25. The wet cake was dissolved in 100 ml of 0.01 M Tris-HCl buffer, pH 8.0. The insoluble part is separated by centrifugation (20 min, 3000 rpm, 6-8 ° C) and discarded. A clear solution was added to a column (4 × 130 cm) with Sephadex G-25 balanced with a 0.05 M solution of ammonium bicarbonate, pH 8.0. Elute at a rate of 100 ml / hour with the same buffer and measure the optical fraction of the eluate at 276 nm. The fractions corresponding to the first protein peak are combined and lyophilized. The powder yield is 1 g.

Ion exchange chromatography. 1 g of powder is dissolved in 20 ml of 0.01 M sodium acetate buffer, pH 4.5 on a magnetic stirrer for 30 minutes. The insoluble part is separated by centrifugation (20 min, 3000 rpm, 6-8 ° C), a clear solution is introduced into a column (2.4 × 26 cm) with carboxymethylsephadex C-25, balanced with 0.01 M sodium acetate buffer, pH 4.5 (15 ml / hour, fractions 5 ml). The passing solution is collected and eluted first with 0.01 M sodium acetate buffer, pH 4.5, then, after the release of unsorbed polypeptides, elute using a linear concentration gradient and pH of sodium acetate buffer from 0.01 M, pH 4.5 to 2 M pH 7.0 (total volume 800 ml). The optical density of the eluate fraction was measured at 276 nm. Eluate fractions containing unsorbed polypeptides are combined with the passing solution, lyophilized and designated as “fraction A”. The fractions of the polypeptides corresponding on the densitogram to the second protein peak are combined, lyophilized and designated as "fraction B".

Desalination on a column with Sephadex G-25.

Fraction B is dissolved in distilled water and introduced into a column (2 × 130 cm) with Sephadex G-25 with distilled water. Elute with distilled water at a rate of 15 ml / hour, the volume of fractions is 5 ml. Ultraviolet absorption is measured at 276 nm. The peak material with Rf 0.5 is combined and lyophilized. The output of the hemostatic stimulant powder is 50 mg.

Determination of biological activity.

The study of the biological activity of the hemostatic drug included three series of experiments on rats: on intact animals, on a model of acute posthemorrhagic anemia, and on a model of hemolytic anemia.

The results of studies on intact rats with a single intramuscular injection are presented in tables 1-7. We studied doses of the drug from 10 ^-8 μg to 10 μg per 1 kg of animal weight.

The amount of hemoglobin in 1-3 days increased from 13.5 g / dl to 14.5-16.0 g / dl, while the average daily increase was 2.47-14.81%.

Elevated hemoglobin levels persisted for 14-30 or more days.

The number of red blood cells increases within 1-3 days from 4.8 * 10 ¹² / l to (7.1-9.3) * 10 ¹² / l, and the average daily increase was 15.97-93.75 %%.

The number of reticulocytes in relation to the control increases by 1.5-3 times, which indicates the effect of the drug on erythropoiesis.

A stimulating effect on the amount of hemoglobin, red blood cells, and reticulocytes was manifested in all doses studied. At doses of 10 ^-10 and 10 ^-12 μg / kg of animal weight, a decrease in the effect of the drug was observed (these data are not shown in the tables).

Tables 8-14 show the effect of the hemostatic stimulator on the model of hemorrhagic anemia caused by bloodletting 1/3 of the total blood volume through a capillary inserted into the conjunctival sac of the rat eye. The hemostatic stimulator was administered every other day for 14 days (7 injections). At all studied doses of 10 ^-2 , 10 ^-4 , 10 ^-6 and 10 ^-8 μg / kg body weight, hemoglobin values returned to normal by 7 days, while in the control (Table 8, group 1) hemoglobin recovered by 30 days. The number of red blood cells reaches normal on the 3rd day, and in the control - on the 21st day. The effect of the hemostatic stimulator and the antianemic drug Tardiferon containing iron sulfate is compared (Tables 13 and 14). As can be seen from the tables, during treatment with a hemostatic stimulator, the average daily increase in hemoglobin is 9.29-9.86 g / l, which is more than four times higher than in the control (2.17 g / l), and two times higher than for Tardiferon (4.79 g / l).

Tables 15-20 show the therapeutic effect on a model of experimental hemolytic anemia caused by hydrochloric acid phenylhydrazine. A haemostimulant was administered intramuscularly every other day for 14 days (7 injections) in doses of 10 ^-2 , 10 ^-4 , 10 ^-6 and 10 ^-8 μg / kg of animal weight. As can be seen from the tables, the hemoglobin level was restored on the 14th day, and the content of red blood cells - on the 7th day. In the control group (table 15), hemoglobin and red blood cells recovered by day 40 and 30, respectively. The average daily increase in hemoglobin was 3.78-3.93 g / l, and in the control - 1.25 g / l. The average daily increase in red blood cells was 15-19%, and in the control - 4.29%.

Tardiferon, administered daily intragastrically (14 doses) at a dose of 7.2 mg / kg, normalized hemoglobin levels on day 21, and the number of red blood cells on day 14. The average daily gains of hemoglobin and red blood cells were 1.5 and 2 times lower than during treatment with a hemostatic stimulator. It should be noted that we also studied doses of 10 ^-10 and 10 ^-12 μg / kg body weight on anemia models. The growth rates of hemoglobin and red blood cells in this case are much lower, although they were higher than in the control.

Desalting fraction A.

From fraction A containing an immunostimulant, after desalination on a column with Sephadex G-25, an immunostimulant (thymosin fraction-7) is obtained in 200 mg yield.

The biological activity of the immunostimulant was studied in mice with age-related involution of the thymus and was evaluated by an increase in the number of nucleated (NSC) thymus cells.

The immunoactive substance restored the number of UCS of the thymus of old mice at a dose of 10 ^-6 μg per mouse (5 × 10 ^-4 μg per 1 kg of body weight) to the level of UCS of mature animals 48 hours after injection. The number of YSC increased in this 2-4 times compared with the control (as in the prototype).

Isolated by the proposed method hemostimulating and immunostimulating substances are not pyrogenic, because increase the temperature of rabbits by 0.2 ° C.

Example 2. Analogously to example 1. Crystalline ammonium sulfate is added at the rate of 30 g for every 100 ml of solution (23.1% salt solution is obtained). The output of a hemostimulating substance is 40 mg, of an immunoactive substance is 180 mg.

Example 3. Analogously to example 1. Ammonium crystalline sulfate is added at the rate of 40 g for every 100 ml of solution (a 28.6% salt solution is obtained). The output of the hemostimulating substance is 45 mg, the immunoactive substance is 170 mg.

Example 4. Analogously to example 1. Ion exchange chromatography on carboxymethyl Sephadex C-25 is carried out in 0.01 M sodium acetate buffer, pH 4.0. The output of the hemostimulating substance is 42 mg, the immunoactive substance is 180 mg.

Example 5. Analogously to example 1. Ion exchange chromatography on carboxymethyl Sephadex C-25 is carried out in 0.01 M sodium acetate buffer, pH 5.0. The output of a hemostimulating substance is 45 mg, of an immunoactive substance - 200 mg.

Example 6. Analogously to example 1. Hemostimulating substance and immunoactive substance are obtained from freshly frozen thymus glands of lambs. The output of a hemostimulating substance is 60 mg from 1 kg of glands, an immunoactive substance is 250 mg.

Example 7. Analogously to example 1. Hemostimulating substance and immunoactive substance are obtained from freshly frozen thymus glands of seals. The output of a hemostimulating substance is 40 mg from 1 kg of glands, and an immunoactive substance is 170 mg.

The positive effect is as follows.

1. The proposed method allows to obtain a fraction of peptides with high hemostimulating activity, manifested in doses of 10 ^-8 μg / kg of animal weight.

2. Along with a hemostatic stimulator, the method allows you to select immunostimulating peptides with the same biological activity as in the prototype, but with a yield twice as high.

3. Integrated technology provides a more efficient processing of feedstock - mammalian thymus.

Table 1
The dynamics of rat peripheral blood indices during a single intramuscular injection of a hemostatic stimulator at a dose of 10 μg / kg to intact animals (M ± m; n = 10). Group 1. Indicators Study time from the moment of drug administration, days Exodus one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 14.3 ± 0.4 14.7 ± 0.6 15.3 ± 0.6 15.3 ± 0.5 15.0 ± 0.6 14.3 ± 4.0 14.8 ± 0.5 15.0 ± 0.5 Red blood cells, 10 ¹² / L 4.8 ± 0.3 9.3 ± 0.5 7.2 ± 0.4 6.3 ± 0.5 9.3 ± 0.4 9.1 ± 0.4 8.7 ± 0.6 6.5 ± 0.8 6.3 ± 0.4 Hematocrit% 47 ± 2.0 56 ± 2.5 58 ± 2.6 53 ± 2.0 62 ± 2.9 60 ± 2.0 58 ± 2,3 56 ± 2.5 52 ± 2.0 MCV, μm ³ 98 60 81 84 67 66 67 86 83 SIT, pg 28 fifteen twenty 24 16 16 16 23 24 ICSU, g / dl 29th 26 25 29th 25 25 25 26 29th Reticulocytes,% 4,5 ± 0,3 8.2 ± 0.5 15.4 ± 0.8 5.7 ± 0.3 10.0 ± 0.4 6.0 ± 0.3 6.3 ± 0.4 5.5 ± 0.4 4.6 ± 0.4 Platelets, 10 ⁹ / L 620 ± 20 850 ± 20 785 ± 16 950 ± 22 620 ± 18 770 ± 16 1050 ± 18 690 ± 18 700 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 13.0 ± 1.2 13.0 ± 1, 11.8 ± 0.3 13.0 ± 1.1 12.0 ± 1.0 12.6 ± 1.2 12.8 ± 1.3 12.0 ± 1.0 P <0.05 with respect to baseline.

table 2
The dynamics of rat peripheral blood indices during a single intramuscular injection of a hemostatic stimulator at a dose of 1 μg / kg to intact animals (M ± m; n = 10). Group 2 Indicators Study time from the moment of drug administration, days Exodus one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 14.0 ± 0.4 16.0 ± 0.7 14.5 ± 0.5 14.5 ± 0.5 14.2 ± 0.4 13.9 ± 0.4 14.5 ± 0.4 140 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 8.3 ± 0.5 7.1 ± 0.4 5.3 ± 0.5 8.5 ± 0.4 8.2 ± 0.7 9.4 ± 0.6 6.5 ± 0.8 5.0 ± 0.4 Hematocrit% 47 ± 2.0 56 ± 2.5 58 ± 2.6 54 ± 2.0 62 ± 2.9 54 ± 2.0 58 + 2,3 56 ± 2.5 50 ± 2.0 MCV, μm ³ 98 67 82 102 73 66 61 86 one hundred SIT, pg 28 17 23 27 17 17 fifteen 22 28 ICSU, g / dl 29th 25 28 27 23 26 24 26 28 Reticulocytes,% 4,5 ± 0,3 7.4 + 0.5 9.3 ± 0.8 6.5 ± 0.3 7.5 ± 0.4 6.0 ± 0.3 5.5 ± 0.3 5.5 ± 0.4 3.8 ± 0.4 Platelets, 10 ⁹ / L 620 + 20 840 ± 20 800 ± 16 700 ± 16 800 ± 18 820 ± 16 950 ± 26 775 ± 18 700 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 13.0 ± 1.2 13.0 ± 1, 11.8 ± 0.3 13.0 ± 1.1 12.0 ± 1.0 12.6 ± 1.2 12.8 ± 1.3 12.0 ± 1.0 P <0.05 with respect to baseline.

Table 3
The dynamics of changes in rat peripheral blood with a single intramuscular injection of hemostatic
at a dose of 10 ^-2 μg / kg to intact animals (M ± m; n = 10). Group 3 Indicators Study time from the moment of drug administration, days Exodus one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 14.8 ± 0.4 15.7 ± 0.7 14.7 ± 0.5 14.7 ± 0.4 13.9 ± 0.3 13.8 ± 4.0 14.8 ± 0.4 150 ± 5.2 Red blood cells, 10 ¹² / L 4.8 ± 0.3 8.9 ± 0.6 8.3 ± 0.4 6.9 ± 0.5 7.7 ± 0.4 6.0 ± 0.4 8.0 ± 0.6 7.5 ± 0.5 7.0 ± 0.5 Hematocrit% 47 ± 2.0 56 ± 2.5 58 ± 2.6 54 ± 2.0 56 ± 2.9 54 ± 2.0 56 + 2,3 56 ± 2.5 52 ± 2.0 MCV, μm ³ 98 63 70 78 73 90 70 75 74 SIT, pg 28 17 19 21 19 23 17 twenty 21 ICSU, g / dl 29th 26 27 27 26 26 25 26 29th Reticulocytes,% 4,5 ± 0,3 7.4 ± 0.4 5.3 ± 0.3 6.4 ± 0.3 8.2 ± 0.6 5.2 ± 0.4 7.3 ± 0.4 5.5 ± 0.4 5.0 ± 0.4 Platelets, 10 ⁹ / L 620 ± 20 1000 ± 30 850 ± 16 800 + 20 600 ± 14 600 ± 16 700 ± 18 690 ± 18 600 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 13.0 ± 1.2 13.0 ± 1, 11.8 ± 0.3 13.0 ± 1.1 12.0 ± 1.0 12.6 + 1.2 12.8 ± 1.3 12.0 ± 1.0 P <0.05 with respect to baseline.

Table 4
The dynamics of changes in rat peripheral blood with a single intramuscular injection of hemostatic
at a dose of 10 ^-4 μg / kg to intact animals (M ± m; n = 10). Group 4. Indicators Study time from the moment of drug administration, days Exodus one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 15.5 ± 0.5 14.5 ± 0.5 14.7 ± 0.5 14.2 ± 0.4 14.5 ± 0.4 13.5 ± 0.3 15.8 ± 0.4 15.0 ± 5.2 Red blood cells, 10 ¹² / L 4.8 ± 0.3 8.0 ± 0.5 7.5 + 0.4 6.4 ± 0.5 8.3 ± 0.5 6.8 ± 0.4 7.7 ± 0.5 6.0 ± 0.8 6.3 ± 0.4 Hematocrit% 47 ± 2.0 54 ± 2.5 56 ± 2.6 54 ± 2.0 60 ± 2.9 56 ± 2.0 55 ± 2,3 53 ± 2.5 52 ± 2.0 MCV, μm ³ 98 68 75 84 72 82 71 88 83 SIT, pg 28 19 19 23 17 21 eighteen 26 24 ICSU, g / dl 29th 29th 26 27 24 26 25 thirty 29th Reticulocytes,% 4,5 ± 0,3 7.4 ± 0.5 10.6 ± 0.8 7.1 ± 0.4 6.7 ± 0.4 8.3 ± 0.3 6.0 ± 0.4 5.5 ± 0.4 4.4 ± 0.4 Platelets, 10 ⁹ / L 620 ± 20 1000 ± 30 900 ± 26 750 ± 16 628 ± 14 675 ± 14 1000 ± 18 900 ± 28 700 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 13.0 ± 1.2 13.0 ± 1, 11.8 ± 0.3 13.0 ± 1.1 12.0 ± 1.0 12.6 ± 1.2 12.8 ± 1.3 12.0 ± 1.0 P <0.05 with respect to baseline.

Table 5
The dynamics of changes in rat peripheral blood with a single intramuscular injection of hemostatic
at a dose of 10 ^-6 μg / kg to intact animals (M ± m; n = 10). Group 5. Indicators Study time from the start of drug administration, days Exodus one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 135 ± 0.3 13.5 ± 0.4 14.5 ± 0.4 14.3 ± 0.3 15.2 ± 0.5 14.5 ± 0.6 15.0 ± 0.4 14.6 ± 0.5 14.0 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 5.4 ± 0.3 7.1 ± 0.4 6.6 ± 0.5 10.5 ± 0.8 6.5 ± 0.4 7.6 ± 0.65 6.0 ± 0.4 5.3 ± 0.4 Hematocrit% 47 ± 2.0 50 ± 2.5 54 ± 2.6 54 ± 2.0 64 ± 2.9 54 ± 2.0 56 ± 2,3 54 ± 2.5 52 ± 2.0 MCV, μm ³ 98 93 76 82 61 83 74 90 98 SIT, pg 28 25 twenty 22 fourteen 22 twenty 24 26 ICSU, g / dl 29th 27 27 26 24 27 27 27 27 Reticulocytes,% 4,5 ± 0,3 35.2 ± 1.5 17.8 ± 0.8 8.8 ± 0.6 10.0 ± 0.4 8.7 ± 0.6 6.0 ± 0.4 5.5 ± 0.3 4.6 ± 0.4 Platelets, 10 ⁹ / L 620 ± 20 900 ± 28 800 ± 16 700 ± 16 700 ± 16 700 ± 16 850 ± 18 800 ± 18 700 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 13.2 ± 1.2 13.6 ± 1, 12.8 ± 1.3 13.0 ± 1.1 12.0 ± 1.0 12.6 ± 1.2 11.8 ± 1.0 12.0 ± 1.0 P <0.05 with respect to baseline.

Table 6
The dynamics of changes in rat peripheral blood with a single intramuscular injection of hemostatic
at a dose of 10 ^-8 μg / kg to intact animals (M ± m; n = 10). Group 6. Study time from the moment of drug administration, days Exodus one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 135 ± 0.3 14.5 ± 0.4 14.5 ± 0.6 16.0 ± 0.6 15.3 ± 0.5 14.0 ± 0.6 13.5 ± 4.0 14.5 ± 0.4 14.0 ± 5.2 Red blood cells, 10 ¹² / L 4.8 ± 0.3 8.7 ± 0.6 7.0 ± 0.4 10.5 ± 0.5 7.3 ± 0.5 8.8 ± 0.6 8.7 ± 0.6 6.5 ± 0.5 6.0 ± 0.4 Hematocrit% 47 ± 2.0 56 ± 2.5 58 ± 2.6 64 ± 2.0 61 ± 2.9 58 ± 2.0 58 ± 2,3 56 ± 2.5 52 ± 2.0 MCV, μm ³ 98 64 83 61 84 66 67 86 87 SIT, pg 28 17 21 fifteen 21 16 16 22 23 ICSU, g / dl 29th 26 25 25 25 24 23 26 27 Reticulocytes,% 4,5 ± 0,3 24.5 ± 1.5 18.3 ± 1.4 17.0 ± 1.0 9.2 ± 0.6 7.7 ± 0.5 6.7 ± 0.4 5.5 ± 0.4 4.9 ± 0.4 Platelets, 10 ⁹ / L 620 ± 20 900 ± 26 1470 ± 36 650 ± 14 900 ± 18 750 ± 16 1 900 ± 28 800 ± 18 700 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 13.0 ± 1.2 13.0 ± 1.1 11.8 ± 0.3 13.0 ± 1.1 12.0 ± 1.0 12.6 ± 1.2 12.8 ± 1.3 12.0 ± 1.0 P <0.05 with respect to baseline.

Table 7
The average daily increase in the number of hemoglobin and the number of red blood cells in intact rats with a single intramuscular injection of a hemostatic stimulator Doses of the drug, mcg / kg Hemoglobin Red blood cells Day * Hemoglobin, g / l The average daily ** increase in hemoglobin Day * Red blood cells, 10 ¹² / L The average daily increase in red blood cells ** g / l % 10 ¹² / l % Hemostimulator, 10 7-21 153-150 2,57 1.90 1-21 9.3-9.1 4,5 93.75 Hemostimulator, 1 3-21 160-142 8.33 6.17 1-30 8.3-9.4 3,5 72.92 Hemostimulator, 10 ^-2 3-14 157-147 7.33 5.43 1-30 8.9-8.0 4.1 85.42 Hemostimulator, 10 ^-4 1-21 155-145 20.00 14.81 1-30 8.0-7.7 3.2 66.67 Hemostimulator, 10 ^-6 3-30 145-150 3.33 2.47 3-30 7.1-7.6 0.77 15.97 Hemostimulator 10 ^-8 1-14 145-153 10.00 7.40 1-30 8.7-8.7 3.9 81.25 Exodus - 135 - - - 4.8 - - * The periods during which an increased level of hemoglobin and red blood cells were observed. ** The average daily increase is calculated on the first day of a significant increase in the indicator.

Table 8
The dynamics of changes in peripheral blood indices in rats with acute posthemorrhagic anemia, control. (M ± m; n = 10). Group 1. Indicators Intact Anemia outcome Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 6.8 ± 0.2 7.0 ± 0.1 8.1 ± 0.3 9.4 ± 0.4 10.1 ± 0.6 12.6 ± 0.6 13.3 ± 0.6 13.5 ± 0.5 13.8 ± 0.6 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.8 ± 0.2 2.9 ± 0.2 3.2 ± 0.2 3.7 ± 0.3 4.2 ± 0.3 4.8 ± 0.4 5.1 ± 0.4 5.0 ± 0.4 5.0 ± 0.4 Hematocrit% 47 ± 2.0 22 ± 1,1 25 ± 2.0 28 ± 2,4 32 ± 3.0 39 ± 3.0 44 ± 3.0 46 ± 3.0 47 ± 3.0 48 ± 3.0 MCV, μm ³ 98 79 86 88 86 93 92 90 94 96 SIT, pg 28 24 24 25 25 24 26 26 27 28 ICSU, g / dl 29th 31 28 29th 29th 26 29th 29th 29th 29th Reticulocytes,% 4,5 ± 0,3 8.4 ± 0.3 6.2 ± 0.2 7.3 ± 0.4 5.5 ± 0.3 5.0 ± 0.4 4.6 ± 0.5 4.1 ± 0.3 4.4 ± 0.3 4.6 ± 0.3 Platelets, 10 ⁹ / L 620 ± 18 800 ± 18 830 ± 18- 785 ± 20 740 ± 17 715 ± 18 700 ± 17 715 ± 18 612 ± 19 650 ± 18 White blood cells, 10 ⁹ / L 11.8 ± 1.0 14.8 ± 1.3 14.6 ± 1.3 10.8 ± 1.0 12.5 ± 1.1 12.6 ± 1.2 11.0 ± 1.4 11.9 ± 1.0 12.0 ± 1.0 12.2 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.6 ± 0.2 3.3 ± 0.3 3.0 ± 0.3 2.9 ± 0.3 2.5 ± 0.3 2.2 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 28.0 ± 2.0 27.4 ± 1.5 26.5 ± 1.8 28.0 ± 1.5 27.8 ± 1.2 29 ± 1.6 28 ± 1,5 28.4 ± 1.4 27.8 + 2.1 eosinophils,% 2.0 ± 0.2 5.0 ± 0.3 5.0 ± 0.3 4.7 ± 0.4 3.7 ± 0.4 3.5 ± 0.3 3.1 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.2 3.5 ± 0.2 3.0 ± 0.2 2.8 ± 0.2 2.4 ± 0.2 2.2 ± 0.2 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.1 lymphocytes,% 64.2 ± 3.5 61.4 ± 4.0 60.8 ± 4.2 62.8 ± 3.4 62.6 ± 3.1 63.8 ± 3.1 63.5 ± 3.1 65.0 ± 3.2 64.6 ± 3.0 65.2 ± 3.5 P <0.05 with respect to baseline.

Table 9
The dynamics of the peripheral blood indices of rats with acute posthemorrhagic anemia treated with a hemostatic stimulator at a dose of 10 ^-2 μg / kg, intramuscular injection. (M ± m; n = 10). Group 2 Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 6.8 ± 0.2 7.7 ± 0.4 9.4 ± 0.4 13.3 ± 0.4 15.1 ± 0.5 14.1 ± 0.4 13.8 ± 0.5 14.0 ± 0.5 14.0 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.8 ± 0.2 3.7 ± 0.3 4.7 ± 0.3 6.7 ± 0.3 5.5 ± 0.4 5.6 ± 0.4 5.2 ± 0.6 5.0 ± 0.5 5.5 ± 0.4 Hematocrit% 47 ± 2.0 22 ± 1,1 37 ± 5.0 47 ± 3.0 60 ± 3.0 56 ± 5.0 55 ± 5 50 ± 5 48 ± 5 48 ± 4 MCV, μm ³ 98 79 one hundred one hundred 90 102 98 96 96 87 SIT, pg 28 24 21 twenty twenty 27 25 27 28 25 ICSU, g / dl 29th 31 21 twenty 22 27 26 28 29th 29th Reticulocytes,% 4,5 ± 0,3 8.4 ± 0.3 6.1 ± 0.2 7.4 ± 0.4 6.9 ± 0.4 6.5 ± 0.5 8.3 ± 0.5 4.9 ± 0.3 4.6 ± 0.3 5.0 ± 0.3 Platelets, 10 ⁹ / L 620 ± 18 800 ± 18 895 ± 26 763 ± 18 725 ± 19 1010128 838 ± 22 715 ± 18 685 ± 17 700 ± 18 White blood cells, 10 ⁹ / L 11.8 ± 1.0 14.8 ± 1.3 13.0 ± 1.2 12.8 ± 1.1 10.8 ± 1.2 10.2 ± 1.1 10.4 ± 1.1 11.2 ± 1.0 12.6 ± 1.1 12.8 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.6 ± 0.2 2.9 ± 0.3 2.7 ± 0.3 2.4 ± 0.2 2.2 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 28.0 ± 2.0 26.5 ± 1.5 25.0 ± 1.3 26.2 ± 1.3 27.0 ± 1.3 28.7 ± 1.5 28.5 ± 1.6 27.4 ± 1.6 27 ± 1,5 eosinophils,% 2.0 ± 0.2 5.0 ± 0.3 4.0 ± 0.3 3.5 ± 0.3 2.8 ± 0.3 2.3 ± 0.2 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.2 2.7 ± 0.3 2.8 ± 0.3 2.3 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 lymphocytes,% 64.2 ± 3.5 61.4 ± 4.0 63.9 ± 3.2 66.0 ± 3.3 66.3 ± 3.2 66.5 ± 3.2 64.3 ± 3.1 64.5 ± 3.1 65.6 ± 3.2 66.0 ± 3.3 P≤0.05 with respect to the control.

Table 10
The dynamics of peripheral blood indices of rats with acute posthemorrhagic anemia treated with a hemostatic stimulator
at a dose of 10 ^-4 μg / kg, intramuscular injection (M ± m; n = 10). Group 3. Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 6.8 ± 0.2 8.0 ± 0.2 10.2 ± 0.3 13.7 ± 0.4 14.1 ± 0.5 14.3 ± 0.4 14.0 ± 0.5 13.8 ± 0.5 14.0 ± 0.5 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.8 ± 0.2 3.9 ± 0.2 4.7 ± 0.3 6.1 ± 0.4 6.6 ± 0.4 5.9 ± 0.4 5.8 ± 0.4 4.8 ± 0.5 5.5 ± 0.4 Hematocrit% 47 ± 2.0 22 ± 1,1 34 ± 0.4 37 ± 1,0 48 ± 1,0 51 ± 2.0 55 ± 4.0 50 ± 5.0 47 ± 4.0 50 ± 2.0 MCV, μm ³ 98 79 87 79 79 77 86 86 98 96 SIT, pg 28 24 21 22 22 21 24 24 29th 26 ICSU, g / dl 29th 31 24 28 29th 28 28 28 29th 28 Reticulocytes,% 4,5 ± 0,3 8.4 ± 0.3 5.9 ± 0.4 8.0 ± 0.5 7.0 ± 0.4 6.6 ± 0.4 5.2 ± 0.5 4.9 ± 0.4 4.6 ± 0.4 5.3 ± 0.4 Platelets, 10 ⁹ / L 620 ± 18 800 ± 18 800 ± 25 838 + 20 725 ± 19 780 ± 17 720 ± 20 675 ± 19 750 ± 19 715 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 14.8 ± 1.3 14.6 ± 1.4 11.2 ± 1.1 10.6 ± 1.0 10.0 ± 1.4 11.2 ± 1.4 10.6 + 1.2 11.6 ± 1.0 11.0 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.6 ± 0.2 2.8 ± 0.4 2.6 ± 0.2 2.5 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 28.0 ± 2.0 29.0 ± 1.3 27.0 ± 1.3 28.0 ± 1.3 28 ± 2.0 28.0 + 2.0 27.5 ± 1.5 27.4 ± 1.6 27.6 ± 1.6 eosinophils,% 2.0 ± 0.2 5.0 ± 0.3 2.8 ± 0.3 2.7 ± 0.3 2.7 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.2 2.8 + 0.2 2.5 ± 0.2 2.7 ± 0.3 2.6 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 lymphocytes,% 64.2 ± 3.5 61.4 ± 4.0 62.6 + 3.1 65.2 ± 3.2 64.1 ± 3.2 64.4 ± 3.0 65.0 ± 5.4 65.5 ± 5.5 65.6 ± 3.4 65.4 ± 4.4 p≤0.05 in relation to the control.

Table 11
The dynamics of peripheral blood indices of rats with acute posthemorrhagic anemia treated with a hemostatic stimulator
at a dose of 10 ^-6 mcg / kg, intramuscular injection (M ± m; n = 10). Group 4 Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 6.8 ± 0.2 8.4 ± 0.2 10.8 ± 0.3 13.6 ± 0.4 14.4 ± 0.5 14.4 ± 0.4 14.2 ± 0.5 13.9 ± 0.5 14.1 ± 0.5 Red blood cells, 10 ^{12 /} L 4.8 ± 0.3 2.8 ± 0.2 3.9 ± 0.2 4.9 ± 0.3 6.0 ± 0.4 6.4 ± 0.4 5.8 ± 0.4 5.6 ± 0.4 4.9 ± 0.5 5.3 ± 0.4 Hematocrit% 47 ± 2.0 22 ± 1,1 32 ± 0.4 38 ± 1,0 47 ± 1,0 52 ± 2.0 56 ± 4.0 50 ± 5.0 47 ± 4.0 50 ± 2.0 MCV, μm ³ 98 79 82 78 78 81 97 89 96 94 SIT, pg 28 24 22 22 23 23 25 25 28 27 ICSU, g / dl 29th 31 26 28 29th 28 26 28 thirty 28 Reticulocytes,% 4,5 ± 0,3 8.4 ± 0.3 6.9 ± 0.4 8.2 ± 0.5 7.2 ± 0.4 6.8 ± 0.4 5.6 ± 0.5 4.8 ± 0.4 4.7 ± 0.4 5.0 ± 0.4 Platelets, 10 ⁹ / L 620 ± 18 800 ± 18 798 ± 25 826 ± 20 740 ± 19 760 ± 17 740 ± 20 685 ± 19 700 ± 19 725 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 14.8 ± 1.3 14.9 ± 1.4 11.8 ± 1.1 10.9 ± 1.0 10.8 ± 1.4 11.8 ± 1.4 10.7 ± 1.2 11.4 ± 1.0 11.2 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.6 ± 0.2 2.4 ± 0.4 2.7 ± 0.2 2.3 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 28.0 ± 2.0 29.0 ± 1.3 27.0 ± 1.3 28.0 ± 1.3 28.2 ± 2.0 28.0 ± 2.0 27.4 ± 1.5 27.5 ± 1.6 27.6 ± 1.6 eosinophils,% 2.0 ± 0.2 5.0 ± 0.3 2.4 ± 0.3 2.5 ± 0.3 2.6 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.2 2.4 ± 0.2 2.5 ± 0.2 2.7 ± 0.3 2.2 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 lymphocytes,% 64.2 ± 3.5 61.4 ± 4.0 63.8 ± 3.1 65.3 ± 3.2 64.4 ± 3.2 64.6 ± 3.0 65.0 ± 5.4 65.6 ± 5.5 65.5 ± 3.4 65.4 ± 4.4 p≤0,05 in relation to the control

Table 12
The dynamics of changes in the peripheral blood indices of rats with acute post-hemorrhagic anemia treated with a hemostatic stimulator at a dose of 10 ^-8 μg / kg, intramuscular injection (M + n; n = 10). Group 5. Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 6.8 ± 0.2 9.8 ± 0.2 12.9 ± 0.3 13.4 ± 0.4 14.5 ± 0.5 14.0 ± 0.4 13.7 ± 0.5 14.0 ± 0.5 14.1 ± 0.5 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.8 ± 0.2 4.1 ± 0.2 4.8 ± 0.4 6.7 ± 0.4 6.3 ± 0.4 6.0 ± 0.4 5.5 ± 0.4 5.0 ± 0.5 4.8 ± 0.4 Hematocrit% 47 ± 2.0 22 ± 1,1 41 ± 0.4 48 ± 1,0 54 ± 1,0 56 ± 2.0 50 ± 4.0 50 ± 5.0 48 ± 4.0 47 ± 2.0 MCV, μm ³ 98 79 one hundred one hundred 81 89 83 91 96 98 SIT, pg 28 24 24 27 twenty 23 23 25 28 29th ICSU, g / dl 29th 31 24 27 25 26 28 27 29th thirty Reticulocytes,% 4,5 ± 0,3 8.4 ± 0.3 6.6 ± 0.4 7.4 ± 0.5 7.5 ± 0.4 6.1 ± 0.4 7.2 ± 0.5 5.1 ± 0.4 4.6 ± 0.4 5.0 ± 0.4 Platelets, 10 ⁹ / L 620 ± 18 800 ± 18 800 ± 25 838 ± 20 775 ± 19 750 ± 17 720 ± 16 767 + 19 650 ± 19 615 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 14.8 ± 1.3 14.6 ± 1.4 12.3 ± 1.1 11.6 ± 1.0 11.4 ± 1.4 11.8 ± 1.4 10.6 ± 1.2 11.6 ± 1.0 12.0 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.6 ± 0.2 2.8 ± 0.2 2.6 ± 0.2 2.5 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 28.0 ± 2.0 29.0 ± 1.3 27.0 ± 1.3 28.0 ± 1.3 28 ± 2.0 28.0 ± 2.0 27.5 ± 1.5 27.4 ± 1.6 27.6 ± 1.6 eosinophils,% 2.0 ± 0.2 5.0 ± 0.3 2.8 ± 0.3 2.7 ± 0.3 2.7 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.2 2.8 ± 0.3 2.5 ± 0.2 2.7 ± 0.3 2.6 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 lymphocytes,% 64.2 ± 3.5 61.4 ± 4.0 62.6 ± 3.1 65.2 ± 3.2 64.1 ± 3.2 64.4 ± 3.0 65.0 ± 5.4 65.5 ± 5.5 65.6 ± 3.4 65.4 ± 4.4 p≤0,05 in relation to the control

Table 13
The dynamics of the peripheral blood indices of rats with acute posthemorrhagic anemia treated with Tardiferon
at a dose of 7.2 mg / kg, intragastrically. (M ± m; n = 10). Group 6. Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 6.8 ± 0.2 10.1 ± 0.4 11.7 ± 0.6 12.5 ± 0.4 13.5 ± 0.6 14.0 ± 0.5 13.5 ± 0.5 13.4 ± 0.5 13.3 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.8 ± 0.2 3.2 ± 0.2 3.6 ± 0.3 4.6 ± 0.3 4.8 ± 0.4 5.0 ± 0.4 4.8 ± 0.4 5.0 ± 0.4 4.8 ± 0.4 Hematocrit% 47 ± 2.0 22 ± 1,1 35 ± 1,0 40 ± 2.0 44 ± 2.6 47 ± 3.0 48 ± 3.0 47 ± 3.0 47 ± 3.0 46 ± 3.0 MCV, p 98 79 109 111 96 98 96 98 94 96 SIT, pg 28 24 32 33 27 28 28 28 27 28 ICSU, g / dl 29th 31 29th 29th 28 29th 29th 29th 29th 29th Reticulocytes,% 4,5 ± 0,3 8.4 ± 0.3 7.0 ± 0.7 7.5 ± 0.7 7.2 ± 0.7 6.6 ± 0.6 5.3 ± 0.4 5.5 ± 0.4 5.2 ± 0.3 5.1 ± 0.3 Platelets, 10 ⁹ / L 620 ± 18 800 ± 18 865 ± 23 840 ± 25 850 ± 17 700 ± 16 720 ± 15 650 ± 14 670 ± 15 645 ± 16 White blood cells, 10 ⁹ / L 11.8 ± 1.0 14.8 ± 1.3 14.8 ± 1.3 13.9 ± 1.1 13.6 ± 1.5 13.7 ± 1.4 12.4 ± 1.0 12.5 ± 1.4 12.6 ± 1.0 12.7 ± 1.1 Neutrophils: stab core,% 2.0 ± 0.2 2.6 ± 0.2 2.8 ± 0.3 2.3 ± 0.2 2.5 ± 0.2 2.1 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 28.0 ± 2.0 27.0 ± 2.0 29.0 ± 1.0 28.5 ± 1.5 28.7 ± 1.3 27.5 ± 5.2 28.3 ± 2.2 27.8 ± 2.5 27.6 ± 2.0 eosinophils,% 2.0 ± 0.2 5.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 lymphocytes,% 64.2 ± 3.5 61.4 ± 4.0 65.2 ± 5.5 63.7 ± 6.3 64.0 ± 5.0 64.2 ± 5.8 65.5 ± 5.5 64.7 ± 5.3 65.2 ± 5.4 65.4 ± 5.2 p≤0.05 in relation to the control.

Table 14
The average daily increase in the number of hemoglobin and the number of red blood cells in rats with acute posthemorrhagic anemia who took the drug hemostimulator and Tardiferon Drugs, doses, mcg / kg Hemoglobin Red blood cells Day to normal Hemoglobin, g / l The average daily increase in hemoglobin Day to normal Red blood cells, 10 ¹² / L The average daily increase in red blood cells g / l % 10 ¹² / l % The control thirty 133 2.17 3.19 21 4.8 0,095 3.39 Hemostimulator 10 ^-2 7 133 9.29 13.66 7 6.7 0.56 19.89 Hemostimulator 10 ^-4 7 137 9.86 14.50 3 4.7 0.633 22.62 Hemostimulator 10 ^-6 7 136 9.71 14.29 3 4.9 0.700 25.00 Hemostimulator 10 ^-8 7 134 9.43 13.87 3 4.8 0.667 23.82 Tardiferon fourteen 135 4.79 7.04 fourteen 4.8 0.143 5.11 Anemia outcome - 68 - - - 2,8 - - Intact 135 ± 3.0 4.8 ± 0.3

Table 15
The dynamics of peripheral blood indices of rats with hemolytic anemia, control. (M + m; n = 10). Group 1. Indicators Intact Anemia outcome Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 8.0 ± 0.2 8.7 ± 0.3 8.2 ± 0.1 9.1 ± 0.1 9.3 ± 0.3 10.2 ± 0.2 11.6 ± 0.2 13.0 ± 0.2 13.2 ± 0.2 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.1 ± 0.2 2.3 ± 0.1 2.4 ± 0.1 2.8 ± 0.1 3.2 ± 0.2 3.7 ± 0.3 4.8 ± 0.3 5.0 ± 0.3 4.7 ± 0.3 Hematocrit% 47 ± 2.0 25 ± 2.1 26 ± 2.0 30 ± 2.2 36 ± 2,4 40 ± 3.1 42 ± 3,5 48 ± 3,2 48 ± 3.0 48 ± 3.0 MCV, μm ³ 98 119 113 125 129 125 113 one hundred 96 102 SIT, pg 28 38 38 34 33 29th 28 24 26 28 ICSU, g / dl 29th 32 33 27 25 23 24 24 27 28 Reticulocytes,% 4,5 ± 0,3 8.9 ± 0.5 8.8 ± 0.5 8.8 ± 0.5 4.9 ± 0.2 4.8 ± 0.2 4.8 ± 0.2 5.0 ± 0.3 4.0 ± 0.3 4.2 ± 0.3 Platelets, 10 ⁹ / L 620 ± 18 816 ± 16 725 ± 20 800 ± 21 700 ± 20 600 ± 18 650 ± 18 640 ± 20 700 ± 18 650 ± 19 White blood cells, 10 ⁹ / L 11.8 + 1.0 15.6 + 1.4 15.8 ± 1.5 15.8 ± 1.3 11.9 ± 1.0 11.6 ± 1.0 11.8 ± 1.0 12.8 ± 1.0 11.9 ± 1.0 12.6 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 23.0 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 28.8 ± 1.2 23.0 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 eosinophils,% 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 monocytes,% 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 lymphocytes,% 64.2 ± 5.8 70.0 ± 1.6 69.3 ± 2.7 69.5 ± 1.5 69.3 ± 2.7 65.9 ± 1.6 64.2 ± 5.8 70.0 ± 1.6 69.3 ± 2.7 69.5 ± 1.5 p≤0,05 in relation to the initial indicators.

Table 16
The dynamics of changes in the peripheral blood indices of rats with hemolytic anemia treated with a hemostatic stimulator at a dose of 10 ^-4 μg / kg, intramuscular injection. (M ± m; n = 10). Group 2 Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 8.0 ± 0.2 10.5 ± 0.2 10.5 ± 0.2 12.0 ± 0.4 13.5 ± 0.4 14.0 ± 0.5 15.5 ± 0.5 15.0 ± 0.5 14.8 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.1 ± 0.2 3.0 ± 0.1 3.8 ± 0.1 4.4 ± 0.2 5.5 ± 0.4 6.1 ± 0.3 6.5 ± 0.3 6.4 ± 0.3 5.4 ± 0.3 Hematocrit% 47 ± 2.0 25 ± 2.1 34 ± 2.0 38 ± 2.2 43 ± 3.0 54 ± 4.0 54 ± 3.0 56 + 3.0 60 ± 4.0 52 ± 4.0 MCV, μm ³ 98 119 113 one hundred 98 98 89 86 94 96 SIT, pg 28 38 35 28 27 24 23 24 23 27 ICSU, g / dl 29th 32 31 28 28 25 26 28 25 28 Reticulocytes,% 4,5 ± 0,3 8.9 ± 0.8 5.6 ± 0.4 6.0 ± 0.4 5.5 ± 0.1 5.7 ± 0.3 7.3 ± 0.4 7, ± 0.3 6.2 ± 0.4 4,5 ± 0,2 Platelets 10 / L 620 ± 18 816 ± 20 740 ± 18 715 ± 16 700 ± 20 725 ± 25 800 ± 18 750 ± 20 800 ± 20 900 ± 20 White blood cells, 10% 11.8 ± 1.0 15.6 ± 1.4 15.0 ± 1.3 15.2 ± 1.1 12.0 ± 11 11.8 ± 1.0 12.0 ± 1.0 12.2 ± 1.1 11.6 ± 1.0 11.8 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 + 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 23.0 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 28.8 ± 1.2 23.0 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 23.5 ± 1.5 23.7 ± 1.3 eosinophils,% 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 monocytes,% 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 lymphocytes,% 64.2 ± 5.8 70.0 ± 1.6 69.3 ± 2.7 69.5 ± 1.5 64.2 ± 5.8 70.0 ± 1.6 69.3 ± 2.7 69.5 ± 1.6 69.5 ± 1.5 69.3 ± 2.7 p≤0.05 in relation to the control.

Table 17
The dynamics of changes in peripheral blood indices of rats with hemolytic anemia treated with a hemostatic stimulator at a dose of 10 ^-6 μg / kg, intramuscular injection. (M ± m; n = 10). Group 3. Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 8.0 ± 0.2 9.5 ± 0.2 10.2 ± 0.2 11.2 ± 0.4 13.3 ± 0.4 13.6 ± 0.5 15.4 ± 0.5 15.2 ± 0.5 13.8 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.1 ± 0.2 2.9 ± 0.1 3.8 ± 0.1 4.6 ± 0.2 5.3 ± 0.4 5.6 ± 0.3 6.3 ± 0.3 6.4 ± 0.4 5.5 ± 0.3 Hematocrit% 47 ± 2.0 25 ± 2.1 29 ± 2.0 37 ± 2.2 44 ± 3.0 51 ± 4.0 54 ± 3.0 61 ± 3.0 60 ± 5.0 53 ± 3.8 MCV, μm ³ 98 119 one hundred 97 96 96 96 97 94 96 SIT, pg 28 38 33 27 24 25 24 24 24 25 ICSU, g / dl 29th 32 33 28 25 26 25 25 25 26 Reticulocytes,% 4,5 ± 0,3 8.9 ± 0.8 5.7 ± 0.4 6.4 ± 0.4 5.8 ± 0.1 6.7 ± 0.3 7.4 ± 0.4 7.2 ± 0.3 6.4 ± 0.4 4.6 ± 0.2 Platelets 10% 620 ± 18 816 ± 20 746 ± 18 745 ± 16 709 ± 20 724 ± 25 850 ± 18 756 ± 20 806 ± 20 780 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 15.6 ± 1.4 15.1 ± 1.3 15.2 ± 1.1 12.6 ± 1.1 11.6 ± 1.0 12.2 ± 1.0 12.0 ± 1.1 11.8 ± 1.0 11.8 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 23.0 ± 1.5 23.6 ± 1.3 23.5 ± 1.5 24.5 ± 1.2 26.0 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 26.5 ± 1.5 26.7 ± 1.3 eosinophils,% 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 monocytes,% 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 lymphocytes,% 64.2 ± 5.8 70.0 ± 1.6 69.4 ± 2.7 69.5 ± 1.5 68.5 ± 5.8 67.0 ± 1.6 69.3 ± 2.7 69.5 ± 1.6 66.5 ± 1.5 66.3 ± 2.7 p≤0.05 in relation to the control.

Table 18
The dynamics of changes in the peripheral blood indices of rats with hemolytic anemia treated with a hemostatic stimulator at a dose of 10 ^-8 μg / kg, intramuscular injection. (M ± m; n = 10). Group 4. Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 8.0 ± 0.2 9.6 ± 0.2 10.7 ± 0.2 12.2 ± 0.4 13.4 ± 0.4 14.6 ± 0.5 15.4 ± 0.5 14.1 ± 0.5 13.8 ± 0.4 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.1 ± 0.2 3.0 ± 0.1 4.1 ± 0.1 4.9 ± 0.2 5.8 ± 0.4 6.0 ± 0.4 6.2 ± 0.3 6.0 ± 0.3 5.5 ± 0.3 Hematocrit% 47 ± 2.0 25 ± 2.1 35 + 2.0 42 ± 2.2 46 ± 3.0 57 ± 4.0 58 ± 3.0 61 ± 3.0 60 ± 3.0 57 ± 4.0 MCV, μm ³ 98 119 117 102 94 98 97 98 one hundred 104 SIT, pg 28 38 32 26 25 23 24 25 24 25 ICSU, g / dl 29th 32 27 25 27 24 25 25 24 24 Reticulocytes,% 4,5 ± 0,3 8.9 ± 0.8 7.7 ± 0.4 7.4 ± 0.4 6.8 + 0.1 6.9 ± 0.3 7.5 ± 0.4 7.0 ± 0.3 6.2 ± 0.4 4.6 ± 0.2 Platelets, 10 ⁹ / L 620 ± 18 816 ± 20 846 ± 18 785 ± 16 769 ± 20 744 ± 25 850 + 18 756 ± 20 800 ± 20 782 ± 20 White blood cells, 10 ⁹ / L 11.8 ± 1.0 15.6 ± 1.4 15.4 ± 1.3 15.6 ± 1.1 12.8 ± 1.1 11.8 ± 1.0 12.4 ± 1.0 12.1 ± 1.1 11.5 ± 1.0 11.6 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 23.0 ± 1.5 23.5 ± 1.3 23.6 ± 1.5 24.2 ± 1.2 26.0 ± 1.5 23.8 ± 1.3 23.5 ± 1.5 26.4 ± 1.5 26.6 ± 1.3 eosinophils,% 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 monocytes,% 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 lymphocytes,% 64.2 ± 5.8 70.0 ± 1.6 69.5 + 2.7 69.4 ± 1.5 68.8 ± 5.8 67.0 ± 1.6 69.2 ± 2.7 69.5 ± 1.6 66.6 ± 1.5 66.4 ± 2.7 p≤0.05 in relation to the control.

Table 19
The dynamics of changes in the peripheral blood indices of rats with hemolytic anemia treated with Tardiferon at a dose of 7.2 mg / kg, intragastrically. (M ± m; n = 10). Group 5. Indicators Intact Anemia Outcome, Group 1 Study time, day one 3 7 fourteen 21 thirty 40 60 Hemoglobin, g / dl 13.5 ± 0.3 8.0 ± 0.2 9.2 ± 0.3 10.0 ± 0.4 11.2 ± 0.4 12.5 ± 0.4 13.6 ± 0.5 13.6 ± 0.5 13.5 ± 0.5 13.4 ± 0.5 Red blood cells, 10 ¹² / L 4.8 ± 0.3 2.1 ± 0.2 2.8 ± 0.2 3.0 ± 0.3 4.3 ± 0.3 4,5 ± 0,4 4.8 ± 0.4 5.1 ± 0.4 4.8 ± 0.4 4.8 ± 0.4 Hematocrit% 47 ± 2.0 25 ± 2.1 30 ± 2.2 36 ± 2,4 46 ± 3.0 48 ± 3.0 50 ± 4.0 51 ± 4.0 50 ± 5.0 48 ± 3.8 MCV, μm ³ 98 119 107 120 107 107 104 one hundred 104 one hundred SIT, pg 28 38 33 33 26 28 28 27 28 28 ICSU, g / dl 29th 32 31 28 24 26 27 27 27 28 Reticulocytes,% 4,5 ± 0,3 8.9 ± 0.5 9.5 ± 0.4 7.8 ± 0.7 6.2 ± 0.7 8.6 ± 0.4 6.5 ± 0.4 4.3 ± 0.4 4,5 ± 0,3 4.6 ± 0.3 Platelets, 10 ⁹ / L 620 ± 18 816 ± 16 800 ± 16 775 ± 18 725 ± 18 695 ± 14 675 ± 15 760 ± 20 676 ± 15 700 ± 16 White blood cells, 10 ⁹ / L 11.8 ± 1.0 15.6 ± 1.4 16.2 ± 0.7 15.6 ± 0.8 13.4 ± 0.8 13.2 ± 0.7 13.0 ± 0.9 12.6 ± 0.5 11.8 ± 1.0 12.0 ± 1.0 Neutrophils: stab core,% 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 1.8 ± 0.2 2.4 ± 0.2 2.1 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 segmented,% 28.8 ± 1.2 23.5 ± 1.5 23.7 ± 1.3 23.5 ± 1.5 25.0 ± 1.0 29.0 ± 1.0 29.2 ± 1.1 30.0 ± 1.7 28.0 ± 1.6 28.2 ± 1.7 eosinophils,% 2.0 ± 0.2 2.0 ± 0.3 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 2.5 ± 0.2 3.0 ± 0.2 2.8 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 monocytes,% 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 3.0 ± 0.3 2.2 ± 0.2 2.0 ± 0.2 2.1 ± 0.2 2.0 ± 0.2 2.0 ± 0.2 lymphocytes,% 64.2 ± 5.8 70.0 ± 1.6 69.3 ± 2.7 69.5 ± 1.5 68.2 ± 5.8 63.9 ± 3.1 63.7 ± 3.2 63.1 ± 3.1 65.0 ± 3.0 64.8 ± 3.0 p≤0.05 in relation to the control.

Table 20
The average daily increase in the number of hemoglobin and the number of red blood cells in rats with hemolytic anemia, taking the drug hemostimulator and Tardiferon Drugs, doses, mcg / kg Hemoglobin Red blood cells Day to normal Hemoglobin, g / l The average daily increase in hemoglobin Day to normal Red blood cells, 10 ¹² / L The average daily increase in red blood cells g / l % 10 ¹² / l % The control 40 130 1.25 1,56 thirty 4.8 0.09 4.29 Hemostimulator 10 ^-4 fourteen 135 3.93 4.91 7 4.4 0.328 15,64 Hemostimulator 10 ^-6 fourteen 133 3.78 4.73 7 4.6 0.357 17.01 Hemostimulator 10 ^-8 fourteen 134 3.86 4.82 7 4.9 0.4 19.05 Tardiferon 21 136 2.67 3.34 fourteen 4,5 0.171 8,143 Anemia outcome - 80 - - - 2.1 - - Intact 135 ± 3 4.8 ± 0.3

Claims (1)

A method of obtaining immunostimulating thymic polypeptides affecting hematopoiesis, namely, that the raw materials are homogenized in a 0.15 M sodium chloride solution, tissue particles are precipitated by centrifugation, the resulting supernatant is heated at 80 ° C for 20 min, the precipitate of thermolabile proteins is separated by centrifugation, and the supernatant is filtered, chilled acetone is added to the filtrate in a ratio of 1: 5, the precipitate formed is washed with acetone and dried, the resulting powder is dissolved in 0.01 M sodium phosphate buffer solution, pH 7.0, respectively 1: 10, the insoluble part is separated by centrifugation, the supernatant is adjusted to pH 4.0 and stirred by adding crystalline ammonium sulfate to a concentration of 23-28.6%, ion-exchange chromatography is performed on carboxymethyl Sephadex C-25, polypeptides are eluted using a linear concentration gradient and The pH of the sodium acetate buffer is from 0.01 M, the pH is 4.0-5.0 to 2 M, the pH is 7.0, the polypeptides obtained are lyophilized and desalted on a Sephadex G-25 column.