RU2480236C1 - Drug possessing regenerative activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to pharmacology and cell engineering. What is presented is the use of hyaluronate-endo-β-N-acetylhexosaminidase immobilized on the low-molecular water-soluble carrier by ionising radiation as a drug possessing regenerative activity.

EFFECT: invention enables extending the indications for the use of hyaluronate-endo-β-N-acetylhexosaminidase immobilized on a low-molecular water-soluble carrier by ionising radiation which possess evident regenerative activity and therapeutic properties in degenerative diseases: diabetes mellitus, hepatic cirrhosis, pulmonary fibrosis and encephalopathy.

9 tbl, 7 ex

Description

The invention relates to medicine, specifically to pharmacology and cell technology.

The high incidence and low effectiveness of the therapy of degenerative diseases [1, 2] are the basis for the development of new agents with pronounced regenerative activity.

Known agents with regenerative activity (sodium nucleinate, methyluracil, etc.) [3].

The disadvantages of these funds in most cases is their low efficiency [1, 2].

There is a hyaluronidase (hyaluronate-endo-β-N-acetylhexosaminidase) immobilized by means of ionizing radiation, which has the ability to increase the reserve of stem cells in the bone marrow [4] and has a hypoglycemic [5] and hemostimulating [6] effect.

We have for the first time revealed pronounced regenerative activity of hyaluronate-endo-β-N-acetylhexosaminidase immobilized with ionizing radiation in models of various degenerative diseases.

The proposed tool does not have adequate analogues.

The problem solved by the present invention is to expand the indications for the use of hyaluronate-endo-β-N-acetylhexosaminidase immobilized by means of ionizing radiation.

The task is achieved by using immobilized using ionizing radiation hyaluronate-endo-β-N-acetylhexosaminidase as a means of regenerative activity.

New in the present invention is the use of immobilized using ionizing radiation hyaluronate-endo-β-N-acetylhexosaminidase as a means of regenerative activity.

The original product used by us, which was immobilized using ionizing radiation (electron beam synthesis), hyaluronate-endo-β-N-acetylhexosaminidase was developed and obtained by LLC “Scientific Future Management” (Novosibirsk) together with the Scientific Research Institute of Pharmacology SB RAMS (Tomsk) .

A distinctive link in the pathogenesis of degenerative diseases is a violation of the ability of individual body tissues to recover and compensate by realizing their own regenerative potential and the development of dystrophic processes against the background of proliferation of sclerotic tissue [7, 8]. Moreover, the pharmacological effect of the agents existing for their treatment is based primarily on the stimulation or replacement of the functions of the preserved cellular elements. However, the concept of compensation for impaired activity and damage to target organs due to the indicated adaptive processes in a significant number of cases is untenable [1, 2, 8]. The presented facts indicate the need for the development of a strategy of regenerative medicine, that is, the replacement of dead or dysfunctioning cellular elements with new ones, as its main and main task.

The implementation of this approach can be achieved using pharmacological approaches to cell therapy, including by prescribing modifiers of stem cell functions [1, 2, 8].

Hyaluronate-endo-β-N-acetylhexosaminidase is an enzyme that affects the metabolism of hyaluronic acid, which is the most common glycosaminoglycan in the body, and is part of the intercellular matrix of tissues, as well as the glycocalyx of cells and their receptors for various biologically active substances [9, 10]. The ability of preparations of this enzyme, including those immobilized with the help of nanotechnology of electron-beam synthesis of hyaluronidase, to increase the permeability of various tissue barriers to exogenous and endogenous substances, provide a therapeutic effect against contractures, scars, etc., stimulate hematopoiesis and others is known [5, 6 ]. The possibility of increasing the content of stem cells (SC) in the bone marrow using hyaluronate-endo-β-M-acetylhexosaminidase is also known [4]. However, the further fate of SC, the possibility of their participation in the processes of cellular self-renewal, the likelihood of the realization of the growth potential and participation in the regeneration of damaged tissues and organs lacking the ability to repair due to their own potential, remain unknown. The experiment showed unpredictable results.

The fact of using immobilized hyaluronate-endo-β-N-acetylhexosaminidase (imGEAGA) with the achievement of a new technical result, which consists in significant regenerative effects in degenerative diseases, is not obvious to a specialist.

New properties do not follow explicitly from the prior art in this field and are not found in the patent and scientific literature.

The present invention can be used in medicine.

Based on the foregoing, it should be considered the claimed technical solution in accordance with the criteria of "Novelty", "Inventive step", "Industrial applicability".

The experiments were performed on 247 CBA / CaLac mice and 94 outbred rats. The animals were obtained from the nursery of the experimental biomedical modeling department of the Scientific Research Institute of Pharmacology SB RAMS.

The studies were carried out in accordance with the rules of laboratory practice (GLP), Order of the Ministry of Health of the Russian Federation No. 267 dated 06/19/2003 “On approval of the rules of laboratory practice”, Federal Law 61 “On the circulation of medicines”, “Guidelines for the experimental (preclinical) study of new pharmacological substances ”(Moscow, 2005) [11].

As pathological conditions, disease models were selected that share the main features of pathogenesis with most degenerative nosologies: type 1 diabetes mellitus, cirrhosis of the liver, pulmonary fibrosis, and posthypoxic encephalopathy; which allows extrapolating the results obtained on these models to most known pathological conditions of a degenerative nature.

Example 1

A 10% aqueous solution of polyethylene glycol with a molecular weight of 400 Da was irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. The treatment was carried out by bremsstrahlung generated by the ILU-6 accelerator with an electron energy of 2.5 MeV, an absorbed dose of 2 kGy, and a dose rate of 1.65 kGy / hour. Hyaluronate-endo-β-N-acetylhexosaminidase (Scientific Future Management LLC, Novosibirsk) was added to the irradiated solution to a final concentration of 70 PIECES of hyaluronate-endo (β-N-acetylhexosaminidase in 1 ml of 10% polyethylene glycol. The mixture was stirred 10 minutes and received the final immobilized preparation in the form of a slightly opalescent solution.The yield of the finished product is 96.2%.

Example 2

A 5% aqueous solution of hydroxyethyl starch with a molecular weight of 1.5 kDa was irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. The treatment was carried out by bremsstrahlung generated by the ILU-10 accelerator with an electron energy of 2.5 MeV, an absorbed dose of 10 kGy, and a dose rate of 1.65 kGy / hour. Hyaluronate-endo-β-N-acetylhexosaminidase (Scientific Future Management LLC, Novosibirsk) was added to the irradiated solution to a final concentration of 1 U of hyaluronate-endo-β-N-acetylhexosaminidase in 1 ml of 5% hydroxyethyl starch. The mixture was stirred for 10 minutes and the final immobilized preparation was obtained in the form of a slightly opalescent solution. The yield of the finished product is 97.3%.

Example 3

In a 10.0% aqueous solution of polyvinylpyrrolidone with a molecular weight of 4 kDa, hyaluronate-endo-β-N-acetylhexosaminidase (Scientific Future Management LLC, Novosibirsk) was added to a final concentration of 500 IU of hyaluronate-endo-β-N-acetylhexosamine 1 ml The mixture was then stirred for 30 minutes and irradiated with gamma radiation at a dose of 1.0 Mrad. The treatment was carried out by bremsstrahlung generated by the ILU-10 accelerator with an electron energy of 2.5 MeV, an absorbed dose of 5 kGy, and a dose rate of 1.65 kGy / hour. The mixture was stirred for 10 minutes and the final immobilized preparation was obtained in the form of a slightly opalescent solution. The yield of the finished product is 95.7%.

Example 4

The antidiabetic effect of immobilized hyaluronate-endo-β-N-acetylhexosaminidase was investigated.

The experimental model of diabetes mellitus was alloxan diabetes [12]. In mice, diabetes was modeled by subcutaneous administration of 1-aqueous alloxan according to the following scheme: for 4 days, 300 mg / kg daily, then again in the same dose on the 7th day after the last injection in a volume of 0.2 ml / mouse . From 3 days after the start of diabetes modeling, experimental animals were injected intragastrically once a day for 3 days with 50 U / kg of imGEAG obtained in Example 1. Control animals were injected with distilled water in the same volume in the same way.

The state of the endocrine apparatus of the pancreas of animals was assessed by recording the level of glucose in peripheral blood and using a morphological study of the organ. Glucose was determined on an empty stomach in the morning on the 11th and 21st days of the experiment using the Optilite glucometer (Hungary) and the Optilite test strip attached to it. For morphological studies on the 8th, 11th, 15th, 21st day of the experiment, the part of the pancreas adjacent to the spleen was fixed in a 10% formalin solution and embedded in paraffin. Deparaffinized sections with a thickness of 5 μm were stained with hematoxylin and eosin. The sections were used to determine the area of 10 consecutive islets of Langerhans by means of a graphical computer analysis and the total number of cells, the number of pyknotized cell elements were calculated in them, and the cell content per unit area of the island and the percentage of pyknotized cells were calculated. In order to study the mechanism of action of the drug, the state of various stem cell pools was studied: the number of multipotent stem cells (MSCs) in the bone marrow and peripheral blood on the 8th day of the experiment [2] and the dynamics of the content of regional stem cells in the pancreas on 8, 11, 15 21st day [13]. The results were processed by the method of variation statistics using Student's t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

During the experiment, the administration of alloxan led to pycnosis of a significant part of the cells of the islets of Langerhans, observed at all stages of the study. In addition, pronounced phenomena of edema and hyperemia of the endocrine apparatus with the development of significant lymphomacrophageal tissue infiltration were noted (Table 1). A natural reflection of morphological changes in the pancreas was a significant increase in glucose in peripheral blood and the development of hyperglycemia (Table 2)

Moreover, the course administration of imGEAG after modeling alloxan diabetes led to a significant decrease in the percentage of pycnotized cells in the islets of Langerhans on the 11th, 15th day of the experiment against the background of a decrease in the total number of cells per unit area of the islet as a result of a significant decrease in islet infiltration (Table 1). The described pathomorphological picture was accompanied by normalization of blood glucose level (Table 2)

Table 1 The dynamics of the content of pycnotized cells in the islet of Langerhans in% (A) and the total number of cells per unit area of the islet (B), (X ± m) Study time (days) Control (alloxan diabetes) Used remedy BUT B BUT B intact 3.88 ± 0.23 0.75 ± 0.04 3.88 ± 0.23 0.75 ± 0.04 8th 16.18 ± 0.89 * 0.75 ± 0.03 15.2 ± 0.9 * 0.78 ± 0.05 11th 14.35 ± 0.94 * 0.77 ± 0.03 5.3 ± 0.5 * # 0.54 ± 0.03 # 15th 12.8 ± 1.06 * 0.74 ± 0.03 5.9 ± 0.2 * # 0.7 ± 0.08 21st 11.8 ± 1.97 * 0.9 ± 0.07 6.1 ± 0.4 * 0.69 ± 0.1 # * - the reliability of relatively intact animals was noted at p <0.05. # - authenticity was noted relative to animals with alloxan diabetes at p <0.05.

table 2 The level of glucose in peripheral blood, mmol / l (X ± m) Study time (days) Control (alloxan diabetes) Used remedy intact 3.16 ± 0.39 11th 17.11 ± 1.15 * 5.8 ± 0.12 # 21st 13.03 ±, 9 * 3.6 ± 0.6 ## * - the reliability of relatively intact animals was noted at p <0.05. # - authenticity was noted relative to animals with alloxan diabetes at p <0.05.

In the study of the possible mechanisms of action of the revealed high efficiency of imGEAG in the experimental treatment of diabetes mellitus in the control group, there were no changes in the bone marrow and circulating pools of true (multipotent) stem cells. Moreover, throughout the experiment, a marked decrease in the content of regional stem cells in the pancreas was noted (Table 3). At the same time, a study of the possible stimulation of these compensatory-adaptive mechanisms when modeling diabetes mellitus using the introduction of imGEAG revealed a significant increase in MSCs in the peripheral blood, indicating their mobilization from depot tissues, and an increase in the number of regional stem cells (Table 3).

Table 3 The number of mesenchymal stem cells in the bone marrow (by 10 ⁶ myelocaryocytes) (A), in the peripheral blood (by 10 ⁶ mononuclear cells) (B), and the dynamics of the content of regional stem cells in the pancreas (by 10 ⁵ nuclears) (C), ( X ± m) Study time (days) Control (alloxan diabetes) Used remedy BUT B AT BUT B AT intact 7 ± 2 4 ± 2 15.17 ± 1.3 7 ± 2 4 ± 2 15.17 ± 1.3 8th 10 ± 2 5 ± 2 8.83 ± 0.54 * 12 ± 2 # 18 ± 2 * # 21.0 ± 0.2 * # 11th - - 6.17 ± 0.87 * - - 18.4 ± 0.7 * # 15th - - 8.17 ± 1.25 * - - 12.8 ± 0.7 # 21st - - 6.5 ± 0.76 * - - 10.2 ± 0.4 * * - the reliability of relatively intact animals was noted at p <0.05. # - authenticity was noted relative to animals with alloxan diabetes at p <0.05.

Thus, immobilized hyaluronate-endo-β-N-acetylhexosaminidase allows for the effective treatment of diabetes mellitus by stimulating endogenous stem cells, accompanied by the repair of the organ's insulin-producing apparatus and normalization of the level of glucose in peripheral blood.

Example 5

The study of therapeutic effects in cirrhosis.

The experiments were conducted on outbred rats. Cirrhosis was caused by the joint intragastric administration of a 50% solution of CCl ₄ ((hepatotropic poison) in olive oil at a dose of 2 ml / kg for 3 weeks 2 times a week (6 times)). Moreover, instead of water, animals received a 10% solution of ethanol throughout the entire period of the experiment [11]. Experimental animals, starting from the day after the last administration of tetrachlorocarbon, were orally administered IMGEAG (obtained according to Example 2) at a dose of 20 U / kg orally every other day for 10 days. To the control animals, distilled water was injected in an equivalent volume in the same way.

To study the state of the liver, rat death was assessed, biochemical studies of serum levels of aspartate and alanine aminotransferases (AcAT, AlAT) were performed on the 40th day, as well as a morphological study of the liver on the 40th day of the experiment. The activity of serum enzymes was determined by conventional methods using a Cormay semi-automatic biochemical analyzer and standard kits for it. Blood for the study was obtained from the femoral artery through a catheter. On histological liver specimens stained with hematoxylin and eosin, the number of infiltrate cells was determined using an Avtandilov ocular network containing 25 test points [14]. In 20 fields of view, the number of cells reaching the test points of the grid was counted. The relative area of infiltration was calculated as the ratio of the grid points per cell of the infiltrate to all grid points in 20 fields of view. The area of connective tissue was determined using computer graphics processing. For this, the area of structures stained with picrofuchsin was measured on a standard area of a liver section (successive microphotographs of 10 fields of view performed by a Digital micro video camera with an image transfer program to a computer from Elecard, Tomsk) and the percentage of the selected standard area was calculated. The results were processed by the method of variation statistics using Student's t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

The experiments showed a significant death of rats in the control (47.6%), while with the introduction of ImGEAG against the background of cirrhosis modeling, virtually no deaths were observed (3.7%). Biochemical studies of blood serum revealed an increase in the activity of AlAT, and AsAT and alkaline phosphatase on the 40th day of the experiment after the start of CCl ₄ administration in the control group (Table 4). At the same time, the use of imGEAG was accompanied by a significant decrease in the enzymatic activity of blood serum.

In the study of histological preparations of the liver in the control group of rats, a marked violation of the lobular structure of the organ was noted. There was a deformation of the terminal hepatic venules, arterioles, and bile ducts. Fields of granulation tissue were replaced by dead hepatocytes, in which neoplasms of the vessels and hepatic ducts occurred, fibrous cords and micro nodes (pseudo-fractions) were visible. The latter were a group of hepatocytes surrounded by patches of fibrosis. In preserved hepatocytes, pronounced large-drop fatty degeneration was observed. The introduction of imGEAG led to a significant regression of morphological signs of cirrhosis. At the same time, restoration of the lobular structure of the liver was observed, although signs of small-drop fatty degeneration and portal infiltration persisted until the end of the observation period.

Calculation of the cell content of the infiltrate and the area of the connective tissue showed a sharp drop in these indicators in the group of rats treated with HEMAAG (Table 5).

Thus, immobilized hyaluronate-endo-β-N-acetylhexosaminidase has a pronounced anti-cirrhotic effect. Moreover, the mechanism of its action, obviously, is the stimulation of liver SC and the mobilization of bone marrow progenitor elements into peripheral blood with their further deterministic homing into the liver tissue and differentiation into tissue-specific elements [1, 2, 8].

Table 4 The effect of drugs on the biochemical parameters of male rats with cirrhosis (1) and with the introduction of immobilized hyaluronate-endo-β-N-acetylhexosaminidase against the background of modeling liver damage (2) on the 40th day of the experiment, X ± m Groups AlAT (mkkat / l) AsAT (mkkat / l) Alkaline phosphatase (E / l) background 0.22 ± 0.01 0.24 ± 0.03 231.0 ± 23.4 one 0.69 ± 0.03 * 0.70 ± 0.04 * 415.9 ± 43.6 * four 0.21 ± 0.01 # 0.29 ± 0.07 # 229.1 ± 16.5 # * - the significance of the difference between the indicator and its background value is noted at p <0.05. # - the significance of the difference of the indicator from its control value at p <0.05 is noted.

Table 5 The effect of drugs on the morphological parameters of the liver of male rats with cirrhosis (1) and with the introduction of immobilized hyaluronate-endo-β-N-acetylhexosaminidase against the background of modeling liver damage (2) on days 21 and 40 of the experiment, X ± m Groups The relative area of the infiltrate (%) The relative area of the compound. tissue (%) background 1.56 ± 0.3 3.78 ± 0.3 one 31.4 ± 3.2 * 29.7 ± 1.44 * 2 7.6 ± 0.58 * # 8.2 ± 0.56 * # * - the significance of the difference between the indicator and its background value is noted at p <0.05. # - the significance of the difference of the indicator from its control value at p <0.05 is noted.

Example 6

The therapeutic effect of immobilized hyaluronate-endo-β-N-acetylhexosaminidase in pulmonary fibrosis was investigated.

Lung fibrosis was simulated in mice by a single intratracheal injection of 80 μg of bleomycin (Bleomycetin, OJSC Lanspharm) in 30 μl of saline [15]. Experimental animals, starting from the 7th day of the experiment, were orally administered for 10 days with imGEAG (obtained in Example 3) at a dose of 100 U / kg. To the control animals, distilled water was injected in an equivalent volume in the same way.

On the 7th, 14th, 21st and 25th and 40th days after administration of bleomycin, the morphological picture of the lungs was studied. For histological examination, lung pieces were fixed in 10% formalin. According to the standard histological method, material was posted, the objects of research were poured into paraffin blocks, from which histological sections were made with a thickness of 5 μm. Deparaffined sections were stained with hematoxylin and eosin and, according to Van Gieson, on connective tissue [14]. Using computer graphical analysis on the standard area of the histological section was measured the area of collagen fibers in lung tissue. The results were processed by the method of variation statistics using Student's t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

The introduction of bleomycin led to the development of morphological changes characteristic of toxic fibrosing alveolitis in the lungs of mice. From the 7th day of the experiment, edema of the alveolar septa, infiltration of the interstitium of the alveoli with neutrophils, lymphocytes, macrophages and plasma cells was noted. The 14th and 21st days were characterized by increased edema and increasing diffuse infiltration of the interstitium of the alveoli and alveolar passages by inflammatory cells, which led to a significant violation of the histoarchitectonics of the lung tissue. At the same time, venous plethora and hemorrhages were observed in the walls of the alveoli. On the 25th day of the experiment, cell infiltration of the lung tissue was most pronounced. In the central parts of the organ, there were areas where the pulmonary pattern was absent, part of the alveoli was emphysema dilated. Strengthening cell lung infiltration led to a thickening of the alveolar interstitium, compression of the capillaries and disorganization of the alveolar structures. In the interstitium of the alveoli, there was an increase in the formation of collagen fibers and a progressive proliferation of connective tissue. On the 7th day of the experiment, the connective tissue was located mainly perivascular and peribronchial, by the 14th day there was a diffuse spread of fibrotic masses, and on the 25th and 40th day the picture of pneumofibrosis was most pronounced.

The introduction of imGEAG was accompanied by a significant normalization of the morphological picture of lung tissue. Edema of the interalveolar septa and infiltration of the interstitium of the alveoli by inflammatory cells, venous congestion and hemorrhage were significantly less pronounced than in the control. At the same time, the histoarchitectonics of the lung tissue was preserved until the end of the observation period, and the analysis of the graphic data of histological preparations revealed a significant decrease in the area of connective tissue in the lungs (Table 6).

Table 6 The effect of drugs on the morphological parameters of the lungs of mice with cirrhosis (1) and with the introduction of immobilized hyaluronate-endo-β-N-acetylhexosaminidase against the background of modeling pulmonary fibrosis (2), X ± w Study time (days) The relative area of the compound. tissue (%) The control Suggested remedy background 2.42 ± 0.4 2.42 ± 0.4 7th 5.6 ± 0.9 * 6.1 ± 0.3 * 14th 6.6 ± 0.78 * 6.3 ± 0.6 * # 21st 8.1 ± 1.2 * 6.4 ± 0.32 * # 25th 12.7 ± 0.78 * 5.9 ± 0.27 * # 40th 13.2 ± 0.5 * 5.1 ± 0.15 * # * - the significance of the difference between the indicator and its background value is noted at p <0.05. # - the significance of the difference of the indicator from its control value at p <0.05 is noted.

Thus, immobilized hyaluronate-endo-β-N-acetylhexosaminidase has a pronounced therapeutic (antisclerotic) effect in pulmonary fibrosis.

Example 7

In an experiment in mice, the cerebroprotective effects of imGEAG were studied in a model of posthypoxic encephalopathy. Assessment of the state of the central nervous system of animals was carried out by registering indicators of neuropsychiatric status: conditioned reflex activity and tentatively research behavior of animals in an open field.

Encephalopathy was simulated using a 500 ml heat chamber. The mice were placed in a heat chamber, the lid was closed, and the mice remained there until an atonal convulsive seizure or respiratory arrest, as determined visually, for 10-15 seconds. After extracting from the heat chamber and restoring spontaneous breathing, after 5-10 minutes, the mice were again placed in the heat chamber also before the atonal state (generalized seizure or respiratory arrest) [16]. On the 7th day after modeling encephalopathy in animals, a conditioned reflex of passive avoidance was developed. Experimental animals, starting from the 7th day of the experiment, were orally administered for 2 days with imGEAG (obtained according to Example 1) at a dose of 10 U / kg. To the control animals, distilled water was injected in an equivalent volume in the same way. Checking the safety of the reflex and orienting research behavior in the open field was carried out on the 14th, 21st day of the experiment. The results were processed by the method of variation statistics using Student's t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

During the experiment, hypoxic exposure led to the formation of encephalopathy. An increase in the total motor activity of mice in the open field, the number of horizontal movements, and an increase in the coefficient of asymmetry of movements were observed. At the same time, there was a sharp drop in the level of reproduction of the conditioned reflex of passive avoidance and spontaneous mortality of animals throughout the observation period, reaching 38.3% on the 21st day.

With the introduction of imGEAG, a pronounced therapeutic (cerebroprotective) effect was observed. The use of the drug completely abolished the appearance of signs of encephalopathy (Tables 7, 8).

Table 7 Conditioned reflex activity and mortality of CBA / CaLac mice with encephalopathy caused by herbal volume hypoxia (1), and with the introduction of immobilized hyaluronate-endo-β-N-acetylhexosaminidase after animals were kept in a heat chamber (2), in conventional units, ( X ± m) Study time, days The share of dead animals,% Reflex Play Level ¹ 14th IR 0 0.9 ± 0.07 one 25.8 * 0.3 ± 0.1 * 2 2.1 * # 0.7 ± 0.08 # 21st IR 0 0.8 ± 0.13 one 38.3 * 0.1 ± 0.1 * 2 2.1 * 0.8 ± 0.03 # ¹ - the proportion of animals with a preserved reflex. IR - here and in tables 8 and 9, the corresponding indicators in intact animals. * - the significance of the difference of the indicator from its value was noted in intact animals at p <0.05. # - the significance of the difference of the indicator from its control value at p <0.05 is noted.

Table 8 Dynamics of indicative and exploratory behavior of CBA / CaLac mice in an open field with encephalopathy caused by herbal volume hypoxia (1) and with the introduction of immobilized hyaluronate-endo-β-N-acetylhexosaminidase after animals were kept in a heat chamber (2), in conventional units . (X ± m) Study time, days Asymmetry coefficient Total motor activity Horizontal motor activity 14th IR 0.49 ± 0.06 12.4 ± 1.4 7.3 ± 0.98 one 0.82 ± 0.04 * 38.8 ± 4.2 * 28.6 ± 3.3 * 2 0.41 ± 0.04 # 15.3 ± 0.6 # 8.4 ± 1.2 # 21st IR 0.53 ± 0.04 17.6 ± 3.31 10.7 ± 1.21 one 0.85 ± 0.03 * 32.57 ± 2.15 * 27.5 ± 2.7 * 2 0.51 ± 0.1 # 14.3 ± 0.86 # 11.4 ± 1.1 # * - the significance of the difference of the indicator from its value was noted in intact animals at p <0.05. # - the significance of the difference of the indicator from its control value at p <0.05 is noted.

In addition, using the cultural methods [17] on the 10th day of the experiment, the content of neural precursors in the paraventricular region of the brain was determined. In the course of the studies, a significant increase in their number was found in animals treated with imGEAG (Table 9).

Table 9 The content of neural precursors in the paraventricular region of the brain of mice with encephalopathy (1) and with the introduction of immobilized hyaluronate-endo-β-N-acetylhexosaminidase after animals are in a heat chamber (2), (X ± m) Study time, days Neural precursor cells, at 10 ⁵ nuclears 7th IR 3.7 ± 0.05 one 5.8 ± 0.3 * 2 14.9 ± 2.4 * # * - the significance of the difference between the indicator and its background value is noted at p <0.05. # - the significance of the difference of the indicator from its control value at p <0.05 is noted.

Thus, the imGEAGA drug had a pronounced therapeutic effect in relation to posthypoxic encephalopathy, and its therapeutic effect was manifested at the beginning of the administration of the agent after the formation of the pathological condition. Moreover, the effect of the drug was associated with the stimulation of the mechanisms of regeneration of the “deep” reserve, determined by the functional activity of endogenous stem cells of the body.

The results obtained indicate that the agent, which is a hyaluronate-endo-β-N-acetylhexosaminidase immobilized by means of ionizing radiation, has pronounced regenerative activity and therapeutic properties in degenerative diseases: diabetes mellitus, liver cirrhosis, pulmonary fibrosis and encephalopathy. Moreover, the mechanisms of action of the drug is the stimulation of the body's own regenerative reserves, determined by endogenous stem cells of various classes [17].

The proposed tool can be used in regenerative medicine to implement a non-invasive strategy for conducting cell therapy based on the principle of pharmacological stimulation of endogenous stem cells [1, 2, 8].

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Claims (1)

The use of hyaluronate-endo-β-N-acetylhexosaminidase immobilized on a low molecular weight water-soluble carrier using ionizing radiation as a means of regenerative activity.