RU2634572C1 - Means for correction of violations in hemogenesis erythroid lineage caused by cystostatic impact - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: application of a liquid alcohol extract of rowan tree fruit (Sorbus aucuparia L.) - for pharmacological correction of disturbances in the hemogenesis erythroid lineage caused by cytostatic impact.

EFFECT: extract effectively corrects disturbances in the hemogenesis erythroid lineage caused by cytostatic impact.

2 tbl, 1 ex

Description

The invention relates to medicine, specifically to hematology, and can be used for pharmacological correction of erythropoiesis disorders developing during cytostatic effects.

According to recent clinical studies, anemia is one of the main manifestations of various pathological processes (1, 2). So, anemia is one of the most important syndromes developing in patients with malignant neoplasms (3, 4). The risk of developing severe anemic conditions in cancer patients increases even more during antitumor chemotherapy, which is associated with the high sensitivity of the erythron to the toxic effect of most known cytostatic drugs (5, 6). It is known that the erythroid hematopoiesis is the most sensitive to the inhibitory effect of cytostatics, in particular, antitumor antibiotics of the anthracycline series (doxorubicin) and platinum preparations (carboplatin) (7, 8).

The main method of treating anemia is blood transfusion, when used, there is a risk of developing infections, as well as allergic and immunological reactions (9, 10). An alternative way to correct erythropoiesis disorders caused by the use of cytostatics can be the use of nucleic acid precursors, B vitamins, zymosan, recombinant erythropoietin, immunomodulators, etc. (11). Despite the high effectiveness of these pharmacopeia drugs, they can cause side effects. So, when using erythropoietin in clinical practice, a hypertensive crisis with the phenomena of encephalopathy, an increase in the number of platelets, a decrease in iron metabolism, allergic manifestations in the form of a rash, itching, urticaria, reactions at the injection site, and individual intolerance to the drug are possible (12).

The problem solved by the present invention is to expand the arsenal of herbal remedies for use with the purpose of pharmacological correction of disorders in the erythroid germ of hematopoiesis caused by cytostatic effects.

To solve this problem, use liquid alcoholic extract of the fruits of mountain ash (Sorbus aucuparia L.), obtained by the method of dynamic fractional maceration. To obtain the extract, we used a laboratory reactor with a steam jacket (Radleys, Germany) connected to a submerged circulating temperature control unit MO1 (Termeks, Russia), an overhead drive mixer RZR 2020 (Heidolph, Germany) and a ball refrigerator. The fruits of mountain ash were crushed to a particle size of 0.3-0.5 cm in diameter. The extract was prepared in a ratio of 1: 5 on 95% acidified ethyl alcohol. During the extraction, 500 ml of a liquid extract was obtained. Acidified ethyl alcohol was prepared at the rate of 6.49 g of concentrated hydrochloric acid per 200 ml of 95% ethyl alcohol. The finished extract was defended at a temperature of no higher than 10 ° C for at least 2 days until a clear liquid was obtained, then it was filtered and stored at a temperature of no higher than 5 ° C in a dark place.

The 95% acidified alcohol extract is a dark cherry liquid with a specific fruity odor of a sour-bitter taste.

The dry residue of the liquid extract of the fruits of mountain ash was determined using a weight hydrometer MS-70 ("AND", Japan). The dry residue of the studied extract is 14 ± 0.22%.

According to the data of chemical analysis carried out by conventional methods (13), the main active ingredients of the extract are phenolic acids and anthocyanins. Quantitative determination of these BAS groups in the studied extract was carried out by direct spectrophotometry (14, 15). The content of the sum of phenolic acids (in terms of chlorogenic acid) in the extract of mountain ash is 2.97 ± 0.15%, and the amount of anthocyanins (in terms of cyanide-3-o-glucoside) is 4.30 ± 0.20%.

New is that as a means of preventing the development of depression of the erythroid germ of hematopoiesis caused by the use of cytostatics, liquid alcoholic extract of the fruits of mountain ash is used.

The use of liquid alcoholic extract of fruits of mountain ash for a new purpose was made possible due to the new properties identified by the authors that prevent depression in relation to the erythroid germ of hematopoiesis against the background of cytostatic effects.

The new property does not explicitly follow from the prior art in this field, is not obvious to a specialist and is not found in the patent and medical literature. Proposed as an invention, the tool can be used in clinical hematology and oncology.

The present invention meets the conditions of patentability "Novelty", "Inventive step" and "Industrial applicability".

This tool has undergone an experimental study in the oncopharmacology laboratory of the Scientific Research Institute of Physiology and Physics E.D. Goldberg (Tomsk) and the department of pharmacognosy with courses in botany and ecology FSBEI HE Siberian State Medical University of the Ministry of Health of Russia.

New properties of the liquid alcoholic extract of the fruits of mountain ash were discovered experimentally. The study was conducted on female mice of the C57BL / 6 line weighing 20-22 g at the age of 3 months. According to the Guidelines for Preclinical Studies of Medicines (16), the model of inhibition of the erythroid germ of hematopoiesis was cytostatic myelosuppression caused by the administration of intact animals with doxorubicin (anthracycline antibiotic) at the maximum tolerated dose (MTD) of 6 mg / kg.

For the experiment, animals (female mice of the C57BL / 6 line) were divided into groups:

1. Intact control - healthy mice.

2. Doxorubicin - mice were once intraperitoneally injected with doxorubicin at a dose of 6 mg / kg in 0.1 ml of physiological saline. Animals received 0.2 ml of distilled water intragastrically, 24 hours after injection of the cytostatic drug daily for 14 days.

3. Doxorubicin + mountain ash extract - mice were once intraperitoneally injected with doxorubicin at a dose of 6 mg / kg in 0.1 ml of physiological saline. Animals received extract of fruits of mountain ash ordinary at a dose of 5 ml / kg in 0.2 ml of distilled water intragastrically, 24 hours after injection of the cytostatic drug daily for 14 days.

The specific activity of drugs that selectively stimulate bone marrow erythropoiesis was evaluated using the following indicators (16):

1) the determination of the number of red blood cells and reticulocytes in the peripheral blood, the content of hemoglobin, hematocrit was performed using a hematological analyzer "Muchic 18vet" (Cormay, France).

2) counting the total cellularity of the bone marrow and myelograms.

In healthy mice (intact control), as well as animals treated with doxorubicin (on the 3rd, 5th, 7th, 10th, 12th and 15th day after cytostatic administration), peripheral blood parameters (reticulocytes, erythrocytes, hemoglobin, hematocrit) and bone marrow ( the total number of karyocytes, immature and mature neutrophilic granulocytes, eosinophils, monocytes, lymphocytes, erythroid cells) using standard hematological methods (17, 18).

Statistical processing of the results was carried out by calculating the mean (X) and standard error (m) using nonparametric Wilcoxon-Mann-Whitney (U) methods. The difference was considered significant at P <0.05 (19).

In order to identify the pharmacological effect of the liquid alcoholic extract of the mountain ash fruits on the erythroid hematopoietic sprout under the conditions of doxorubicin-induced anemic syndrome in healthy C57BL / 6 mice, the following experiment was performed.

Example 1

In the experiment, it was found that a single intraperitoneal administration of doxorubicin at a dose of 6 mg / kg has a depressing effect on the erythroid germ of hematopoiesis. The dynamics of the content of reticulocytes was characterized by the development of reticulocytopenia (3-7 days), with subsequent recovery by 10 days of the study. The maximum drop in the number of reticulocytes was observed on the 5th day of the experiment: their number in the peripheral blood of mice treated with doxorubicin decreased by 3.3 times (P <0.01) relative to this indicator in healthy animals. On days 3, 5, and 7 of the experiment, the number of red blood cells, as well as the hemoglobin content in the peripheral blood of mice of the monochemotherapy group, were significantly lower than these values in intact animals. A decrease in the number of red blood cells was recorded 12 days after the administration of cytostatic. The hematocrit level throughout the experiment was significantly lower than the values in healthy mice (table 1).

Note: hereinafter, before the significance level P, the numbers of the compared groups are indicated.

The course application of the extract of fruits of mountain ash ordinary at a dose of 5 mg / kg had both a protective and stimulating effect on the process of erythropoiesis. So, on the 3rd, 5th and 12th day of the experiment, the number of reticulocytes in mice that received the plant extract after administration of doxorubicin turned out to be 1.3 (P <0.01), 1.9 (P <0.05) and 1.1 ( P <0.05) times higher than those in animals of the monochemotherapy group. A significant increase in the number of red blood cells, the level of hemoglobin and hematocrit in the peripheral blood of animals treated with rowanberry extract against doxorubicin compared with these parameters in mice treated only with a cytostatic was noted at the same time of the study (table 1).

Analysis of animal myelograms showed that the development of hypoplasia of bone marrow hematopoiesis under the influence of cytostatics was due to the deep suppression of all germs of hematopoiesis. So, after a single intraperitoneal injection of doxorubicin to mice at a dose of 6 mg / kg, a reduced number of karyocytes was recorded from 3 to 7 days, reaching the values of animals in the intact control group by 10 days (table 2).

A significant decrease in the number of mature non-core granulocytes by 1.3 times on the 3rd and 5th day of the study was noted. The number of immature (at 7, 12, 15 days) and mature (at 12, 15 days) forms of neutrophilic leukocytes exceeded that of healthy mice. A significant decrease in the number of bone marrow lymphoid elements was observed up to 12 days with a maximum decrease in their number (1.7 times, P <0.01) by day 3 of the study (table 2).

The administration of doxorubicin to mice also led to the development of deep depression of the erythroid germ of hematopoiesis. So, on 3, 5, 7, 10, 12 days after cytostatic injection, the number of erythroid cells was reduced by 3.2; 2.0; 2.4; 2.1; 1.5 times (P <0.01), respectively, relative to this indicator in animals of intact control (table 2).

When using the extract of fruits of mountain ash ordinary during the administration of doxorubicin, an increased number of karyocytes in the bone marrow of mice was observed on the 5th, 7th and 12th days of the experiment compared with that in mice of the monochemotherapy group. The analysis of myelograms showed that the extract of the fruits of the mountain ash ordinary has protective properties, and also stimulates the regeneration of erythroid and granulocytic hematopoietic sprouts. The number of erythroid cells in the bone marrow of mice treated with a herbal preparation exceeded 1.5 by 3, 5, and 12 days; 1.5 and 1.4 times the value of this indicator in animals receiving only cytostatic (table 2).

On the 5th and 12th days of the experiment, an increase in the content of mature and / or immature forms of neutrophilic granulocytes was recorded in the bone marrow of mice treated with doxorubicin extract of the mountain ash fruits compared to those in animals of the monochemotherapy group. The plant extract also contributed to the restoration of the cellularity of the hematopoietic lymphoid germ after cytostatic damage: on the 5th and 7th day of the study, the number of bone marrow lymphoid cells was significantly higher (1.3 times) in mice treated with doxorubicin (table 2).

Thus, in an experiment on female mice of the C57BL / 6 line with doxorubicin-induced anemic syndrome, the ability of the alcohol extract of the fruits of mountain ash to prevent the development of depression of the erythroid germ of hematopoiesis due to its protective properties for 3, 5 days of research and stimulation of the regeneration of erythropoiesis cells in more late dates (12 days).

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Claims (1)

The use of liquid alcohol 95% acidified extract of fruits of mountain ash (Sorbus aucuparia L.) as a means for pharmacological correction of disorders in the erythroid germ of hematopoiesis caused by cytostatic effects.