RU2482869C1 - Hemostimulant and pharmaceutical composition and method for hemopoiesis stimulation - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical composition and medicine, and represents a hypoglycemic, hypocholesteremic, hypolipidemic and (or) antioxidant combination of bis-(γ-L-glutamyl)-L-cysteinyl-glycine in the form of disodium salt and lipoic acid in the form of sodium salt, and coordination compounds formed by palladium, copper and γ-L-glutamyl-L-cysteinyl-glycine wherein the molar ratio of bis-(γ-L-glutamyl)-L-cysteinyl-glycine of disodium salt : palladium : copper is found within the range of 100-10000 : 1-10:1-10.

EFFECT: invention provides hemopoiesis stimulation with no negative effect on the endogenic production of autologous hemopoiesis.

8 cl, 4 ex, 14 tbl

Description

FIELD OF THE INVENTION

This invention relates to bio-inorganic chemistry and medicine, in particular to the field of production of drugs, and can be used in bio-inorganic chemistry, pharmacology, medicine and veterinary medicine.

State of the art

Total hemodepression of varying severity occurs in almost a hundred percent of cases during chemotherapy in oncology, antibiotic therapy of chronic purulent diseases in surgery, during hemodialysis, in case of massive lesions in natural and man-made disasters. Medicines for the treatment of hemodepression: drugs based on erythropoietin and colony stimulating factors, thrombopoietins, are high-molecular products of biotechnological synthesis of a peptide nature. One of the drawbacks of these drugs is side effects that are dangerous to health, including the negative effect on the endogenous production of their own hematopoietic stimulants, the need to treat them mainly in a hospital, and low-temperature storage, which is practically unacceptable for emergency supplies. The production of stimulants of erythropoiesis (erythropoietins), leukopoiesis (neupogen), thrombopoiesis requires complex technological methods. Erythropoietins, neupogen, thrombopoietins are not suitable for creating means of an inviolable reserve in case of mass injuries in natural and man-made disasters.

Therefore, there is a need to develop a hematopoietic stimulator with therapeutic efficacy not lower than that of selective hematopoietic stimulation agents: erythropoietin, colony-stimulating factors, thrombopoietin, which provide an opportunity to use an emergency reserve in case of emergencies with a large number of injured and affected people who need hemostatic stimulators .

SUMMARY OF THE INVENTION

The objective of the invention was to provide a means to stimulate hematopoiesis of all blood sprouts, which does not adversely affect the endogenous production of its own hematopoiesis, and also the physiologically established interaction of endogenous hematopoiesis stimulants and blood sprouts. Such a tool should preferably have a long shelf life.

This problem is solved by the fact that a combination of bis- (γ-L-glutamyl) -L-cysteinyl-glycine of dilithium salt and coordination compounds formed by palladium, copper and (γ-L-glutamyl) -L-cysteinyl-glycine (a tool for invention).

It is advisable when in such a combination the molar ratio of bis- (γ-L-glutamyl) -L-cysteinyl-glycine of dilithium salt: palladium: copper is in the range of 100-10000: 1-10: 1-10, for example 1000: 1: 1 .

In such a combination, cis-diaminodichloropalladium can be taken as a source of palladium, and copper dichloride as a source of copper.

The proposed combination can be obtained by oxidation of the lithium salt - (γ-L-glutamyl) -L-cysteinyl-glycine using cis-diaminodichloropalladium and copper dichloride.

Such a combination can be used in therapy, in particular as a stimulator of hematopoiesis.

Also provided is a pharmaceutical composition comprising said combination in an effective amount and pharmaceutically acceptable excipients.

Such a composition has the activity of a hematopoietic stimulant.

A method for stimulating hematopoiesis is also provided in which a combination or pharmaceutical composition of the invention is administered to a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The proposed combination is able to stimulate all blood sprouts, does not adversely affect the endogenous production of its own hematopoiesis stimulants, as well as the physiologically established interaction of endogenous hematopoiesis stimulants and blood sprouts. The specified combination can be stored for a long time at room temperature.

Glutathione, gamma-glutaminyl-cysteinyl-glycine, is a tripeptide formed by the residues of three amino acids: glutamic acid, cysteine and glycine.

Glutathione is found in all living organisms and is important for redox reactions due to the ability of the sulfhydryl group (SH-) of cysteine to enter a reversible reaction:

Reduced glutathione (GSH) is referred to herein as (γ-L-glutamyl) -L-cysteinyl-glycine. The oxidized form of glutathione (GSSG) is designated as bis- (γ-L-glutamyl) -L-cysteinyl-glycine.

Unless otherwise indicated, glutathione in this description means reduced glutathione.

Glutathione is able to form salts with cations. GSSGLi ₂ denotes the dilithium salt of glutathione.

The combination of the invention contains d-metals. The sources of el-metals, palladium and copper can be their various salts, which, when dissolved in water, lead to the formation of complex compounds. In particular, when simple copper (II) salts, such as CuCl ₂ or CuBr ₂ , are introduced into the aqueous solution, they are subjected to aquotation, followed by subsequent hydrolysis and the formation of copper (II) oligonuclear aquahydroxo complexes in the solution. Palladium can also be added in the form of its suitable salts, in particular cis-diaminodichloropalladium.

The reaction of obtaining the agent according to the invention GSSGLi ₂ / Pd / Cu = 1000/1/1 corresponds to the equation:

In the catalytic oxidation of RSH thiols, one of the most important functions of palladium is the formation of coordination compounds - products of the addition of RS ^- thiolate ions to the palladium ion, followed by their oxidation and destruction of the coordination polyhedron.

, водные растворы, содержащие Pd-Cu катализаторы, по данным ¹H ЯМР, ИК спектроскопии и ВЭЖХ устойчивы к процессам разложения.Experimental studies of catalytic systems using copper-palladium catalysts show their significantly greater catalytic efficiency compared to the binuclear thiolabatic palladium (II) compounds. Unlike aqueous solutions of oxidized glutathione GSSG containing copper thiolate complexes

, aqueous solutions containing Pd-Cu catalysts, according to ¹ H NMR, IR spectroscopy and HPLC, are resistant to decomposition processes.

Studies show that in Pd-Cu catalysts, the Pd: Cu ratios optimally range from 1: 0.2 to 1: 2.

For the processes of mild selective oxidation of GSH to GSSG, we can conclude that the binuclear thiolabridge palladium (II) complexes perform the main function of oxidation catalysts, and the copper (I) thiolate complexes should be attributed to chemical sites that change their catalytic activity or, in other words, managing their catalytic activity.

, с бидентатномостиковой координацией глутатиона с управляющим сайтом типа

, образующимся из солей меди СиХ₂ (где Х=Сl^- или Вr^-), превращающихся в среде тиолов в соответствующие тиолатные производные комплексов меди(I), показывает один из наиболее эффективных подходов к управлению активностью каталитических систем на основе комплексов палладия

и

.Thus, a combination of functional binuclear palladium coordination compounds, for example, the formula

, with bidentate bridging glutathione coordination with a site type control

formed from copper salts CuX ₂ (where X = Cl ^- or Br ^- ), which are converted in the environment of thiols into the corresponding thiolate derivatives of copper (I) complexes, shows one of the most effective approaches to controlling the activity of catalytic systems based on palladium complexes

and

.

The formation of complex compounds, including Cu, Pd and GSH in comparable amounts, provides the biological effect of the proposed combination.

To obtain the pharmaceutical compositions of the invention, pharmaceutically acceptable excipients are used. In particular, these are inorganic or organic carriers. Lactose, corn starch or its derivatives, talc, stearic acid or its salts and the like. can be used, for example, as such carriers for tablets, coated tablets, dragees and hard gelatine capsules. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Suitable carriers for solutions and syrups are, for example, water, polyols, sucrose, invert sugar, glucose and the like. Suitable carriers for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.

In addition, pharmaceutical compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for regulating the osmotic pressure, buffers, masking agents or antioxidants and other necessary components.

In general, all products and methods of the invention may alternatively include, consist of or essentially consist of any suitable components and steps disclosed herein or known to those skilled in the art, and such products or methods of the invention may further or alternatively exclude any component, or stage, or object that is used in a product or method known from the prior art, or which is not necessary to achieve the technical result of the present invention.

Specific Embodiments

Example 1. Obtaining the means of the invention bis- (γ-L-glutamyl) -L-cysteinyl-glycine dilithium salt: cis-diaminodichloropalladium: copper dichloride (GSSGLi ₂ / Cu = 1000/1/1)

To carry out the reaction, 330 g (1.074 mol) of reduced glutathione are suspended in 3100 ml of water. To the resulting suspension, 39.67 g (0.537 mol) of lithium carbonate are added with constant stirring. After completion of the carbon dioxide evolution, the reaction mass is cooled and 0.1135 g (0.537 mmol) of cis-diaminodichloropalladium and 0.0915 g (0.537 mmol) of 2-aqueous copper chloride are added. To the resulting solution, with constant stirring, add 304.5 g of 6% solution (0.537 mol) of hydrogen peroxide, controlling the temperature, the values of which should not exceed 15 ° C. After adding peroxide, the resulting solution was kept at room temperature for 1.5 hours, filtered through a 0.22µ filter and lyophilized. The yield of bis (γ-L-glutamyl) -L-cysteinyl-glycine dilithium salt: cis-diaminodichloropalladium: copper dichloride (in a ratio of 1000: 1: 1) is 99%.

Example 2. The effect of the agent according to the invention on the state of hematopoiesis during cyclophosphamide-induced hemosuppression

The agent of the invention provides a combination of lithium cations and glutathione disulfide anions in combination with copper and palladium bioelement complexes. Compounds forming the product of chemical synthesis characterize a wide range of biological activity, including hematopoiesis stimulation (myelogenesis and lymphopoiesis).

Purpose of the study. Determining the ability of the agent according to the invention to stimulate hematopoiesis.

Preparation of solutions of the test compounds for the study.

The synthesized substance was stored at 4 ° C; immediately before the start of the experiment, the substance was dissolved in physiological saline. The solution was sterilized by passing through 0.22 μm Millex-GS filters (Millipore) in a sterile laminar box.

A solution of the agent of the invention was labeled V-002.

As a means of comparison, we used the erythropoiesis stimulator epocrine, myelopoiesis stimulant leycomax, and the recommended drugs for the treatment of toxic leukopenia pentoxil and methyluracil.

Study Models

The study was conducted on white randomly bred male rats weighing 140-160 g. Hematopoiesis stimulation was studied using radiation, toxic and traumatic hemodepression models.

Radiation pancytopenia model

To irradiate rats with ionizing radiation, the IGUR-1 gamma installation was used. The absorbed dose of gamma radiation per animal was 3.5 Gy, with a dose rate of 1.4 Gy / min.

The animals were irradiated with gamma rays at the IGUR-1 facility. The absorbed dose was 2.3 Gy (LD30 / 45), with a dose rate of 1.4 Gy / min.

Cyclophosphamide myelosuppression model

Cyclophosphamide was administered once under the skin of the back at a dose of 100 mg / kg (the solvent is water for injection).

Lead Anemia Model

Male adult rats weighing 170-180 g daily for 7 days are injected subcutaneously with a solution of lead acetic acid (lead acetate) at a dose of 50 mg / kg. After 10 days, hemoglobin and red blood cells are reduced by an average of 70-80% from the initial level. Drugs begin to be administered from the 11th day from the start of the experiment.

Benzene Leukopenia Model

Adult male rats weighing 170-180 g daily for 7 days are injected subcutaneously with benzene in doses of 0.1 mg / kg. After 8 days, the number of leukocytes decreases by 2-2.5 times, and granulocytes - by 10 times compared with the initial level. Drugs begin to be administered from the 9th day from the start of the experiment.

Post-Traumatic Anemia Model (Prolonged Compression Syndrome)

Compression injury. In order to inflict compression trauma, the soft tissues of the hind limbs of white rats and mice were subjected to compression using a special design vise. The compression lasted 2.5-3 hours. In white mice, before setting up the main experiment, the compression time was determined at which the death of mice in the subsequent 3 days after injury was about 50%; this time was in the range of 2.5-3 hours. For rats, the compression time required for the development of anemia, but not causing the death of animals, was 3 hours

The agent of the invention (V-002) was administered intramuscularly (thigh) once a day for 10 days at a dose of 10 mg / kg. Animals were divided into the following groups: intact animals (once subcutaneous saline), control group (animals exposed, but not treated), experimental groups (animals exposed, exposed, treated with the agent according to the invention or comparison drug). Each group included 10 animals.

Hematological studies were carried out by standardized standard methods.

results

Description of the results of studies proving the effectiveness of the proposed development on experimental models

The results of experimental studies are presented in tables 1-6.

Table 1 Complete blood count of male rats with radiation cytopenia 3 days after gamma irradiation (3.5 Gy) Research indicators Biological control Exposure control Leikomaks + radiation V-002 + exposure Hemoglobin, g / l 15.2 ± 0.16 13.0 ± 0.2 12.9 ± 0.22 12.5 ± 0.25 Red blood cells, 10 ¹² / L 6.7 ± 0.16 6.2 ± 0.19 6.2 ± 0.23 5.8 ± 0.15 ESR, mm / h 2.5 ± 0.85 2 ± 0.37 3.3 ± 0.80 3.2 ± 0.54 Platelets, 10 ⁹ L 368 ± 21.5 370 ± 35.3 387 ± 17.4 502 ± 36.4 ** Reticulocytes,% 1.8 ± 0.2 0.08 ± 0.005 * 0.3 ± 0.1 ** 0.5 ± 0.1 ** White blood cells, 10 ⁹ / L 9.2 ± 0.35 0.58 ± 0.026 * 1.38 ± 0.11 * 1.49 ± 0.11 * Stab 0.3 ± 0.1 0 2.5 ± 0.3 0 neutrophils,% Segmented 16 ± 2.5 (1472) 18.7 ± 2.4 (108) * 42 ± 1.6 (580) ** 38 ± 1.6 (566) ** neutrophils,% Eosinophils,% 2 ± 0.3 0 0 0 Monocytes,% 5.7 ± 0.5 11.6 ± 0.6 10.3 ± 0.5 10.6 ± 0.6 Lymphocytes,% 75.5 ± 1.4 (6945) 71 ± 1.8 (411) * 45 ± 1.6 (621) 51.3 ± 0. (764) ** - in brackets - the absolute number of cells; - * - p 0.05 - compared with biological control; - ** - p 0.05 - compared with the irradiated control.

table 2 Complete blood count of male rats with radiation cytopenia 10 days after gamma irradiation (3.5 Gy) Research indicators Biological control Exposure control Leikomaks + radiation V-002 + exposure Hemoglobin, g / l 15.2 ± 0.16 13.8 ± 0.56 13.2 ± 0.68 13.9 ± 0.31 Red blood cells, 10 ¹² / L 6.7 ± 0.16 6.0 ± 0.25 6.2 ± 0.18 6.1 ± 0.08 ESR, mm / h 2.5 ± 0.85 1.8 ± 0.31 3.2 ± 0.80 2.0 ± 0.37 Platelets, 10 ³ / L 368 ± 21.5 167 ± 19.3 * 126 ± 10.0 * 228 ± 13.8 ** Reticulocytes,% 1.8 ± 0.2 1.0 ± 0.1 1.5 ± 0.2 1.33 ± 0.2 White blood cells, 10 ⁹ / L 9.2 ± 0.35 2.5 ± 0.15 * 3.41 ± 0.09 * 4.88 ± 0.18 ** Stab 0.3 ± 0.1 0 2 ± 0.3 2 ± 0.2 neutrophils,% Segmented 16 ± 2.5 (1472) 32 ± 1.9 (800) * 50.4 ± 0.6 (1719) ** 63.3 ± 1.6 (3456) neutrophils,% ** Eosinophils,% 2 ± 0.3 1.7 ± 0.3 1.7 ± 0.2 0.3 ± 0.1 Monocytes,% 5.7 ± 0.5 8.7 ± 0.4 5.3 ± 0.5 5.7 ± 0.4 Lymphocytes,% 75.5 ± 1.4 (6945) 57.7 ± 1.1 (1443) * 41 ± 0.9 (1398) * 22.6 ± 0.8 (877) * - in brackets - the absolute number of cells - * - p 0.05 - compared with biological control - ** - p 0.05 - compared with the irradiated control

Table 3 Complete blood count of male rats with radiation cytopenia 15 days after gamma irradiation (3.5 Gy) Research indicators Biological control Exposure control Leikomaks + radiation V-002 + exposure Hemoglobin, (g / l) 15.2 ± 0.16 13.3 ± 0.68 11.6 ± 0.63 12.5 ± 0.52 Red blood cells, 10 ¹² / L 6.7 ± 0.16 5.7 ± 0.17 5.7 ± 0.26 5.2 ± 0.12 ESR, mm / h 2.5 ± 0.85 2.3 ± 0.61 2.7 ± 0.49 4.8 ± 0.70 Platelets, 10 ³ / L 368 ± 21.5 273 ± 56.9 290 ± 38.3 498 ± 72.9 ** Reticulocytes,% 1.8 ± 0.2 1.2 ± 0.2 1.7 ± 0.3 2 ± 0.2 White blood cells, 10 ⁹ / L 9.2 ± 0.35 2.9 ± 0.04 3.6 ± 0.22 5.6 ± 0.19 ** Stab 0.3 ± 0.1 0 0 0.7 ± 0.3 neutrophils,% Segmented 16 ± 2.5 (1472) 14.3 ± 1.6 (415) 20.3 ± 2.4 (731) 26 ± 2.2 (1456) neutrophils,% Eosinophils,% 2 ± 0.3 0 0.6 ± 0.2 0 Monocytes,% 5.7 ± 0.5 12.3 ± 0.6 6.2 ± 0.8 9.0 ± 0.7 Lymphocytes,% 75.5 ± 1.4 73.4 ± 1.8 (2129) 72.5 ± 1.4 (2610) 64.3 ± 0.9 (3600) - in brackets - the absolute number of cells

Table 4 Bone marrow analysis of male rats with radiation cytopenia 3 days after gamma irradiation (3.5 Gy) Research indicators Biological control Exposure control Leikomaks + radiation V-002 + exposure The number of JAX in the leg 10 ⁶ 10.0 ± 0.49 0.7 ± 0.08 2.6 ± 0.64 ** 2.7 ± 0.75 ** Erythroblasts,% 0.8 ± 0.05 0.4 ± 0.02 0.5 ± 0.02 0.5 ± 0.02 Pronormocytes,% 0.6 ± 0.02 0.5 ± 0.03 0.5 ± 0.02 0.4 ± 0.05 Basophilic 7.3 ± 0.03 6.2 ± 0.13 5.8 ± 0.19 5.7 ± 0.21 normocytes,% Polychromatophilic 8.4 ± 0.04 8.3 ± 0.07 8.3 ± 0.09 8.5 ± 0.09 normocytes,% Oxyphilic 5.7 ± 0.3 8.5 ± 0.11 8.9 ± 0.1 9.0 ± 0.08 normocytes,% The number of mitoses red 0.5 ± 0.02 0.2 ± 0.06 0.1 ± 0.03 0.15 ± 0.03 blood% Myeloblasts,% 2.4 ± 0.2 1.1 ± 0.09 1.4 ± 0.06 1.6 ± 0.07 ** Promyelocytes,% 4.3 ± 0.21 2.1 ± 0.05 2.4 ± 0.04 2.5 ± 0.06 Myelocytes,% 0.2 ± 0.02 6.0 ± 0.13 6.2 ± 0.07 6.1 ± 0.04 Metamyelocytes,% 7.2 ± 1.01 7.4 ± 0.13 7.5 ± 0.13 7.5 ± 0.07 Stab 11.8 ± 0.04 8.3 ± 0.08 9.6 ± 0.22 9.3 ± 0.21 neutrophils,% Segmented 21.5 ± 1.3 33.8 ± 0.49 31.9 ± 0.23 32.6 ± 0.6 neutrophils,% Eosinophils,% 7.5 ± 0.03 5.4 ± 0.1 5.7 ± 0.14 5.8 ± 0.6 The number of white mitoses 0.5 ± 0.01 0.1 ± 0.04 0.2 ± 0.06 0.3 ± 0.05 ** blood% Lymphocytes,% 8.2 ± 0.08 7.6 ± 0.18 7.2 ± 0.1 7.1 ± 0.06 Plasma cells, 1.5 ± 0.01 1.0 ± 0.07 1.1 ± 0.03 1.0 ± 0.03 % Monocytes,% 0.9 ± 0.2 1.1 ± 0.14 1.1 ± 0.04 1.2 ± 0.04 Reticulocytes,% 2.5 ± 0.2 0.4 ± 0.06 0.4 ± 0.05 0.4 ± 0.03 Megacaryoblasts and 0.4 ± 0.01 0.6 ± 0.04 0.8 ± 0.07 ** 0.8 ± 0.05 ** megakaryocytes,% Undifferentiated 0.5 ± 0.02 0.8 ± 0.09 0.6 ± 0.04 0.5 ± 0.02 blasts,%

Table 5 Bone marrow analysis of male rats with radiation cytopenia 10 days after gamma irradiation (3.5 Gy) Research indicators Biological control Exposure control Leikomaks + radiation V-002 + exposure The number of YSC in the lower leg, 10 ⁶ 10.0 ± 0.49 0.9 ± 0.08 4.2 ± 0.46 ** 6.6 ± 0.47 ** Erythroblasts,% 0.8 ± 0.05 0.6 ± 0.03 0.6 ± 0.03 0.6 ± 0.02 Pronormocytes,% 0.6 ± 0.02 0.5 ± 0.03 0.5 ± 0.02 0.6 ± 0.04 Basophilic normocytes, 7.3 ± 0.03 7.1 ± 0.14 7.2 ± 0.09 7.0 ± 0.06 % Polychromatophilic 8.4 ± 0.04 8.4 ± 0.1 8.6 ± 0.11 8.6 ± 0.13 normocytes,% Oxyphilic normocytes, 5.7 ± 0.3 7.3 ± 0.08 8.9 ± 0.09 8.9 ± 0.06 % The number of mitoses red 0.5 ± 0.02 0.3 ± 0.01 0.3 ± 0.006 0.3 ± 0.05 blood% Myeloblasts,% 2.4 ± 0.2 1.6 ± 0.07 2.5 ± 0.04 ** 2.6 ± 0.04 ** Promyelocytes,% 4.3 ± 0.21 2.0 ± 0.06 3.1 ± 0.03 ** 3.2 ± 0.06 ** Myelocytes,% 0.2 ± 0.02 5.2 ± 0.11 6.4 ± 0.07 ** 6.6 ± 0.07 ** Metamyelocytes,% 7.2 ± 1.01 8.2 ± 0.13 7.3 + 0.1 7.5 ± 0.09 Stab 11.8 ± 0.04 11.1 ± 0.33 9.5 ± 0.2 9.8 ± 0.3 neutrophils,% Segmented 21.5 ± 1.3 30.1 ± 0.64 25.9 ± 0.56 25.7 ± 0.4 neutrophils,% Eosinophils,% 7.5 ± 0.03 4.5 ± 0.3 5.7 ± 0.16 5.6 ± 0.15 The number of white mitoses 0.5 ± 0.01 0.2 ± 0.09 0.31 ± 0.04 0.38 ± 0.07 blood% Lymphocytes,% 8.2 ± 0.08 7.9 ± 0.19 8.2 ± 0.09 7.9 ± 0.16 Plasma cells, 1.5 ± 0.01 1.3 ± 0.05 1.3 ± 0.08 1.2 ± 0.03 % Monocytes,% 0.9 ± 0.2 1.6 ± 0.08 1.5 ± 0.08 1.4 ± 0.1 Reticulocytes,% 2.5 ± 0.2 1.4 ± 0.05 1.4 ± 0.08 1.9 ± 0.04 ** Megacaryoblasts and 0.4 ± 0.01 1.4 ± 0.02 0.5 ± 0.02 0.7 ± 0.03 ** megakaryocytes,% Undifferentiated 0.5 ± 0.02 0.6 ± 0.02 0.8 ± 0.03 ** 0.8 ± 0.03 ** blasts,%

Table 6 Bone marrow analysis of male rats with radiation cytopenia 15 days after gamma irradiation (3.5 Gy) Research indicators Biological control Exposure control Leikomaks + radiation + V-002 + radiation The number of JAX in the leg 10 ⁶ 10.0 ± 0.49 1.0 ± 0.1 6.2 ± 1.4 ** 7.6 ± 1.03 ** Erythroblasts,% 0.8 ± 0.05 0.7 ± 0.01 0.8 ± 0.03 0.8 ± 0.02 Pronormocytes,% 0.6 ± 0.02 0.5 ± 0.03 0.6 ± 0.02 0.7 ± 0.03 Basophilic 7.3 ± 0.03 7.2 ± 0.2 7.4 ± 0.03 7.2 ± 0.05 normocytes,% Polychromatophilic 8.4 ± 0.04 8.5 ± 0.1 8.4 ± 0.05 8.5 ± 0.15 normocytes,% Oxyphilic 5.7 ± 0.3 7.0 ± 0.06 8.8 ± 0.04 ** 8.8 ± 0.09 ** normocytes,% The number of mitoses red 0.5 ± 0.02 0.4 ± 0.06 0.4 ± 0.04 0.4 ± 0.01 blood% Myeloblasts,% 2.4 ± 0.2 2.3 ± 0.07 3.0 ± 0.04 ** 3.0 ± 0.05 ** Promyelocytes,% 4.3 ± 0.21 4.2 ± 0.05 4.5 ± 0.05 4.6 ± 0.14 Myelocytes,% 0.2 ± 0.02 6.3 ± 0.12 6.9 ± 0.03 ** 7.0 ± 0.12 ** Metamyelocytes,% 7.2 ± 1.01 8.5 ± 0.14 8.1 ± 0.02 8.2 ± 0.17 Stab 11.8 ± 0.04 12.4 ± 0.2 11.9 ± 0.11 12.4 ± 0.16 neutrophils,% Segmented 21.5 ± 1.3 21.3 ± 0.7 19.5 ± 0.19 18.8 ± 0.78 neutrophils,% Eosinophils,% 7.5 ± 0.03 6.0 ± 0.18 5.1 ± 0.09 5.3 ± 0.07 The number of white mitoses 0.5 ± 0.01 0.3 ± 0.07 0.4 ± 0.04 0.4 ± 0.01 blood% Lymphocytes,% 8.2 ± 0.08 8.3 ± 0.12 8.1 ± 0.03 7.8 ± 0.16 Plasma cells, 1.5 ± 0.01 1.3 ± 0.13 1.3 ± 0.03 1.4 ± 0.05 % Monocytes,% 0.9 ± 0.2 1.7 ± 0.13 1.4 ± 0.02 1.7 ± 0.08 Reticulocytes,% 2.5 ± 0.2 2.1 ± 0.18 2.5 ± 0.03 2.5 ± 0.11 Megacaryoblasts and 0.4 ± 0.01 0.5 ± 0.03 0.6 ± 0.05 * 0.9 ± 0.06 ** megakaryocytes,% Undifferentiated 0.5 ± 0.02 1.0 ± 0.06 0.8 ± 0.04 0.8 ± 0.04 blasts,%

Studies have shown that both drugs have hemostimulating activity and contribute to the correction of post-radiation myelosuppression. It is noteworthy that already on the 3rd day after irradiation of experimental animals with the introduction of Leukomax and V-002, the number of peripheral blood leukocytes increases by an average of 3 times compared with the level of irradiated control. At the same time, a similar dynamics was revealed in the number of nucleated cells of the bone marrow of these animals. An increase (on average 2 times) in the number of white blood mitoses and the number of segmented neutrophils was also revealed. On the 10th day after radiation exposure, both studied drugs equally have a stimulating effect on the myeloid germ, which is manifested in an increase in the number of leukocytes. However, it should be noted that the number of segmented neutrophils in the peripheral blood of irradiated animals treated with the drugs increases (in absolute terms, by 2–3 times. V-002 has the greatest activity for this period of study. In addition, the use of this drug promotes the activation of megakaryoblasts, which found reflection in preventing a decrease in the number of platelets in the peripheral blood of irradiated animals (10 days of the study). By the 15th day after radiation exposure, the platelet count is not only it fills background values, but also exceeds it by an average of 1.5 times, during this period a significant increase (almost 2 times compared with the irradiated control) of the number of leukocytes in peripheral blood in the group of animals treated with V-002, as well as an increase (on average 3 times) the number of segmented neutrophils and lymphocytes.

The effect of the drug V-002 on the characteristics of peripheral blood and myelograms when administered to intact animals and animals with cyclophosphamide cytopenia is shown in tables 7 and 8.

Table 7 Complete blood count of male rats with cytopenia with prior administration of cyclophosphamide at a dose of 100 mg / kg and treatment with V-002 Research indicators Intact (saline) Cyclophosphamide V-002, 10 mg / kg Cyclophosphamide + V-002.10 mg / kg 10th day Hemoglobin, g / dl 12.9 ± 04 11.0 ± 0.4 * 14.0 ± 02 12 9 ± 0.4 Hematocrit, g% 47.3 ± 1.0 32.4 ± 0.9 * 52.7 ± 1.1 46.7 ± 10 Red blood cells, 10 ¹² / L 6.2 ± 0.3 3.2 ± 0.4 * 6.5 ± 0.3 5.4 ± 03 Color indicator (SIT), pg 17.5 ± 0.3 15.1 ± 0.4 * 18.5 ± 0.5 16.2 ± 0.2 ESR, mm / h 5, 5 ± 0.2 7.4 ± 0.3 * 5.6 ± 0.2 5.8 ± 0.2 Platelets, 10 ⁹ / L 680 ± 54 420 ± 42 * 730 ± 65 703 ± 56 Reticulocytes,% 2.1 ± 0.3 0 * 2.1 ± 0.1 2.0 ± 05 White blood cells, 10 ⁹ / L 7.1 ± 02 1.8 ± 03 * 7.4 ± 0 3 5.2 ± 0.3 * Band neutrophils,% 0.5 ± 0.3 25 ± 07 0 0.6 ± 0.3 Segmented neutrophils,% 12.3 ± 3.0 54.4 ± 57 * 15.3 ± 2.9 27.3 ± 2.7 Basophils,% 0 0 0 0 Lymphocytes,% 79.2 ± 3.5 33.6 ± 2.5 * 80.3 ± 3.9 58.0 ± 3.8 Monocytes,% 2.5 ± 0.2 7.9 ± 0.7 * 1.8 ± 0.7 3.8 ± 0.5 Eosinophils,% 2.3 ± 0.3 0 2.1 ± 0.3 2.0 ± 0.5 Plasma cells,% 0.5 ± 0.2 0 0 2.9 ± 0.3 * Normocytes,% 0 0 0 1.0 ± 0.4 Polychromatophiles,% 0 1.2 ± 0.5 0 0.7 ± 0.3 15th day Hemoglobin, g / dl 13.4 ± 0.3 10.4 ± 0.3 * 14.1 ± 0.3 13.2 ± 0.3 Hematocrit g% 48.5 ± 0.9 31.3 ± 1.1 * 51.1 ± 0.8 48.2 ± 0.6 Red blood cells, 10 ¹² / L 6.3 ± 0.2 3.3 ± 0.3 * 6.2 ± 0.2 5.8 ± 0.3 Color indicator (SIT), Pg 17.6 ± 0.3 14.6 ± 0.4 * 18.2 ± 0.4 17.0 ± 0.4 ESR, mm / h 5.4 ± 0.2 7.6 ± 0.3 * 5.1 ± 0.3 5.5 ± 0.3 Platelets, 10 ⁹ / L 695 ± 60 340 ± 47 * 724 ± 54 724 ± 42 Reticulocytes,% 20 ± 0.2 0.3 ± 0.3 2.4 ± 0.4 1.3 ± 0.4 White blood cells, 10 ⁹ / L 7.6 ± 0.3 1.4 ± 02 * 8.2 ± 0.2 5.4 ± 0.3 * Band neutrophils,% 05 ± 03 1.9 ± 0.6 0 0.9 ± 0., 2 Segmented neutrophils,% 13.2 ± 2.7 51.3 ± 4.4 * 14.0 ± 2.1 34.8 ± 2.3 Basophils,% 0 0 0 0 Lymphocytes,% 781 ± 2.4 35.5 ± 2.8 * 78.5 ± 2.6 58.3 ± 3.3 * Monocytes,% 2.1 ± 0.2 6.3 ± 0.5 * 2.4 ± 0.3 3.0 ± 0.3 Eosinophils,% 2.0 ± 0.4 0.3 ± 0.2 1.7 ± 0.1 0 Plasma cells,% 0.6 ± 0.2 0 0.5 ± 0.2 2.2 ± 0.4 * Normocytes. % 0 0 0 0 Polychromatophiles,% 0 2.2 ± 1.1 0 1.3 ± 0.7 Note: the 't) icon indicates significant differences from the intact group at a confidence level of P> 0.95.

Table 8 Myelogram of male rats with cytopenia with prior administration of cyclophosphamide at a dose of 100 mg / kg and treatment of V-002 at doses of 5 and 10 mg / kg Research indicators Intact (saline) Cyclophosphamide V-002, 10 mg / kg Cyclophosphamide + V-002, 10 mg / kg 10th day The number of YSC cells, 10 ⁶ / l 73.2 ± 4.6 34.7 ± 5.0 * 76.4 ± 5.0 60.4 ± 6.6 Undifferentiated blasts,% 0.5 ± 0.1 0 * 0.5 ± 0.2 0.4 ± 0.2 Proerythroblasts,% 0 0 0.1 ± 0.05 0.1 ± 0.05 Erythroblasts,% 0.8 ± 0.1 0.4 ± 0.1 1.0 ± 0.2 1.7 ± 0.3 * Pronormoblasts,% 0.6 ± 0.1 0 * 0.7 ± 0.1 0.6 ± 0.1 Basophilic normoblasts,% 7.4 ± 0.8 2.8 ± 0.4 * 7.3 ± 0.6 6.4 ± 0.4 Polychromatophilic normoblasts,% 8.3 ± 1.0 3.2 ± 1.2 * 8.6 ± 1.5 11.0 ± 1.9 * Eosinophilic normoblasts,% 5.8 ± 1.2 2.4 ± 0.8 5.2 ± 1.3 8.0 ± 1.4 The number of mitoses of red blood,% 0.56 ± 0.10 0.19 ± 0.08 * 0.60 ± 0.12 0.75 ± 0.16 Myeloblasts,% 2.2 ± 0.5 1.2 ± 0.2 * 2.4 ± 0.3 2.0 ± 0.6 Promyelocytes,% 4.0 ± 0.4 1.8 ± 0.5 * 4.3 ± 0.6 4.0 ± 0.6 Myelocytes,% 6.2 ± 0.7 3.4 ± 1.1 * 6.1 ± 0.7 5.5 ± 0.9 Metamyeloiditis,% 8.1 ± 1.2 4.5 ± 0.8 * 8.3 ± 1.0 6.8 ± 1.4 Band neutrophils,% 11.8 ± 1.4 4.2 ± 1.0 * 12.0 ± 1.4 7.7 ± 1.3 Segmented neutrophils,% 16.6 ± 3.2 35.2 ± 4.3 * 17.2 ± 4.1 32.5 ± 4.8 * Eosinophils,% 7.5 ± 1.1 3.2 ± 1.0 * 7.6 ± 1.1 7.0 ± 1.3 Basophils,% 0 0 0 0.1 ± 0.05 The number of mitoses of white blood,% 0.45 ± 0.13 0.17 ± 0.04 * 0.44 ± 0.12 0.37 ± 0.13 Prolymphocytes,% 0.1 ± 0.1 0 0 0.1 ± 0.1

Research indicators Intact (saline) Cyclophosphamide V-002, 10 mg / kg Cyclophosphamide + V-002, 10 mg / kg Lymphocytes,% 8.0 ± 1.0 2.1 ± 0.5 * 8.5 ± 1.3 6.1 ± 0.8 Plasma cells,% 1.4 ± 0.3 0 1.2 ± 0.3 0.8 ± 0.2 * Promonocytes,% 0 0 0 0 Monocytes,% 0.7 ± 0.2 0.2 ± 0.05 * 0.8 ± 0.2 0.4 ± 0.2 Reticulocytes,% 2.4 ± 0.5 1.0 ± 0.3 2.2 ± 0.4 2.3 ± 1.0 Megakaryocytes and megakaryoblasts,% 0.43 ± 0.08 0.13 ± 0.05 * 0.53 ± 0.00 0.34 ± 0.09 Number of cells, 10 ¹¹ / L 71.6 + 4.5 35.5 + 4.8 * 74.6 ± 5.6 63.6 ± 6.2 Undifferentiated blasts,% 0.5 ± 0.1 0 * 0.5 ± 0.1 0.6 ± 0.3 Proerythroblasts,% 0 0 0.1 ± 0.05 0.1 ± 0.05 Erythroblasts,% 0.9 ± 0.2 0.3 ± 0.1 * 1.2 ± 0.2 1.8 ± 0.3 * Pronormoblasts,% 0.7 ± 0.1 0.2 ± 0.1 0.7 ± 0.1 0.5 ± 0.1 Basophilic normoblasts,% 7.5 ± 0.5 2.3 ± 0.5 * 7.7 ± 0.8 6.6 ± 1.0 Polychromatophilic normoblasts,% 8.8 ± 1.2 3.0 ± 0.8 * 8.8 ± 1.2 10.3 ± 1.6 * Eosinophilic normoblasts,% 5.7 ± 1.0 2.8 ± 0.9 5.4 ± 1.4 7.0 ± 1.5 The number of mitoses of red blood,% 0.57 ± 0.11 0.20 ± 0.10 * 0.63 ± 0.12 0.74 ± 0.12 Myeloblasts,% 2.1 ± 0.2 1.4 ± 0.4 2.2 ± 0.4 2.1 ± 0.8 Promyelocytes,% 40 ± 0.6 1.5 ± 0.3 * 4.0 ± 0.8 3.7 ± 0.7 Myelocytes,% 6.1 ± 0.6 3.3 ± 0.5 * 6.2 ± 0.5 5.3 ± 0.6 Metamyelocytes,% 8.4 ± 1.0 3.5 ± 0.9 * 8.2 ± 1.1 6.7 ± 1.1 Band neutrophils,% 11.2 ± 1.1 3.5 ± 0.7 * 11.7 ± 1 3 7.9 ± 1.6 Segmented neutrophils,% 16.2 ± 2.7 38.6 ± 48 * 16.4 ± 40 31.1 ± 3.9 * Eosinophils,% 7.4 ± 1.0 3.1 ± 0.9 * 7.7 ± 1.3 7.1 ± 1.3 Basophils,% 0 0 0 0 The number of mitoses of white blood,% 0.46 ± 0.10 0.15 ± 0.04 * 0.45 ± 0.14 0.35 ± 0.10 Prolymphocytes,% 0 0 0.3 ± 0.2 0.2 ± 0.1 Lymphocytes,% 8.1 ± 0.7 2.0 ± 0.4 * 8.2 ± 1.2 6.3 ± 2.0 Plasma cells,% 1.2 ± 0.2 0 1.7 ± 0.4 1.0 ± 0.5 Promonocytes,% 0 0 0.1 ± 0.05 0 Monocytes,% 0.8 ± 0.2 0.1 ± 0.05 * 0.8 ± 0.2 0.5 ± 0.2 Reticulocytes,% 2.3 ± 0.5 1.4 ± 0.3 2.7 ± 0.5 1.9 ± 0.6 Megakaryocytes and megakaryoblasts,% 0.51 ± 0.12 0.18 ± 0.08 * 0.55 ± 0.08 0.47 ± 0.10

The introduction of V-002 has a clear stimulating effect, more pronounced on the red sprout of hematopoiesis. In this case, we can say that erythroid processes replaced leukocyte processes. In addition, there was an expansion of the granulocyte germ. The appearance of immature forms in the blood indicates the activation of bone marrow hematopoiesis.

There is an improvement in general condition, positive dynamics of body weight. No deaths were observed.

The effectiveness of the drug V-002 in a model of lead anemia

The data of analyzes of peripheral blood and myelogamma of animals treated with "V-002" and the comparison drug are presented in tables 9 and 10.

Table 9 Hematological parameters of animals of experimental groups in the model of lead anemia Research indicators Intact Pb Acetate V-002 Acetate Pb + V-002 Pb Acetate + Epocrine Hemoglobin, g / dl 12.9 ± 0.4 5.0 ± 0.4 * 14.0 ± 0.2 - 9.2 ± 0.3 * 10.0 ± 0.4 * Hematocrit, g% 46.6 ± 1.0 19.9 ± 0.9 * 49.1 ± 1.1 30.8 ± 0.9 * 33.0 ± 1.0 * Red blood cells, 10 ¹² / L 6.3 ± 0.3 3.1 ± 0.4 * 6.8 ± 0.3 4.4 ± 0.3 * 4.8 ± 0.3 * Color indicator (SIT), pg 20.5 ± 0.3 16.1 ± 0.4 * 20.6 ± 0.5 18.6 ± 0.2 18.3 ± 0.2 ESR, mm / h 5.5 ± 0.2 17.4 ± 0.3 * 5.6 ± 0.2 13.8 ± 0.3 14.1 ± 0.2 Reticulocytes,% 2.1 ± 0.3 10.1 ± 0.8 2.0 ± 0.5 8.4 ± 0.2 * 8.0 ± 0.5 * Polychromatophiles,% 0.2 ± 0.05 1.2 ± 0.2 * 0.2 ± 0.05 0.7 ± 0.2 0.8 ± 0.2 Erythrocytes with basophilic granularity, 1 / million 140 ± 100 1100 ± 300 * 160 ± 100 400 ± 200 * 520 ± 200 * Platelets, 10 ⁹ / L 680 ± 54 320 ± 42 * 722 ± 86 469 ± 50 * 503 ± 56 * White blood cells, 10 ⁹ / L 7.1 ± 0.2 5.1 ± 0.3 * 7.4 ± 0.3 6.3 ± 0.2 * 6.0 ± 0.3 * Band neutrophils,% 0.5 ± 0.3 0.6 ± 0.7 0.5 ± 0.2 0.6 ± 0.3 0.5 ± 0.4 Segmented neutrophils,% 14.3 ± 3.0 16.4 ± 3.7 14.3 ± 2.9 16.0 ± 3.3 * 17.3 ± 2.7 Basophils,% 0 0 0 0 0 Lymphocytes,% 79.8 ± 2.1 73.5 ± 2.5 78.5 ± 1.9 74.9 ± 2.8 73.5 ± 2.3 Monocytes,% 2.5 ± 0.2 4.9 ± 0.7 * 2.8 ± 0.7 4.8 ± 0.6 * 3.8 ± 0.5 Eosinophils,% 2.3 ± 0.3 4.0 ± 0.3 2.1 ± 0.3 3.2 ± 0.3 4.0 ± 0.5 * Plasma cells,% 0.5 ± 0.2 0.6 ± 0.2 0.3 ± 0.2 0.3 ± 0.2 0.7 ± 0.3 *) - significant differences from the intact group at P> 0.95.

Table 10 The indicators of the myelogram of animals of experimental 5 groups in the model of lead anemia Research indicators Intact Pb Acetate V-002 Acetate Pb + V-002 Pb Acetate + Epocrine Number of cells, 10 ¹¹ / L 73.2 ± 3.6 57.7 ± 4.0 * 76.4 ± 5.0 64.2 ± 4.2 63.4 ± 6.6 Undifferentiated blasts,% 0.1 ± 0.1 1.9 ± 0.6 0.5 ± 0.2 0.2 ± 0.2 0.5 ± 0.2 Proerythroblasts,% 0 0 0.1 ± 0.05 0 0.1 ± 0.05 Erythroblasts,% 0.8 ± 0.1 0.6 ± 0.1 1.0 ± 0.2 1.6 ± 0.3 * 1.7 ± 0.3 * Pronormoblasts,% 0.6 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 0.5 ± 0.2 0.6 ± 0.1 Basophilic normoblasts,% 7.4 ± 0.8 6.9 ± 0.4 7.3 ± 0.6 5.9 ± 0.7 7.0 ± 0.4 Polychromatophilic normoblasts,% 9.3 ± 1.0 8.5 ± 1.2 8.6 ± 1.5 7.2 ± 1.7 8.5 ± 1.9 Eosinophilic normoblasts,% 5.8 ± 1.2 6.3 ± 0.8 5.2 ± 1.3 5.5 ± 1.8 5.4 ± 1.4 The number of mitoses of red blood,% 0.56 ± 0.10 0.35 ± 0.08 * 0.60 ± 0.12 0.71 ± 0.18 0.75 ± 0.16 Myeloblasts,% 2.2 ± 0.5 3.8 ± 0.2 2.3 ± 0.3 2.9 ± 0.8 3.3 ± 0.6 Promyelocytes,% 4.0 ± 0.4 3.8 ± 0.5 4.3 ± 0.6 3.6 ± 0.7 4.0 ± 0.6 Myelocytes,% 6.2 ± 0.7 4.4 ± 1.1 6.1 ± 0.7 5.8 ± 0.8 5.5 ± 0.9 Metamyelocytes,% 9.1 ± 1.2 7.5 ± 0.8 8.3 ± 1.0 7.6 ± 1.4 6.8 ± 1.4 Band neutrophils,% 11.8 ± 1.4 14.2 ± 1.0 12.0 ± 1.4 12.9 ± 1.5 12.7 ± 1.3 Segmented neutrophils,% 16.6 ± 3.2 20.2 ± 4.3 17.2 ± 4.1 18.8 ± 5.5 18.5 ± 4.8 Eosinophils,% 7.5 ± 1.1 6.2 ± 1.0 7.6 ± 1.1 7.2 ± 1.2 7.0 ± 1.3 Basophils,% 0 0 0 0 0.1 ± 0.05 The number of mitoses of white blood,% 0.45 ± 0.13 0.32 ± 0.04 * 0.44 ± 0.12 0.52 ± 0.11 0.48 ± 0.13 Prolymphocytes,% 0.1 ± 0.1 0 0 0.3 ± 0.1 0.2 ± 0.1 Lymphocytes,% 8.3 ± 1.0 6.9 ± 0.5 * 8.5 ± 1.3 7.4 ± 1.1 7.1 ± 0.8 Plasma cells,% 0.8 ± 0.3 0 1.2 ± 0.3 0.7 ± 0.2 0.5 ± 0.2 Promonocytes,% 0 0 0 0.1 ± 0.05 0 Monocytes,% 0.7 ± 0.2 0.6 ± 0.05 0.8 ± 0.2 0.5 ± 0.2 0.7 ± 0.2 Reticulocytes,% 2.4 ± 0.5 4.2 ± 0.3 * 2.2 ± 0.4 3.1 ± 0.8 3.3 ± 1.0 Megakaryocytes and megakaryoblasts,% 0.43 ± 0.08 0.45 ± 0.05 * 0.53 ± 0.09 0.42 ± 0.12 0.34 ± 0.09

Thus, the introduction of the drug "V-002" had a clear stimulating effect, more pronounced on the red sprout of hematopoiesis. In this case, we can say that erythroid processes replaced leukocyte processes. In addition, there was an expansion of the granulocyte germ. The appearance of immature forms in the blood testified to the activation of bone marrow hematopoiesis.

The stimulating effect of the drug was more pronounced against the background of experimental anemia caused by the introduction of lead acetic acid.

In relation to the comparison drug epocrine, the studied drug "V-002" had almost the same efficiency.

Myelostimulatory activity of V-002 in experimental benzene leukopenia

Daily administration of benzene to rats at a dose of 0.1 mg / kg for 7 days has a serious hemo- and myelotoxic effect. At the same time, the number of granulocytic cells, the absolute number of which decreases by 8-10 times compared with the normal level, is reduced to the greatest extent.

It should be noted that in the control group receiving only benzene, four rats died on the 10th, 11th, 12th and 14th day. Almost all the animals in this group were lethargic, asthenic, looked exhausted, their coat had an untidy appearance.

A study of blood and bone marrow conducted in this group 10 days after the cessation of benzene administration showed a significant decrease in the number of all blood cells. The total number of leukocytes was reduced by more than 50%, while the cells of the granulocyte series were most affected. A study of the bone marrow showed a similar picture: the number of cardiomyelocytes was reduced by almost 50% of the norm, the proportion of granulocytes was reduced, and mitotic activity was also reduced.

In the group receiving "V-002", none of the animals died. At the same time, an improvement in the general condition of animals and a positive dynamics of body weight were noted. Hematological studies have shown that the hemoglobin content, as well as the number of all blood cells, although it remained reduced relative to levels in intact animals, was almost at the lower limit of normal. The appearance of immature forms in the blood testified to the activation of bone marrow hematopoiesis. The latter is also confirmed by the results of myelographic research - the total number of myelokaryocytes was closer to normal than in the control, mitotic activity was higher than even in intact animals.

The introduction of pentoxyl and methyluracil also reduced the hemo- and myelotoxic effect of benzene. However, in the groups receiving comparison drugs, the death of animals was noted: pentoxyl - one rat on day 14; methyluracil - two on the 11th and 14th day.

Judging by the results of hematological and myelographic studies, both comparison drugs are inferior in their hematopoietic effect to the new drug "V-002".

The data of analyzes of peripheral blood and myelogamma of animals treated with "V-002" and comparison products in the model of benzene leukopenia are presented in tables 11 and 12.

Table 11 Hematological parameters of animals from experimental groups in a benzene leukopenia model Research indicators Intact Benzene Benzene + V-002 Benzene + Pentoxin Benzene + Methyluracil Hemoglobin, g / dl 12.9 ± 0.4 8.0 ± 0.4 * 11.8 ± 0.2 11.1 ± 0.4 9.9 ± 0.4 * Hematocrit, g% 46.6 ± 1.0 29.5 ± 0.9 * 40.5 ± 0.8 * 39.1 ± 1.4 * 35.8 ± 1.3 * Red blood cells, 10 ¹² / L 6.3 ± 0.3 4.2 ± 0.4 * 5.8 ± 0.3 5.5 ± 0.3 * 5.1 ± 0.4 * Color indicator (SIT), pg 20.5 ± 0.3 10.0 ± 0.4 * 20.3 ± 0.4 20.2 ± 0.4 19.4 ± 0.4 * ESR, mm / h 5.5 ± 0.2 17.4 ± 0.3 * 9.2 ± 0.3 * 10.7 ± 0.7 * 9.4 ± 1.3 * Platelets, 10 ⁹ / L 680 ± 54 415 ± 66 * 493 ± 52 522 ± 43 460 ± 61 * White blood cells, 10 ⁹ / L 7.1 ± 0.2 2.8 ± 0.3 * 5.2 ± 0.3 * 4.6 ± 0.2 * 4.5 ± 0.4 * Band neutrophils,% 0.5 ± 0.3 0.1 ± 0.1 * 0.6 ± 0.2 0.3 ± 0.2 0.4 ± 0.2 Segmented neutrophils,% 14.3 ± 3.0 4.4 ± 5.7 * 10.4 ± 2.4 11.0 ± 2.8 10.2 ± 3.3 Basophils,% 0 0 0 0 0 Lymphocytes,% 79.8 ± 2.1 85.4 ± 3.5 82.4 ± 4.4 80.4 ± 4.4 81.0 ± 2.6 Monocytes,% 2.5 ± 0.2 4.8 ± 0.6 * 2.9 ± 0.7 3.1 ± 0.3 4.1 ± 0.4 * Eosinophils,% 2.3 ± 0.3 3.9 ± 0.3 2.7 ± 0.3 3.1 ± 0.6 3.8 ± 0.2 Plasma cells,% 0.5 ± 0.2 0 0 0.6 ± 0.2 0 Normocytes,% 0 0 0 0 0 Polychromatophiles,% 0 1.2 ± 0.5 0 0.6 ± 0.4 0.3 ± 0.2 *) - significant differences from the intact group at P> 0.95.

Table 12 Myelogram indices of animals of experimental groups in a benzene leukopenia model Research indicators Intact Benzene Benzene + V-002 Benzene + Pentoxin Benzene + Methyluracil Number of cells, 10 ¹¹ / L 73.2 ± 3.6 34.7 ± 5.0 * 52.4 ± 5.0 50.0 ± 6.1 48.5 ± 2.3 * Undifferentiated blasts,% 0.1 ± 0.1 1.1 ± 0.2 * 0.6 ± 0.2 1.0 ± 0.3 0.8 ± 0.3 Proerythroblasts,% 0 0 0.1 ± 0.05 0.1 ± 0.05 0 Erythroblasts,% 0.8 ± 0.1 1.2 ± 0.2 * 1.0 ± 0.2 0.8 ± 0.2 1.5 ± 0.3 Pronormoblasts,% 0.6 ± 0.1 1.4 ± 0.3 * 1.2 ± 0.4 0.9 ± 0.3 1.1 ± 0.3 * Basophilic normoblasts,% 7.4 ± 0.8 9.5 ± 0.6 * 7.8 ± 0.5 8.2 ± 0.7 8.8 ± 0.8 Polychromatophilic normoblasts,% 9.3 ± 1.0 12.2 ± 1.2 * 10.2 ± 0.8 11.4 ± 1.0 11.0 ± 1.3 * Eosinophilic normoblasts,% 5.8 ± 1.2 7.4 ± 0.8 6.4 ± 0.7 7.3 ± 1.1 7.5 ± 1.2 The number of mitoses of red blood,% 0.56 ± 0.10 0.37 ± 0.08 * 0.64 ± 0.10 0.60 ± 0.21 0.52 ± 0.18 Myeloblasts,% 2.210.5 2.2 ± 0.2 1.8 ± 0.3 1.6 ± 0.4 0.8 ± 0.4 * Promyelocytes,% 4.0 ± 0.4 4.4 ± 0.4 3.5 ± 0.6 3.6 ± 0.5 2.6 ± 0.5 * Myelocytes,% 6.2 ± 0.7 6.4 ± 1.1 5.5 ± 0.9 5.8 ± 0.8 4.8 ± 0.6 Metamyelocytes,% 9.1 ± 1.2 10.2 ± 0.8 7.9 ± 1.0 7.9 ± 0.8 9.5 ± 1.0 Band neutrophils,% 11.8 ± 1.4 6.5 ± 1.0 * 12.6 ± 1.4 9.2 ± 1.3 9.7 ± 1.2 * Segmented neutrophils,% 16.6 ± 3.2 9.2 ± 2.1 * 14.1 ± 3.0 12.4 ± 2.4 10.1 ± 1.8 * Eosinophils,% 7.5 ± 1.1 8.1 ± 1.0 * 6.0 ± 1.1 5.6 ± 0.4 4.2 ± 0.9 Basophils,% 0 0 0 0 0 The number of mitoses of white blood,% 0.45 ± 0.13 0.33 ± 0.10 0.68 ± 0.18 0.57 ± 0.16 0.53 ± 0.14 Prolymphocytes,% 0.1 ± 0.1 0 0 0.4 ± 0.2 0 Lymphocytes,% 8.3 ± 1.0 12.4 ± 0.8 * 10.7 ± 1.3 11.2 ± 1.1 * 13.4 ± 1.5 Plasma cells,% 0.8 ± 0.3 1.8 ± 0.3 2.0 ± 0.5 * 2.4 ± 0.4 * 2.8 ± 0.5 * Promonocytes,% 0 0 0 0.1 ± 0.05 0 Monocytes,% 0.7 ± 0.2 1.1 ± 0.1 1.0 ± 0.1 0.4 ± 0.2 0.7 ± 0.3 Reticulocytes,% 2.4 ± 0.5 2.1 ± 0.3 2.1 ± 0.5 1.8 ± 0.4 2.2 ± 0.6 Megakaryocytes and megakaryoblasts,% 0.43 ± 0.08 0.53 ± 0.05 0.47 ± 0.15 0.33 ± 0.11 0.41 ± 0.15

Thus, the introduction of the drug “V-002” against the background of experimental benzene leukopenia had a clear therapeutic effect: in the group receiving the drug, no death of animals was noted, while in the control it was 3 out of 10. The introduction of the drug had an obvious hematopoietic effect, more pronounced on the most suppressed - granulocytic - germ of hematopoiesis.

Moreover, “V-002” was even more effective than the official drugs pentoxyl and methyluracil.

An experimental study of the effect of the drug "V-002" on the body's resistance (survival) and erythropoiesis with prolonged compression syndrome

Research results

The effect of V-002 on body resistance

In white mice subjected to compression trauma for 2.5-3 hours, mortality in the first 3 days of the post-compression period was 43.7%. The introduction of physiological saline according to the treatment-and-prophylactic regimen did not have a statistically significant effect on the survival of animals (death was 33.3%; p> 0.05). At the same time, the administration of V-002 led to a significant increase in the survival of injured animals: in the experimental groups of mice that received V-002, the death was in the range of 5-10% (p <0.05). The results are presented in table.13.

In the next series of experiments performed on white rats, the effectiveness of the therapeutic effect of V-002 was evaluated for anemia that forms in the post-compression period and is mainly caused by inhibition of erythropoiesis (Table 16.). In rats, the number of red blood cells in the peripheral blood was determined before compression, and then 1, 2, 4, 8, 11, and 14 days after the compression injury. It was found that during the first two days the number of red blood cells in the peripheral blood remained within normal limits, although there was a slight tendency to increase it (by 2-3%). However, from the 4th day the number of red blood cells began to decrease, reaching the lowest possible level (65.3% of the initial value - by the 11th day of observation) about a week after the injury. In rats of the control group, which were injected with saline, a reliably low number of red blood cells remained during the rest of the observation time, i.e. up to 14 days At the same time, in animals of the experimental groups treated with V-002, the percentage of erythrocyte reduction was 2 times lower, and by the end of the observation period the number of red blood cells in the peripheral blood increased significantly, reaching 94% of the norm, which is 20-25 % higher than in control.

An analysis of the results presented in this section allows us to conclude that the administration of V-002 at a dose of 10 mg / kg in the form of a course of 3-4 injections to white mice under conditions of compression trauma increases the overall resistance of injured animals. This is confirmed by a significant decrease in the mortality rate in the groups receiving the drug compared with the control (35-45% in the control and 5-10% in the experiment). The drug V-002, when administered intramuscularly at a dose of 10 mg / kg for 14 days, helps to restore erythropoiesis in white rats after compression injury.

Thus, the agent according to the invention, bis- (γ-L-glutamyl) -L-cysteinyl-glycine dilithium salt: cis-diaminodichloropalladium: copper dichloride (GSSGLi ₂ / Pd / Cu = 1000/1/1), characterizes the ability to restore the physiologically necessary the level of hematopoiesis, violations of which arose as a result of radiation, toxic and traumatic effects. The therapeutic efficacy of the action of the agent according to the invention is comparable to the action of agents for the selective stimulation of hematopoiesis of drugs based on erythropoietin, colony stimulating factors, thrombopoietin. In contrast to them, the tool according to the invention characterizes the possibility of long-term storage at room temperature and use as a means of emergency stock in case of emergency with a large number of wounded and injured, in need of hemostatic stimulants.

Example 3. Obtaining the dosage form of the funds according to the invention

In a container for preparing a solution with a capacity of 1.0 l, 0.8 ml of water for injection are added to 13.6 g of sodium acetate 3-aqueous, 60 g of the substance of the agent according to the invention and mixed until dissolved, followed by bringing the total volume to 1.0 L. The pH value is determined using a pH meter and adjusted with 20% acetic acid to a pH of 6.0 ± 0.2. Sterilized filtration is carried out using a sterilizing filter with a pore size of 0.22 μm and the filling is carried out (1 ml - 1 ampoule) in glass ampoules. The result is 970 ampoules (including losses) containing a composition for intravenous or intramuscular administration that meets the requirements of regulatory documents for 3 years and has the following composition:

The tool according to the invention 60 g Sodium Acetate 3-Aq 13.6 g Acetic acid up to pH 6.0 Water up to 1000 ml

Example 4. Obtaining the dosage form of the funds according to the invention

In a container for preparing a solution with a capacity of 1.0 l, 0.8 g of water for injection are added to 13.6 g of sodium acetate 3-aqueous, 30 g of the substance of the agent according to the invention and mixed until dissolved, followed by bringing the total volume to 1.0 l The pH value is determined using a pH meter and adjusted with 20% acetic acid to a pH of 6.0 ± 0.2. Sterilized filtration is carried out using a sterilizing filter with a pore size of 0.22 μm and the filling is carried out (1 ml - 1 ampoule) in glass ampoules. The result is 970 ampoules (including losses) containing a composition for intravenous or intramuscular administration that meets the requirements of regulatory documents for 3 years and has the following composition:

The tool according to the invention 30 g Sodium Acetate 3-Aq 13.6 g Acetic acid up to pH 6.0 Water up to 1000 ml

Claims (8)

1. A combination of bis (γ-L-glutamyl) -L-cysteinyl-glycine dilithium salt and coordination compounds formed by palladium, copper and (γ-L-glutamyl) -L-cysteinyl-glycine, having a molar ratio bis- (γ-L-glutamyl) -L-cysteinyl-glycine dilithium salt: palladium: copper is in the range 100-10000: 1-10: 1-10. 2. The combination according to claim 1, where the molar ratio of bis- (γ-L-glutamyl) -L-cysteinyl-glycine dilithium salt: palladium: copper is 1000: 1: 1. 3. The combination according to claim 1, where cis-diaminodichloropalladium is selected as the source of palladium. 4. The combination according to claim 1, where copper dichloride is selected as the source of copper. 5. The combination according to claim 1, obtained by oxidation of the lithium salt of - (γ-L-glutamyl) -L-cysteinyl-glycine using cis-diaminodichloropalladium and copper dichloride. 6. The combination according to any one of claims 1 to 5 for use in therapy. 7. A pharmaceutical composition having hematopoiesis stimulant activity, comprising a combination of claims 1-6 and pharmaceutically acceptable excipients. 8. A method of stimulating hematopoiesis, in which the patient in need is administered a combination according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7.