RU2850768C1 - Use of 2-methyl-5-vinyl pyridine copolymer and n-vinyl pyrrolidone hydrochloride for restoring haematopoietic function - Google Patents

Abstract

FIELD: chemistry and pharmaceuticals.

SUBSTANCE: use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride with an average molecular weight of 30-55 kDa and a 2-methyl-5-vinylpyridine content of 28-40 mol.% for restoring bone marrow haematopoietic function during chemotherapy or radiotherapy of neoplasms or blood loss.

EFFECT: increase in the effectiveness of restoring bone marrow haematopoietic function after chemotherapy or radiotherapy of neoplasms and in cases of blood loss.

6 cl, 16 tbl, 4 ex

Description

The invention relates to the field of medicine and can be used in oncology as a means of maintaining and restoring the hematopoietic function of human bone marrow during chemotherapy and radiotherapy of neoplasms, as well as in cases of blood loss.

Hematopoietic toxicity is the most common side effect of chemotherapy and manifests itself through the suppression of all hematopoietic processes. The cells that produce leukocytes and platelets are particularly frequently damaged, while the cells responsible for red blood cell development are less frequently affected. Hematopoietic suppression is usually observed in the days following chemotherapy (7-12 days). Some drugs cause delayed toxicity. Risk factors for the development of bone marrow toxicity from chemotherapy include: previous chemotherapy and radiation therapy, patient age over 60 and under 1 year, general condition, and exhaustion [http://www.pror.ru/treatment/complic_chem].

In the case of radiation therapy for neoplasms, the leading link in the pathogenesis is also hematopoiesis disorders and the development of cytopenic syndrome. Leukopenia and thrombocytopenia develop primarily. The number of leukocytes, and especially neutrophils, decreases sharply. The platelet count decreases significantly. Erythremia develops only with bleeding, since the lifespan of erythrocytes is quite long. A decrease in the leukocyte count to 1×10 ⁹ /l is considered agranulocytosis. The bone marrow becomes hypo- or aplastic [Gutsol L.O. et al. Pathogenesis and basic principles of pathogenetic therapy and prevention of post-radiation damage: a tutorial / Irkutsk, Irkutsk State Medical University, 2018, p. 20].

In acute blood loss, the absolute number of erythrocytes, the absolute and percentage content of maturing forms of granulocytic myelokaryocytes, and the neutrophil maturation index decrease in the bone marrow. These changes in the morphological composition of the bone marrow indicate accelerated maturation of granulocytes and accelerated elimination of erythroid and granulocytic myelokaryocytes into the bloodstream. The most likely cause of these changes in the morphological composition of the bone marrow may be changes in blood microcirculation in the red bone marrow caused by acute blood loss, leading to accelerated elimination of myelokaryocytes from it and mobilization of erythroid and granulocytic reserves [Belchenko Dmitry Ivanovich, Volkova O. V. The effect of acute blood loss on bone marrow hematopoiesis in children with chronic idiopathic thrombocytopenic purpura // Hematology and Transfusiology. 2012. No. 5].

The search for new approaches to maintaining and restoring the hematopoietic function of human bone marrow during chemotherapy and radiotherapy of tumors, as well as during blood loss, is one of the most important areas in modern medicine and pharmacy.

The following is known from the prior art:

- Patent RU 2000004 (published 15.02.1993) proposes copolymers of N-vinylpyrrolidone and 2-methyl-5-vinylpyridine with an average viscosity molecular weight in the range of 29-45 kDa, containing 25-40 mol.% 2-methyl-5-vinylpyridine units, which have an immunostimulating effect, as well as a prolonged effect when antibiotics are administered.

- Patent RU 2015993 (published 15.07.1994) shows the production of a group of copolymers with an average viscosity molecular weight of 40 kDa, similar to those described in publication RU 2000004, in which the average number of 2-methyl-5-vinylpyridine units n is 35, and their antitumor effect is also shown.

- Patent RU 2415876 (published 10.04.2011) proposes copolymers based on N-vinylpyrrolidone, represented by the general formula: where the monomer unit M is a fragment of 2-methyl-5-vinyltetrazole (MBT) or 2-methyl-5-vinylpyridine (MVP), the content of monomer units n is 25-90 mol.%, and the average viscosity molecular weight of the copolymer Mμ = 46-150 kDa, if M is MVP, suitable for use as adjuvants in the manufacture of vaccines.

- Patent RU 2430932 (published 10.10.2011) discloses copolymers based on N-vinylpyrrolidone, represented by the general formula: where the monomer unit M is a fragment of 2-methyl-5-vinyltetrazole (MBT) or 2-methyl-5-vinylpyridine (MVP), the content of monomer units n is 25-50 mol.%, and the viscosity-average molecular weight of the copolymer Mµ = 15-28 kDa, if M is MVP, which are activators of the production of interleukin-1, as a result of which they can be useful as anti-cancer agents.

- Patent RU 2430933 (published 10.10.2011) discloses the use of copolymers based on N-vinylpyrrolidone, the structure of which is described by the general formula given above, as phagocytosis activators.

All of the listed N-vinylpyrrolidone-based copolymers containing 2-methyl-5-vinylpyridine fragments are considered by the authors of this invention to be analogs of the present invention. Their main drawback is low solubility in weakly acidic and neutral aqueous media (pH 6-7), which hinders their effective use in the manufacture of injectable dosage forms. Moreover, the solubility of the copolymers in such media, while maintaining a constant viscosity-average molecular weight, decreases quite sharply with an increasing proportion of 2-methyl-5-vinylpyridine fragments. As a result, effective concentrations of copolymers containing over 55 mol% MVP fragments are not achieved in weakly acidic and neutral aqueous solutions.

- In patent RU 2533113 (published 20.11.2014), a copolymer based on N-vinylpyrrolidone in the form of pharmaceutically acceptable acid addition salts is represented by the general formula, where R is hydrogen or methyl; the content of monomer units n is 25-90 mol.%; X is an anion of a pharmaceutically acceptable inorganic or organic acid; k is 1 or 2; m takes values from 0.1 to 1.0; and the viscosity-average molecular weight of the copolymer Mµ is 15-150 kDa. The invention makes it possible to expand the arsenal of biologically active copolymers with improved solubility characteristics and exhibiting greater activity, compared to known similar compounds, as an immunostimulant, prolongator of the action of antibiotics, activator of IL-1 production and phagocytosis.

However, a copolymer based on N-vinylpyrrolidone in the form of pharmaceutically acceptable acid addition salts, possessing improved solubility characteristics and exhibiting greater activity than known similar compounds as an immunostimulant, prolongator of antibiotic action, activator of IL-1 production and phagocytosis, has not yet been considered (used) as a means of restoring the hematopoietic function of the bone marrow after chemo- or radiotherapy of neoplasms.

Neupogen and Betaleukin, recombinant protein-peptide preparations, are known in the art. These preparations are intended for multiple use. Neupogen is a recombinant human granulocyte colony-stimulating factor (G-CSF). The hematopoietic growth factor filgrastim is a highly purified, non-glycosylated protein consisting of 175 amino acids. It is produced by the K12 strain of Escherichia coli, into whose genome the human G-CSF gene has been introduced using genetic engineering. Indications for use of this drug include neutropenia and febrile neutropenia in patients receiving intensive myelosuppressive cytotoxic chemotherapy for malignant diseases (excluding chronic myeloid leukemia and myelodysplastic syndrome), as well as neutropenia and its clinical consequences in patients receiving myeloablative therapy followed by allogeneic or autologous bone marrow transplantation with an increased risk of developing prolonged and severe neutropenia. Disadvantages of this drug include low stability, the potential for increased risk of bone marrow depletion, which requires constant monitoring of hematopoiesis, and a wide range of side effects, including nausea, vomiting, hypotension, etc. Neupogen should be used therapeutically in cases of acute, rapidly developing leukopenia. It does not stimulate platelet production in the bone marrow. It should also be noted that during treatment with Neupogen, spleen size, urine composition, and peripheral blood leukocyte and platelet counts should be monitored closely. Patients with severe renal or hepatic impairment should not use this medication.

Betaleukin is a medicinal form of recombinant human interleukin-1b. Interleukin-1b is a polypeptide produced by macrophages that enhances the T-cell response. Betaleukin's hemostimulatory effect is due to the stimulation of colony-stimulating factor production, as well as enhanced proliferation and differentiation of cells of various hematopoietic lineages. Recombinant interleukin-1b affects hematopoietic cells at various stages of their maturation, including the pluripotent stem cell stage. Therefore, Betaleukin stimulates an increase in the number of not only granulocytes but also platelets and lymphocytes. Disadvantages of Betaleukin include repeated long-term use, the duration of treatment (up to three weeks), and constant monitoring of spleen size, urine composition, and the number of leukocytes and platelets in the peripheral blood.

The disadvantages of the above-mentioned drugs include their complex and expensive production methods. These protein-peptide drugs are produced using recombinant DNA technology in Escherichia coli cells or Chinese hamster ovary (CHO) cells. Therefore, recombinant drugs are expensive.

The authors of the claimed invention unexpectedly discovered the ability of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride to enhance hematopoietic function restoration after chemotherapy and radiotherapy for tumors, as well as during hemorrhagic shock. This property of the copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride can be used in practical healthcare to improve treatment effectiveness.

The technical result of the claimed invention is to increase the efficiency of restoration of the hematopoietic function of the bone marrow after chemotherapy or radiotherapy of neoplasms, in case of blood loss and, as a consequence, to increase the efficiency of the therapy.

The above technical result is achieved by using a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride to restore the hematopoietic function of the bone marrow during chemotherapy or radiotherapy of neoplasms, or blood loss.

In this case, the average molecular weight of the copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride can range from 30 to 55 kDa, and the content of 2-methyl-5-vinylpyridine units can range from 28 to 40% (mol).

As an embodiment of the claimed invention, the copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride can be used in chemotherapy, including the administration of drugs such as Paclitaxel, Cisplatin, Fluorouracil, Doxorubicin, Carboplatin, as well as other cytostatic drugs, the side effects of which include a decrease in the hematopoietic function of the bone marrow.

Alternatively, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone copolymer hydrochloride can be administered to the patient as a 5% solution in water for injection.

Alternatively, the copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride can be administered to the patient's body intramuscularly once at a dose of 1.5-4 mg per 1 kg of the patient's body weight.

The above technical result is also achieved through a method for restoring the hematopoietic function of the bone marrow during chemotherapy or radiotherapy of neoplasms, which includes a single intramuscular administration of the claimed medicinal product at a dose of 1.5-4 mg of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride per 1 kg of the patient's body weight.

Alternatively, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone copolymer hydrochloride may be administered as a 5% solution in water for injection.

The copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride ensures the rapid development of the predominantly myeloid lineage of the hematopoietic system after the toxic effects of chemo- and radiotherapy on proliferating bone marrow cells and, thus, allows for an increase in the survival rate of cancer patients by restoring hematological parameters.

A copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride can be obtained, for example, by the following method.

Obtaining a copolymer of N-vinylpyrrolidone and 2-methyl-5-vinylpyridine (2-M-5-VP) in the form of hydrochloride.

A. Synthesis of the base

The reactor is loaded with the initial reaction mixture containing 1.00 kg (960 ml) of N-VP, 16.8 g (17.6 ml) of 2-M-5-VP, 1.6 g (1.7 ml) of 30% aqueous hydrogen peroxide solution, 109.2 g (140 ml) of cyclohexane and 35 g of azo-bis-isobutyric acid dinitrile (AIBN). The reaction mass is heated to 65-70 °C and the synthesis is carried out for 5 hours at this temperature, periodically taking samples for chromatographic analysis (HPLC, Spherisorb C-18, 5 μm, 4.6×250 mm, EtOH/H2O 6:94, 2.5 ml/min (50 °C), 254 nm). A mixture containing 150 g (144 ml) of N-VP, 57.3 g (55 ml) of 2-M-5-VP, 0.51 g (0.5 ml) of 30% aqueous hydrogen peroxide solution and 7.5 g of AIC is fed. Upon reaching a monomer conversion of 20%, the reaction mass is cooled to 20°C and poured with stirring into 30 l of diethyl ether, left with stirring overnight, the resulting precipitate is isolated by filtration and dried: first at atmospheric pressure at 40-60°C, and then in a vacuum drying oven at 80-90°C. 167.4 g of product are obtained.

B. Obtaining hydrochloride in aqueous solution

Dissolve 100.0 g of the obtained copolymer in 2.0 L of distilled water with stirring, add 0.5 ml of 35% hydrochloric acid, and stir for 1 hour. The target hydrochloride is isolated by evaporating the water under reduced pressure and then dried to constant weight in a vacuum oven at 80-90°C.

B. Obtaining hydrochloride in a non-aqueous solvent

50.0 g of the obtained copolymer are dissolved in 1.0 L of anhydrous chloroform, and dried hydrogen chloride is bubbled in with stirring for 15 minutes at a flow rate of 10 ml/min. The resulting solution is evaporated under reduced pressure, and the solid is dried as described above.

The loadings of the main ingredients used in the production of the 2-methyl-5-vinylpyridine/N-vinylpyrrolidone hydrochloride copolymer vary depending on the desired product characteristics (number-average molecular weight and 2-methyl-5-vinylpyridine content). The loadings are listed in Table 1.

Table 1. Substance loadings and properties of the resulting product

The claimed invention is illustrated by the implementation examples set out below.

EXAMPLE 1. In an animal experiment (pigs), the specific pharmacological activity of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride (hereinafter referred to as the copolymer) was assessed for the restoration of bone marrow hematopoietic function after the administration of antitumor cytostatic drugs. The effect of the copolymer, administered intramuscularly at various doses, on the morphological parameters of red bone marrow in pigs with disorders induced by various cytostatic drugs was assessed.

Preparations and methods of application

Copolymer

Three samples of copolymer with different number average molecular weights and 2-methyl-5-vinylpyridine unit contents were used (Table 2).

Table 2. Studied copolymer samples.

Sample number Average number
molecular weight, kDa Content of 2-methyl-5-vinylpyridine units,
% (mol) C1 30 28 C2 42 34 C3 55 40

Each copolymer sample was administered intramuscularly (IM) once. The concentrations of the copolymer injection solutions were calculated at 143 μL per 1 kg of animal body weight (5 mL of solution for an animal weighing 35 kg). The volumes of copolymer injection solutions (Table 3) were physiologically acceptable for IM administration to animals of the given species.

Cytostatic drugs used

To model toxic hematodepression, the study used antitumor drugs (cytostatics) paclitaxel (PCTS), cisplatin (CPLN), fluorouracil (FTL), doxorubicin (DRN), carboplatin (CPLT). For

The primary toxic effect of each of the above-mentioned drugs is the suppression of myelopoiesis in the red bone marrow, leading to the development of leukopenia, neutropenia, and thrombocytopenia. In all cases, the antitumor drugs were administered to the animals in the experimental groups as a single, slow intravenous (IV) dose at a dose corresponding (according to the mouse-human-pig dose conversion factors) to the maximum tolerated dose for the given animal species. The administered doses are presented in Table 4.

Table 4. Doses of administered cytostatic drugs

Name of the drug (abbreviated) Administered dose, mg/kg PKTS 3.0 TsPLN 2.75 FTL 16.6 DRN 2.5 KPLT 11

For intravenous administration of cytostatic drug solutions (primarily into the caudal epigastric superficial veins or the veins of the medial surface of the thigh), animals were fitted with closed vascular catheters Introcan-W (B Braun Melsungen AG.

Laboratory animals

The experiments were conducted on Danish Hybrid F1 piglets obtained from the Klimovskaya ECO FARM nursery. The animals were housed in specialized compartments (1-2 animals per compartment) in the vivarium of the A.F. Tsyb Medical Research Center. The animals were maintained in accordance with the requirements of Order No. 708n of the Russian Ministry of Health: under natural light and 16-fold forced ventilation, at a temperature of 18-20˚C and a relative humidity of 40-70%, on a hay bedding (the bedding was changed every two days), with the provision of specialized SK-4 briquetted feed (LaboratorSnab LLC, Russia) twice a day according to the standards for the animals' age and body weight, and with free access to drinking water (water in the drinkers was renewed 2-3 times a day). Before the start of the studies, animals from each batch underwent a 10-14-day quarantine.

Experimental work with animals

All animal work was performed in accordance with generally accepted ethical standards and the standard operating procedures adopted at the A.F. Tsyb Medical Research Center, which comply with the European Convention for the Protection of Animals. All studies and animal manipulations were conducted in the equipped experimental clinic of the MRC vivarium.

Animal examination . From admission until the end of the study, each animal was examined twice daily and recorded. General condition, behavioral and spontaneous motor activity, response to auditory and tactile stimuli, and food and water consumption were assessed.

Animal body weight measurement . The body weight of each animal was measured and recorded on the day of admission, at the end of quarantine, before administration of cytostatics, copolymer, or placebo, and before scheduled euthanasia. For this purpose, a staff member wearing protective clothing lifted the animal to chest level and stepped on an MP 150 VDA "Gulliver 12" electronic floor scale (MIDL, Russia). The animal's body weight was then calculated to the nearest 100 g.

Injections of cytostatics and copolymer . To securely immobilize unanesthetized pigs during injections and planned euthanasia, an X-shaped wooden frame was used. This frame, fabricated by vivarium technical staff from planed wooden boards and beams, allowed the animal to be immobilized in both a natural and supine position. Before injections, the animal's skin at the injection site was repeatedly cleaned with sterile surgical drapes soaked in 70% ethanol. Cytostatics were administered into the caudal epigastric superficial veins. Copolymer samples were injected into the hamstrings.

Planned euthanasia was performed by rapid severing of the carotid arteries.

Red bone marrow tissue isolation . Immediately after euthanasia, the right and left femurs were isolated from the animals, cleared of muscle fibers, and sawed distally from the intertrochanteric crest using an HB 740 oscillating section saw (Kugel medical GmbH&Co, Germany). The cut surfaces were then cleared of debris, and 800-1200 mg of red bone marrow (RBM) were collected from the diaphysis of each bone using a surgical spatula and weighed into tall weighing cups. The resulting RBM samples were weighed using an AR-512 analytical balance (Ohaus Corp., USA), and primary RBM suspensions were immediately prepared by adding a 10-fold amount (by weight) of aseptic 0.9% sodium chloride solution (AO Pharmsintez, Russia).

Pathomorphological examination . After isolating the animals' femurs, a macroscopic examination of the cytostatic and copolymer injection sites was performed, as well as the condition of the internal organs of the chest and abdominal cavity.

Methods for assessing the cellularity of red bone marrow

To remove connective and adipose tissue elements from the primary CCM suspensions, they were filtered through a nylon mesh filter with 41 µm pores (Merck KbaA, Germany).

Automated counting of the total karyocyte count in bone marrow suspensions was performed using the Abacus JunVet veterinary hematology analyzer (Austria). For this purpose, the bone marrow suspension was diluted with saline solution by another 4 times (total bone marrow dilution was 44 times), ensuring a karyocyte concentration in the suspension characteristic of blood leukocytes. When analyzing such a sample, the WBC value calculated by the Abacus provides a quantitative estimate of the karyocyte concentration in the suspension. To minimize error, primary bone marrow suspensions from each bone were analyzed in 3-9 replicates.

Statistical processing

The statistical significance of intergroup differences in weight parameters and total RMC cellularity was assessed using the Kruskal-Wallis rank ANOVA test with post-hoc analysis using the Mann-Whitney U test and the Holm-Bonferroni multiple comparison procedure. The significance of linear regression of RMC cellularity parameters measured by different methods was assessed using the ^χ2 test. All statistical calculations, correlation, and regression analyses were performed using the Statistica 7.0 software package (StatSoft Inc., USA).

Clinical observation data

Animals received for the study with a significant difference in time, nevertheless, were extremely close in age and anthropometric parameters.

Upon arrival at the vivarium of the A.F. Tsyb Medical Research Center, the average body weight of the animals was 22-24 kg. The animals quickly adapted to the vivarium conditions, and by the end of the first day of quarantine, they were feeling comfortable within the confines of their enclosure. Water and food consumption, as well as daily weight gain, were consistent with age norms for the species at all stages of the experiment. By the beginning of the experimental manipulations, the average body weight of the animals was 30-31 kg.

Discussion of results

The results of the studies showed that each of the copolymer samples, when administered intramuscularly once in a model of hematodepression in animals induced by various cytostatic drugs, has a statistically significant stimulating effect on hematopoiesis in almost the entire range of doses used (Tables 5, 6, 7, 8, 9).

Table 5. The effect of a single intramuscular injection of the copolymer on the cellularity indices of the CMC of animals previously treated with PCTS at a dose of 3 mg/kg

Groups Sample C1. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Sample C2. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Sample C3. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Biological control 888.7 ± 155.8 883.5 ± 145.6 886.4 ± 156.4 PCTS 3 mg/kg (5th day) 432.4 ± 100.9 ^* 428.4 ± 98.6 ^* 433.6 ± 98.4 ^* PCTS 3 mg/kg (day 5) + copolymer 1 mg/kg (day 4) 633.8 ± 122.9 ^*+ 635.7 ± 120.7 ^*+ 634.5 ± 123.6 ^*+ PCTS 3 mg/kg (day 5) + copolymer 1.2 mg/kg (day 4) 701.4 ± 131.3 ^*+ 699.4 ± 129.5 ^*+ 700.5 ± 130.5 ^*+ PCTS 3 mg/kg (day 5) + copolymer 1.5 mg/kg (day 4) 808.4 ± 117.8 ⁺ 806.3 ± 115.7 ⁺ 805.6 ± 111.6 ⁺ PCTS 3 mg/kg (day 5) + copolymer 1.7 mg/kg (day 4) 815.4 ± 121.1 ⁺ 818.5 ± 119.3 ⁺ 816.5 ± 125.3 ⁺ PCTS 3 mg/kg (day 5) + copolymer 2 mg/kg (day 4) 832.7 ± 123.4 ⁺ 834.7 ± 122.9 ⁺ 833.5 ± 120.6 ⁺ PCTS 3 mg/kg (day 5) + copolymer 3 mg/kg (day 4) 841.5 ± 125.8 ⁺ 839.7 ± 124.5 ⁺ 844.5 ± 123.5 ⁺ PCTS 3 mg/kg (day 5) + copolymer 4 mg/kg (day 4) 850.8 ± 129.6 ⁺ 852.8 ± 124.5 ⁺ 854.5 ± 127.4 ⁺ PCTS 3 mg/kg (day 5) + copolymer 5 mg/kg (day 4) 851.6 ± 143.1 ⁺ 852.3 ± 145.6 ⁺ 851.8 ± 140.7 ⁺ PCTS 3 mg/kg (day 5) + copolymer 10 mg/kg (day 4) 852.1 ± 135.7+ 855.5 ± 136.7+ 853.7 ± 135.6+

* - significant differences from the biological control group; + - significant differences from the PCTS 3 mg/kg group (day 5).

Table 6. The effect of a single intramuscular injection of the copolymer on the cellularity indices of the CMC of animals previously treated with CPLN at a dose of 2.75 mg/kg

Groups Sample C1. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Sample C2. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Sample C3. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Biological control 834.5 ± 132.4 832.6 ± 133.6 836.6 ± 137.6 CPLN 2.75 mg/kg (5th day) 465.2 ± 98.5 ^* 464.7 ± 95.3 ^* 465.5 ± 96.5 ^* CPLN 2.75 mg/kg (5th day) + copolymer 1 mg/kg (4th day) 611.8 ± 103.9 ^*+ 616.6 ± 105.5 ^*+ 616.8 ± 104.5 ^*+ CPLN 2.75 mg/kg (day 5) + copolymer 1.2 mg/kg (day 4) 698.4 ± 145.6 ^*+ 695.6 ± 145.7 ^*+ 695.5 ± 147.7 ^*+ CPLN 2.75 mg/kg (day 5) + copolymer 1.5 mg/kg (day 4) 800.3 ± 115.1 ⁺ 803.4 ± 116.4 ⁺ 803.7 ± 118.3 ⁺ CPLN 2.75 mg/kg (day 5) + copolymer 1.7 mg/kg (day 4) 805.6 ± 119.7 ⁺ 806.5 ± 116.6 ⁺ 806.4 ± 114.5 ⁺ CPLN 2.75 mg/kg (5th day) + copolymer 2 mg/kg (4th day) 822.5± ^103.5+ 819.6±105.5 ⁺ 826.6± ^107.7+ CPLN 2.75 mg/kg (5th day) + copolymer 3 mg/kg (4th day) 847.6 ± 119.4 ⁺ 841.4 ± 117.6 ⁺ 845.6 ± 115.5 ⁺ CPLN 2.75 mg/kg (day 5) + copolymer 4 mg/kg (day 4) 854.5 ± 123.6 ⁺ 856.1 ± 129.4 ⁺ 856.6 ± 126.7 ⁺ CPLN 2.75 mg/kg (5th day) + copolymer 5 mg/kg (4th day) 858.6 ± 147.1 ⁺ 854.3 ± 143.2 ⁺ 856.3 ± 144.8 ⁺ CPLN 2.75 mg/kg (day 5) + copolymer 10 mg/kg (day 4) 857.9 ± 134.6+ 859.7 ± 137.8+ 858.5 ± 137.3+

* - significant differences from the biological control group; + - significant differences from the 2.75 mg/kg CPLN group (day 5).

Table 7. The effect of the copolymer upon a single intramuscular injection on the cellularity indices of the BMC of animals,

previously received FTL at a dose of 16.6 mg/kg

Groups Sample C1. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Sample C2. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Sample C3. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue
(M ± SD) Biological control 863.4 ± 156.4 865.5 ± 157.5 862.5 ± 155.5 FTL 16.6 mg/kg (5th day) 424.5 ± 116.3 ^* 420.8 ± 118.4 ^* 423.4 ± 117.6 ^* FTL 16.6 mg/kg (day 5) + copolymer 1 mg/kg (day 4) 637.3 ± 131.1 ^*+ 634.1 ± 135.1 ^*+ 638.6 ± 134.6 ^*+ FTL 16.6 mg/kg (day 5) + copolymer 1.2 mg/kg (day 4) 706.7 ± 133.5 ^*+ 708.9 ± 137.6 ^*+ 704.5 ± 132.4 ^*+ FTL 16.6 mg/kg (day 5) + copolymer 1.5 mg/kg (day 4) 804.6 ± 123.5 ⁺ 804.6 ± 121.7 ⁺ 805.8 ± 126.5 ⁺ FTL 16.6 mg/kg (day 5) + copolymer 1.7 mg/kg (day 4) 817.5 ± 123.2 ⁺ 821.8 ± 120.5 ⁺ 813.5 ± 124.9 ⁺ FTL 16.6 mg/kg (day 5) + copolymer 2 mg/kg (day 4) 822.4 ± 119.9 ⁺ 826.5 ± 114.1 ⁺ 827.5 ± 121.4 ⁺ FTL 16.6 mg/kg (day 5) + copolymer 3 mg/kg (day 4) 849.6 ± 121.8 ⁺ 854.7 ± 118.9 ⁺ 853.7 ± 124.6 ⁺ FTL 16.6 mg/kg (day 5) + copolymer 4 mg/kg (day 4) 854.4 ± 125.7 ⁺ 856.2 ± 126.4 ⁺ 858.6 ± 126.2 ⁺ FTL 16.6 mg/kg (day 5) + copolymer 5 mg/kg (day 4) 856.1 ± 131.1 ⁺ 855.4 ± 137.2 ⁺ 851.9 ± 134.5 ⁺ FTL 16.6 mg/kg (day 5) + copolymer 10 mg/kg (day 4) 855.6 ± 136.3+ 858.7 ± 134.4+ 855.8 ± 133.5+

* - significant differences from the biological control group; + - significant differences from the FTL 16.6 mg/kg group (5th day).

Table 8. The effect of a single intramuscular injection of the copolymer on the cellularity indices of the CMC of animals previously treated with DRN at a dose of 2.5 mg/kg

Groups Sample C1. Total bone marrow cellularity, 10 ⁶ cells per 1 g of BMC tissue (M ± SD) Sample C2. Total bone marrow cellularity, 10 ⁶ cells per 1 g of BMC tissue (M ± SD) Sample C3. Total bone marrow cellularity, 10 ⁶ cells per 1 g of bone marrow tissue (M ± SD) Biological control 873.5 ± 161.8 872.5 ± 165.5 873.6 ± 163.6 DRN 2.5 mg/kg (5th day) 434.9 ± 103.5 ^* 430.5 ± 99.4 ^* 438.7 ± 103.7 ^* DRN 2.5 mg/kg (5th day) + copolymer 1 mg/kg (4th day) 629.5 ± 120.3 ^*+ 632.6 ± 124.3 ^*+ 627.5 ± 120.7 ^*+ DRN 2.5 mg/kg (day 5) + copolymer 1.2 mg/kg (day 4) 706.2 ± 125.4 ^*+ 707.4 ± 124.6 ^*+ 704.7 ± 125.3 ^*+ DRN 2.5 mg/kg (day 5) + copolymer 1.5 mg/kg (day 4) 800.5 ± 111.9 ⁺ 804.7 ± 116.5 ⁺ 805.2 ± 111.6 ⁺ DRN 2.5 mg/kg (day 5) + copolymer 1.7 mg/kg (day 4) 818.1 ± 128.1 ⁺ 817.5 ± 124.5 ⁺ 814.3 ± 128.5 ⁺ DRN 2.5 mg/kg (day 5) + copolymer 2 mg/kg (day 4) 837.3 ± 115.9 ⁺ 835.7 ± 116.7 ⁺ 834.6 ± 115.4 ⁺ DRN 2.5 mg/kg (day 5) + copolymer 3 mg/kg (day 4) 840.5 ± 124.5 ⁺ 845.6 ± 125.8 ⁺ 846.7 ± 124.6 ⁺ DRN 2.5 mg/kg (day 5) + copolymer 4 mg/kg (day 4) 850.6 ± 134.8 ⁺ 853.8 ± 136.5 ⁺ 854.6 ± 134.7 ⁺ DRN 2.5 mg/kg (day 5) + copolymer 5 mg/kg (day 4) 849.6 ± 141.2 ⁺ 848.4 ± 144.6 ⁺ 845.4 ± 141.4 ⁺ DRN 2.5 mg/kg (day 5) + copolymer 10 mg/kg (day 4) 850.8 ± 137.1+ 854.6 ± 138.3+ 853.7 ± 137.5+

* - significant differences from the biological control group; + - significant differences from the DRN 2.5 mg/kg group (day 5).

Table 9. The effect of a single intramuscular injection of the copolymer on the cellularity indices of the CMC of animals previously treated with CPLT at a dose of 11 mg/kg

Groups Sample C1. Total bone marrow cellularity, 10 ⁶ cells per 1 g of BMC tissue (M ± SD) Sample C2. Total bone marrow cellularity, 10 ⁶ cells per 1 g of BMC tissue (M ± SD) Sample C3. Total bone marrow cellularity, 10 ⁶ cells per 1 g of BMC tissue (M ± SD) Biological control 880.3 ± 143.1 883.5 ± 144.2 884.5 ± 144.7 KPLT 11 mg/kg (5th day) 428.7 ± 99.4 ^* 425.5 ± 97.6 ^* 429.4 ± 98.3 ^* KPLT 11 mg/kg (day 5) + copolymer 1 mg/kg (day 4) 629.5 ± 119.3 ^*+ 631.6 ± 120.6 ^*+ 632.5 ± 117.5 ^*+ CPLT 11 mg/kg (day 5) + copolymer 1.2 mg/kg (day 4) 700.7 ± 125.8 ^*+ 699.7 ± 123.6 ^*+ 705.3 ± 124.6 ^*+ KPLT 11 mg/kg (day 5) + copolymer 1.5 mg/kg (day 4) 808.3 ± 121.7 ⁺ 809.6 ± 124.6 ⁺ 804.6 ± 125.5 ⁺ CPLT 11 mg/kg (day 5) + copolymer 1.7 mg/kg (day 4) 821.4 ± 123.7 ⁺ 821.7 ± 125.8 ⁺ 820.3 ± 126.6 ⁺ KPLT 11 mg/kg (day 5) + copolymer 2 mg/kg (day 4) 836.4 ± 120.9 ⁺ 838.6 ± 126.7 ⁺ 836.6 ± 125.4 ⁺ KPLT 11 mg/kg (day 5) + copolymer 3 mg/kg (day 4) 847.6 ± 123.1 ⁺ 844.6 ± 123.1 ⁺ 847.5 ± 126.5 ⁺ KPLT 11 mg/kg (day 5) + copolymer 4 mg/kg (day 4) 860.5 ± 123.6 ⁺ 866.7 ± 122.7 ⁺ 866.7 ± 125.5 ⁺ KPLT 11 mg/kg (day 5) + copolymer 5 mg/kg (day 4) 859.5 ± 132.9 ⁺ 861.6 ± 133.5 ⁺ 858.3 ± 135.2 ⁺ KPLT 11 mg/kg (day 5) + copolymer 10 mg/kg (day 4) 856.6 ± 145.3+ 853.5 ± 146.7+ 858.6 ± 144.2+

* - significant differences from the biological control group; + - significant differences from the 11 mg/kg KPLT group (day 5).

On the fourth day after administration, statistically significant attenuation of hematodepression caused by the cytostatic drugs was observed in animals treated with all copolymer samples at doses of 1 and 1.2 mg/kg, compared to untreated animals. However, the magnitude of this pharmacological effect was modest: the degree of cytostatic-induced BMC depletion decreased by an average of 50%, but BMC values remained statistically significantly lower than BMC levels in healthy animals. In other words, at these doses, the copolymer attenuated, but did not completely eliminate, the hematotoxic effects of the cytostatic drugs.

In this study, all copolymer samples exhibited a more pronounced effect at doses of 1.5-10 mg/kg. In the corresponding groups, on the fourth day after copolymer administration, no hematotoxic effects of cytostatic drugs were observed in animals. The level of total cellularity in the BMC of animals in these groups, despite prior exposure to subtoxic doses of various cytostatic drugs, was consistent with that of healthy animals.

At a dose of 1 mg/kg, the effect of all copolymer samples was weak and unstable (essentially only one animal out of three responded) and did not have a significant effect on the hematotoxic effect of cytostatics.

A model was used to demonstrate that a single copolymer administration 24 hours after cytostatic drug administration can reduce the hematotoxicity of the drug. This effect is observed at copolymer doses ranging from 1.5 to 10 mg/kg by day 4 post-administration.

Across the studied dose range of all copolymer samples, the optimal dose for the prevention and treatment of hematodepression induced by various cytostatic drugs in animals was 1.5-4 mg/kg. In this case, by day 4 after copolymer administration, animals previously treated with subtoxic doses of cytostatic drugs showed virtually no hematotoxic effects. Further increases in the copolymer dose did not significantly increase the CMC in the animals.

The rate of growth of the total cellularity of the RMC induced by the copolymer (by 1.5-2.5 times) indicates that the leading target of the drug is the myeloid lineage, which is the most numerous in the RMC.

No statistically significant differences were found between the effects of the studied copolymer samples.

Thus, the claimed invention makes it possible to increase the efficiency of restoration of the hematopoietic function of the bone marrow after chemotherapy, and, as a consequence, to increase its effectiveness.

EXAMPLE 2. In an experiment on animals (mice), the specific pharmacological activity of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride (hereinafter referred to as the copolymer) was assessed according to the criterion of restoration of the hematopoietic function of the bone marrow during radiotherapy.

Animals

The experiments were performed on male C57Bl/6j mice aged 1.5–2 months and weighing 18–20 g, kept under standard conditions and on a standard diet based on pelleted feed. The mice were randomly assigned to groups. All work with animals was performed in accordance with the requirements of regulatory legal acts on the procedure for experimental work and the humane treatment of animals [Zapadnyuk I.A., Zapadnyuk V.I., Zakharia E.A., Zapadnyuk B.V. Laboratory animals. Breeding, maintenance, and experimental use. Kyiv: Vishcha shkola, 1983. 383 p.].

The substance being administered

Three samples of copolymer with different number average molecular weights and 2-methyl-5-vinylpyridine unit contents were used (Table 10).

Table 10. Studied copolymer samples

Sample number Average number
molecular weight, kDa Content of 2-methyl-5-vinylpyridine units,
% (mol) C1 30 28 C2 42 34 C3 55 40

Irradiation conditions

Total irradiation of animals was performed in plexiglass containers with ^60Co γ-rays using the Luch therapeutic unit (Russia), once, at doses of 7.0 and 8.5 Gy, with an absorbed dose rate of 34.75 cGy/min. Dosimetric measurements were performed using a clinical dosimeter type 27012 (Veb RFT Messellktronik "Otto Schon", Germany). The confidence interval of the measurement error at a probability of 0.95 does not exceed 9%. For each dose of radiation exposure, 5 groups (10-14 mice each) were formed: intact control, copolymer, irradiation, copolymer + irradiation (protective variant), irradiation + copolymer.

To analyze the radioprotective properties, copolymer samples at a dose of 50 mg/kg were administered to mice intramuscularly in a volume of 0.2 ml 20 minutes after the end of radiation exposure. The hematopoietic function of the copolymer samples was assessed based on 30-day survival and the average lifespan of animals in all experimental groups. Hematopoiesis was also analyzed.

Analysis of quantitative and morphological characteristics of mature peripheral blood cells

An analysis of the quantitative and morphological characteristics of mature peripheral blood cells in laboratory mice from all experimental groups was performed using an Abacus junior vet automated hematology analyzer (Diatron, Austria). For this purpose, 0.25 ml of blood was collected from the sinus of the eye from some mice on the sixth day after irradiation and placed into special disposable EDTA tubes (Aquisel® Tube). The erythrocyte, platelet, and leukocyte counts, the white blood cell count, and the morphological heterogeneity of erythrocytes and platelets in the blood and bone marrow isolated from the femur immediately after euthanasia by cervical dislocation were assessed in all experimental groups.

Statistical analysis of the obtained data

The mean arithmetic values of the indicators, their standard errors, medians and quartiles were determined. To assess the significance of intergroup differences, parametric criteria (Student's t-test, Fisher's F-test) and nonparametric ones (Wilcoxon-Mann-Whitney (U), median chi-square test and Warden's rank test) were used [Zhavoronkov L.P. Fundamentals of Applied Medical and Biological Statistics. Obninsk: FGBU MRRC of the Ministry of Health and Social Development of the Russian Federation, 2012. 60 p.]. Differences between groups were considered statistically significant when the probability integral value p did not exceed 0.05. Statistical analysis was performed using Origin 6.0 (MicroCal Software, USA) and Statistica 6.0 (StatSoft, Inc.) programs.

Results and discussion

As can be seen from the results presented in Table 10, 30 minutes after a single intramuscular injection of the copolymer at a dose of 50 mg/kg, an increase in the total number of leukocytes, primarily due to granulocytes, is observed in the peripheral blood of animals. After 6 days, against a background of persistent leukocytosis, a decrease in the number of granulocytes and an increase in the number of lymphocytes are recorded in mice of this group. With isolated exposure to γ-rays at a dose of 8.5 Gy, profound leukopenia and thrombocytopenia are recorded in irradiated animals on the 6th day, and signs of hypochromic anemia are noted in the erythroid hematopoietic lineage (Table 11). Against this background, pronounced depletion of the bone marrow is noted, recorded by a decrease in bone marrow cellularity.

Radiation exposure at an absorbed dose of 8.5 Gy causes 100% mortality in the irradiated control group (Table 12) within the first 10 days after irradiation. Using the "irradiation + copolymer" regimen, 52.8% survival was recorded in animals on the 11th day after acute γ-ray exposure (with 100% mortality in the "irradiation" group). A slight increase in leukocyte count and a decrease in anemia were observed on the 6th day after radiation exposure.

Isolated radiation exposure at a dose of 7.0 Gy, which kills about 20% of animals, causes less disruption to the state of hematopoiesis and the antioxidant system than a dose of 8.5 Gy (Table 13).

Table 11. Main parameters (M±m) of peripheral blood and bone marrow cellularity in C57Bl/6j mice

when using a copolymer at a radiation dose of 8.5 Gy

Indicators Groups Intact control 6th day after training at a dose of 8.5 Gy Irradiation Irradiation + copolymer (sample C1) Irradiation + copolymer (sample C2) Irradiation + copolymer (sample C3) Leukocytes, ×10 ⁹ /l. 6.67±0.98 0.17±0.01*
p≤0.00004 0.88±0.35*ν
p≤0.00005 0.87±0.36*ν
p≤0.00005 0.84±0.36*ν
p≤0.00005 Lymphocytes, ×10 ⁹ /l. 4.65±0.75 0.06±0.03*
p≤0.00004 0.44±0.11*ν
p≤0.0002 0.42±0.15*ν
p≤0.0002 0.46±0.14*ν
p≤0.0002 Granulocytes, ×10 ⁹ /l. 1.75±0.23 0.07±0.02*
p≤0.00006 0.44±0.10*
p≤0.0001 0.46±0.15*
p≤0.0001 0.47±0.11*
p≤0.0002 Erythrocytes, ×10 ¹² /l. 9.5±0.3 7.5±0.8 8.8±0.5 8.9±0.5 8.4±0.6 Hemoglobin, g/l 145±6 110±13*
p≤0.01 130±6
p≤0.07 133±7
p≤0.07 135±5
p≤0.07 Platelets, ×10 ⁹ /l. 307±64 56±6*
p≤0.004 75±16*
p≤0.007 76±18*
p≤0.007 76±17*
p≤0.008 Thrombocrit, % 0.16±0.01 0.036±0.004*
p≤0.004 0.048±0.005*
p≤0.007 0.044±0.005*
p≤0.007 0.045±0.006*
p≤0.007 Bone marrow cellularity, ×10 ⁶ /hip 18.3±1.5 0.74±0.37*
p<0.0001 0.96±0.08*
p<0.0002 0.97±0.06*
p<0.0002 0.98±0.04*
p<0.0002

* - statistically significant differences compared to the control group; ν - statistically significant differences compared to the irradiation group

Table 12. Evaluation of the effect of copolymer samples on 30-day survival (%)

24 hours after exposure γ-irradiation 8.5 Gy Irradiation + copolymer (sample C1) Irradiation + copolymer (sample C2) Irradiation + copolymer (sample C3) 6 69.2±5.8 91.1±0.6 90.3±0.7 93.8±0.6 7 51.7±15.1 86.3±5.3 86.5±5.1 87.8±4.7 8 29.2±29.2 83.2±2.1 83.6±2.5 84.1±2.8 9 16.7±16.7 73.1±1.8 73.0±1.3 74.4±2.4 10 16.7±16.7 64.7±6.7 64.4±6.5 63.8±6.9 11 0 52.8±5.2 52.0±5.1 51.9±4.9 12 0 48.8±1.2 48.5±1.3 46.8±1.0 13 0 36.9±13.1 37.1±13.6 34.7±13.5 14 0 20.9±20.9 20.7±21.3 21.2±21.2 15 0 12.5±12.5 12.3±12.2 13.7±12.8 16 0 0 0 0 Average life expectancy for the group, days (M±m) 8.6±0.6 11.5±1.2 u 11.8±1.8 u 11.6±1.4 u

u - significant differences according to the Wilcoxon-Mann-Whitney U-test, in groups of 8-10 mice.

Table 13. Main parameters (M±m) of peripheral blood and bone marrow cellularity of C57Bl/6j mice when using the copolymer at a radiation dose of 7 Gy

Indicators Groups Intact control 6th day after training at a dose of 7 Gy Irradiation Irradiation + copolymer
(sample C1) Irradiation + copolymer
(sample C2) Irradiation + copolymer
(sample C3) Leukocytes, ×10 ⁹ /l. 6.29±0.93 0.45±0.08*
p <0.0005 1.14±0.37ν 1.13±0.35ν 1.16±0.34ν Lymphocytes, ×10 ⁹ /l. 4.42±0.70 0.31±0.06*
p≤0.0004 0.78±0.28*
p≤0.0007 0.79±0.25*
p≤0.0007 0.81±0.28*
p≤0.0008 Granulocytes, ×10 ⁹ /l. 1.77±0.28 0.15±0.05*
p≤0.0004 0.46±0.09*
p≤0.0001 0.42±0.11*
p≤0.0001 0.47±0.10*
p≤0.0001 Erythrocytes, ×10 ¹² /l. 9.8±0.5 7.91±0.30* 7.91±0.32 7.98±0.33 7.94±0.37 Hemoglobin, g/l 143±4 125±5*
p≤0.007 112±7*
p≤0.007 115±5*
p≤0.007 114±5*
p≤0.007 Platelets, ×10 ⁹ /l. 309±67 103±42*
p≤0.05 120±13*
p≤0.005 123±14*
p≤0.005 127±13*
p≤0.006 Thrombocrit, % 0.17±0.03 0.10±0.05 0.04±0.01 0.05±0.02 0.07±0.01 Bone marrow cellularity, ×10 ⁶ /hip 18.5±1.2 0.65±0.11*
p <0.0001 2.9±0.43*ν
p <0.0003 3.1±0.46*ν
p <0.0003 3.0±0.44*ν
p <0.0003

* - statistically significant differences compared with the control group; ν - statistically significant differences compared with the irradiation group.

It's worth noting that, with this radiation dose, on the sixth day, the white blood cell count in mice from both experimental groups was more than twice that of the "irradiated" group. Furthermore, the total cellularity of the femoral bone marrow in both experimental groups was 5-6 times higher than that in the "irradiated" group.

No statistically significant differences were found between the effects of the studied copolymer samples.

Thus, the claimed invention makes it possible to increase the efficiency of restoration of the hematopoietic function of the bone marrow after radiation therapy, and, as a consequence, to increase its effectiveness.

EXAMPLE 3.

Evaluation of the biological effect of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride (hereinafter referred to as the copolymer) according to the criterion of restoration of the hematopoietic function of the bone marrow against the background of acute blood loss.

Main tasks

In an experiment on rats, to study the rate of restoration of bone marrow cellularity upon administration of the copolymer in a single dose of 25 mg/kg after acute blood loss.

The substance being administered

Three samples of copolymer with different number average molecular weights and 2-methyl-5-vinylpyridine unit contents were used (Table 14).

Table 14. Studied copolymer samples

Sample number Average number
molecular weight, kDa Content of 2-methyl-5-vinylpyridine units,
% (mol) C1 30 28 C2 42 34 C3 55 40

Research methodology

The study was conducted on 40 female Wistar rats weighing 180-200 g. The animals were divided into 4 groups: a control group (blood loss only) and 3 experimental groups (blood loss + samples (C1-C3)). Blood loss of 35-40% was achieved by collecting blood from the tail vein under specific conditions of warming the animals and local application of anticoagulants. The copolymer was administered intravenously immediately after blood loss at a dose of 25 mg/kg in a volume of 0.45 ml. Changes in the total bone marrow cellularity and survival of the animals were assessed.

Research results

The bone marrow cellularity index in rats on the 16th day after blood loss and administration of the copolymer is presented in Table 15.

Table 15. Total cellularity of bone marrow in rats on the 16th day after blood loss and administration of the copolymer

Groups Bone marrow cellularity, ×10 ⁵ /hip Control 186.6±21 C1 220.3±23 ⁰ C2 225.5±18 ⁰ C3 214±17 ⁰

Note: ⁰ - significant differences (p<0.05) according to Student's T-test of the indicators of the experimental groups with the control.

Data on the dynamics of animal survival in the experiment are presented in Table 16.

Table 16. Survival rate of animals in the experiment

Group Survival per day
experiment, % Survival in the next 16 days of the experiment after
blood loss, % Control 72 57 C1 100 100* C2 86 86* C3 100 86*

Note: * - intergroup differences were identified (according to Fisher, χ ² criterion)

Discussion of results

Based on the obtained results, in animals of the experimental groups that received samples of the copolymer (C1-C3), on the 16th day after blood loss, the value of the total cellularity of the bone marrow turned out to be statistically significantly higher than the corresponding value for animals of the control group.

A study of the survival dynamics of animals in the experiment also revealed differences between animals in the control and experimental groups. On the first day of the experiment, after a single blood draw of 25-30% of the total circulating blood volume, survival rates were 80% and 90%, respectively. Over the next 16 days of the experiment, after blood loss, 50% of animals in the control group survived, while 80% of animals in the copolymer solution-treated group survived (Table 16). These survival differences were statistically significant according to the ^χ2 test.

No statistically significant differences were found between the effects of the studied copolymer samples.

Thus, the claimed invention makes it possible to increase the efficiency of restoring the hematopoietic function of the bone marrow in case of blood loss.

EXAMPLE 4

The preferred forms of administration of the copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride for the restoration of hematopoietic function during chemo- and radiotherapy of neoplasms are injection forms. The above forms can be sterilized and contain additives such as preservatives: sodium metabisulfite, benzoic acid, sodium benzoate, thiomersal, a mixture of methylparaben and propylparaben; stabilizers: apricot and acacia gum, dextrin, starch paste, methylcellulose, tween; salts regulating osmotic pressure (sodium chloride), or buffers. In addition, they can contain other therapeutically useful substances.

Composition per 1 bottle: active substance: copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride - 250 mg, water for injection - up to 5 ml (5%).

To prepare injection forms, the active compound (500 g; the amount required for 1,000 vials) is finely ground and mixed in a mixer with water for injection (10,000 ml). The resulting solution is sterilized-filtered and packaged in 5 ml vials.

Claims (6)

1. The use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride, the average molecular weight of which is 30-55 kDa and the content of 2-methyl-5-vinylpyridine units is 28-40 mol.%, to restore the hematopoietic function of the bone marrow during chemotherapy or radiotherapy of neoplasms or blood loss. 2. The use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride according to claim 1, wherein the chemotherapy comprises the administration of the drug Paclitaxel. 3. The use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride according to claim 1, wherein the chemotherapy comprises the administration of the drug Cisplatin. 4. The use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride according to claim 1, wherein the chemotherapy comprises the administration of the drug Fluorouracil. 5. The use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride according to claim 1, wherein the chemotherapy comprises the administration of the drug Doxorubicin. 6. The use of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone hydrochloride according to claim 1, wherein the chemotherapy comprises the administration of the drug Carboplatin.