RU2421239C1 - Agent intensifying action of biologically active substances and drugs - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention relates to biology and medicine. Hyaluronidase immobilised with using an electron-beam synthesis technology is applied as an agent for intensifying the action of biologically active substances and drugs of various pharmacological groups. ^ EFFECT: application of said hyaluronidase allows improving the efficacy of biologically active substances and drugs. ^ 7 tbl, 6 ex

Description

The invention relates to biology and medicine.

There are tools with the ability to modulate the effect of individual biologically active substances, as well as enhancing the effect of drugs [1, 2].

The disadvantage of these funds is their high specificity. Each of the existing agents is capable of exerting its influence on either a specific endogenous biologically active substance or a limited group of substances, or a drug and its function [2]. In addition, the available funds are not effective enough.

A means is known, which is immobilized using ionizing radiation (nanotechnology electron beam synthesis) hyaluronidase. It was shown that this tool is able to increase the reserve of stem cells in the body [3].

For the first time, the authors revealed its pronounced potentiating activity with respect to the action of biologically active substances (including endogenous hormones, growth factors, and other cytokines) and drugs of various pharmacological groups, which allows them to reduce their effective therapeutic doses.

The problem solved by this invention is to increase the effectiveness of biologically active substances and drugs.

The task is achieved by using immobilized using the technology of electron-beam synthesis of hyaluronidase as a means to enhance the action of biologically active substances and drugs of different pharmacological groups.

New in the invention is the use of immobilized hyaluronidase as a means to enhance the action of biologically active substances and drugs of different pharmacological groups.

Used by the authors, an original tool immobilized with ionizing radiation on a low molecular weight carrier of hyaluronidase was developed and obtained by the Scientific Research Institute of Pharmacology SB RAMS (Tomsk) in conjunction with Scientifical Future Management LLC (Novosibirsk).

Hyaluronic acid (HA) is one of the main components of the intercellular matrix of body tissues. An important role in the metabolism of this glycosaminoglycan (GAG) belongs to the enzyme hyaluronidase, under the influence of which the cleavage of HA to polymers with different molecular weights and biological properties occurs [4, 5].

At the same time, it was shown that hyaluronidase is able to increase the permeability of tissue barriers, thereby changing the pharmacokinetics of a number of drugs, increasing their effectiveness by increasing the concentration of drugs in the body [1]. At the same time, hyaluronidase preparations (Lidaza, Longidaza) are unable to enhance the effects of highly soluble (freely penetrating biological barriers) substances, as well as receptor drugs if they are used in the most effective doses. In addition, native hyaluronidase preparations cannot be used in non-toxic doses [4] as a means to obtain systemic therapeutic effects (associated with the resorptive effect of the drug), which is due to the high content of endogenous hyaluronidase inhibitors in mammalian tissues and blood serum [6], predetermining its rapid inactivation when introduced into the body.

However, it is known that immobilization of hyaluronidase on a polymer carrier using electron beam synthesis technology / nanotechnology (directed flow of accelerated electrons with an electron energy of 2.5 MeV, absorbed dose of 2 to 10 kGy, dose rate of 1.65 kGy / hour ) is accompanied by a significant increase in its physical stability and resistance to proteases, which makes it possible to obtain systemic / resorptive effects of this enzyme / agent [3]. In particular, the possibility of increasing the functional activity of stem cells in the body associated with the degradation of the HA of the intercellular matrix and the formation of a significant amount of its medium and low molecular weight forms was established [3].

At the same time, hyaluronic acid is a part of not only the intercellular matrix, but also the glycocalyx of cells and their receptors for various biologically active substances [7]. However, the dependence of the degree of affinity (affinity) of receptors for ligands on the state of hyaluronic acid, which is part of the corresponding receptors, has not yet been studied.

The fact of using immobilized hyaluronidase (imGD) with the achievement of a new technical result, which consists in enhancing the action of biologically active substances, including endogenous hormones, growth factors, cytokines and other substances, as well as drugs of different pharmacological groups, is not obvious to a specialist.

The claimed essential features in the aggregate showed new properties that are not explicitly derived from the prior art in this field. The present invention can be used in experimental biology and medicine with access to practical health care. An identical set of features was not found in the study of the state of the art in patent and medical literature.

Based on the foregoing, the claimed technical solution should be considered relevant criteria: "Novelty", "Inventive step", "Industrial applicability".

The experiments were conducted on male mice of the CBA / CaLac line in the amount of 268 pieces weighing 18-20 g. Animals were obtained from the nursery of the experimental biomedical modeling department of the Research Institute of Pharmacology SB RAMS.

The present invention is illustrated by the following examples.

Example 1

Assessment of the effect of immobilized hyaluronidase on the ability of progenitor cells of various classes to respond to specific and non-specific growth factors

The model for studying the sensitivity of cellular elements (the ability to react) to hematopoietic growth factor - granulocyte colony stimulating factor (G-CSF), erythropoietin hormone, and polyfunctional and pleiotropic regulator of progenitor cell functions - fibroblast growth factor, served as bone marrow cells of mice.

The model for studying the sensitivity of cellular elements (the ability to react) to an early-acting stimulator of stem cell functions — the stem cell growth factor — was the liver cells of mice. The study used cultural research methods [8].

To study the effect of imGD, the incubated material was preincubated in a medium containing this enzyme. Pre-incubation of whole bone marrow and dissociated liver tissue (2 million / ml) was carried out in a liquid culture medium (DMEM, Sigma, USA) containing 0.1; 0.5 and 1 IU / ml imGD. The pre-incubation time in the solution containing 0.1 IU imGD was 10, 30 and 60 minutes. In the case of using other concentrations of imGD, preincubation was carried out for 60 minutes. After that, the ability of myelokaryocytes to form CFU-F in a semi-viscous medium [8] was studied, in which fibroblast growth factor was added, as well as the ability of parenchymal liver progenitor cells to form tissue-specific colonies in the presence of stem cell growth factor.

Pre-incubation of non-adherent bone marrow myelocaryocytes (2 million / ml) was carried out in a liquid culture medium (DMEM, Sigma, USA) containing 0.1; 0.5 and 1 IU / ml imGD for 60 minutes Then studied the formation of erythroid (CFU-E) and granulomonocytic (CFU-GM) colonies in a semi-viscous medium containing respectively recombinant erythropoietin (Recormon, Hoffman-la-Roche, Switzerland, at a dose of 2 U / ml) and G-CSF ( “Neupogen”, Hoffman-la-Rochecher, Switzerland, at a dose of 5 ng / ml) [8].

For cloning of bone marrow precursors, a medium of the following composition was used: 1% methylcellulose, 80% DMEM medium, 19% fetal calf serum, 280 mg / L L-glutamine, 50 mg / L gentamicin (as well as the above growth factors), and incubated in CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 3 days when studying the growth of CFU-E and 7 days when studying the growth of CFU-GM and CFU-F.

For cloning of liver tissue precursors, the following medium was used: 90% DMEM medium, 10% fetal calf serum, 6 g / L glucose, 280 mg / L L-glutamine, 50 mg / L gentamicin, 8000 IU / L heparin, 25 mg / L insulin, 20 ng / ml stem cell growth factor (Sigma, USA), and incubated in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 10 days.

The controls in all cases were the colony forming ability of cells not treated with hyaluronidase, and such when culturing cellular material in a medium that did not contain growth factor.

During the experiment, it was found that pre-treatment of myelokaryocytes or liver cells with an enzyme is accompanied by a significant increase in the output of CFU-E, CFU-GM, CFU-F and liver progenitor cells (CFU-Pech) in a medium that does not contain additional growth factors. In this case, the colony-stimulating activity of the nutrient medium was determined by the set of biologically active substances of fetal calf serum [8] (Table 1).

Table 1 The growth of CFU-E, CFU-GM, CFU-F and CFU-Pech with the addition of erythropoietin, G-CSF, fibroblast growth factor and stem cell growth factor, respectively, as well as the growth of colonies after pre-incubation of myelokaryocytes in imGD solution, (X ± m) Study time, days CFU-E, for 10 ⁵ myelocaryocytes CFU-GM, on 10 ⁵ myelocaryocytes CFU-F, for 10 ⁵ myelocaryocytes CFU-Pecs, for 10 ⁵ nuclears background the control 3.83 ± 0.31 2.17 ± 0.31 12.67 ± 0.33 5.7 ± 0.56 growth factor 7.83 ± 0.6 * 5.5 ± 0.5 * 27.5 ± 2.17 * 14.67 ± 1.3 * 0.1 units IMGD 10 min the control - - 26.83 ± 1.47 * 13.1 ± 1.1 * growth factor - - 41.0 ± 0.97 * &# 27.6 ± 2.4 * &# 0.1 units imgd 30 min the control - - 22.33 ± 2.69 * 15.6 ± 0.98 * growth factor - - 40.67 ± 3.79 * &# 28.9 ± 2.6 * &# 0.1 units IMGD 60 min the control 10.83 ± 0.79 * & 6.17 ± 0.4 * 21.83 ± 2.52 * 19.4 ± 3.1 * growth factor 16.0 ± 0.37 * &# 11.0 ± 0.86 * &# 43.5 ± 4.4 * # 31.6 ± 0.82 * &# 0.5 units IMGD 60 min the control 10.5 ± 0.89 * & 8.67 ± 1.15 * & 16.17 ± 1.08 * & 14.3 ± 0.6 * growth factor 16.83 ± 0.7 * &# 15.67 ± 1.05 * &# 33.5 ± 1.77 * &# 26.7 ± 0.45 * &# 1 unit imgd 60 min the control 5.17 ± 0.31 * & 7.83 ± 0.98 * 12.5 ± 1.18 & 6.8 ± 0.62 growth factor 7.5 ± 0.43 * # 8.67 ± 0.88 * & 10.17 ± 1.05 & 7.3 ± 0.54 * - the significance of the difference of the indicator from its background value was noted without adding growth factors at p <0.05; & - the significance of the difference of the indicator from its background value with the addition of growth factors at p <0.05; # - the significance of the difference of the indicator from the control value without adding a growth factor at p <0.05 is noted.

In addition, the cellular elements obtained after their incubation with imGD according to different schemes had a significantly more pronounced susceptibility to the response (than intact nuclears) and to specific growth factors (Table 1). Thus, the ability of hematopoietic and mesenchymal progenitor cells to respond to erythropoietin, granulocyte colony stimulating factor and fibroblast growth factor, and parenchymal stem cells of the liver, to stem cell growth factor was significantly enhanced.

Thus, the results obtained indicate a direct effect of imGD on cellular elements, which determines their sensitivity to various biologically active substances and the expression of their functions.

Example 2

The study of the stimulating effect of imGD in relation to the hypoglycemic activity of hypoglycemic drugs

Short-acting insulin preparation (Actropid, Novo Nordisk) and oral glucose-lowering drug Glyukobay (Bayer, Germany) were used as sugar-lowering drugs.

A study of the effect of imGD on the activity of insulin and an oral hypoglycemic was determined using a model of diabetes mellitus alloxan in BALB / c mice [9]. The insulin preparation was used subcutaneously at a dose of 2 IU / kg, and glucobay orally at a dose of 50 mg / kg. Prior to the use of drugs (1 hour), animals of the experimental groups received intragastrically a preparation of immobilized hyaluronidase (imGD) at a dose of 50 U / kg. Peripheral blood glucose was determined 2 hours, 4 hours, 6 hours and 24 hours after the administration of glucose-lowering drugs (SmartScan device (Johnson & Johnson team, USA). Blood was taken from the tail vessels. During the experiment, the animals had free access to water and food.

During the experiment, the course administration of alloxan led to the development of persistent hyperglycemia, accompanied by the death of animals (16% by the end of the experiment) (Table 2).

The introduction of the studied drugs in all cases had a pronounced sugar-lowering effect to varying degrees. Moreover, the presence of hypoglycemic activity was also detected in immobilized HD, the effect of which was manifested during the entire observation period. Moreover, the discovered phenomenon (independent hypoglycemic activity of imGD) was obviously associated with a change / increase in the sensitivity of body tissues under the influence of the enzyme to endogenous insulin.

Parenteral administration of a standard short-acting insulin preparation naturally led to the normalization of glucose in peripheral blood at 2 and 4 hours after its use. Moreover, the corrective effect on the hyperglycemia of this drug was also noted after 6 and 24 hours. However, the preliminary administration of imGD significantly enhanced the specific activity of the hormone. In particular, 2 hours after the use of insulin, all animals developed a hypoglycemic coma (lasted about 8-10 hours, death after 24 hours was 40%). At the same time, the blood sugar concentration during all observation periods was significantly lower than when using only insulin, and reached its lowest values after 4 hours (1.94 mmol / L).

Glucobai possessed the least pronounced hypoglycemic properties. So, the oral use of this substance was accompanied only by the development of a tendency to a decrease in blood glucose in the early stages (2, 4 hours) and a reliable, but not as pronounced, as in previous cases (with the introduction of other drugs), a decrease in this indicator relative to control values by the end of the experiment (6, 24 hours).

At the same time, the combined use of glucoboy with imGD led to a significantly more significant correction of hyperglycemia than when using one oral hypoglycemic agent.

table 2 The dynamics of glucose in peripheral blood (mmol / l) with alloxan diabetes (DM) and with the introduction of immobilized hyaluronidase (HD), insulin, glucobay and the combined use of insulin and glucobay with hyaluronidase against the background of modeling diabetes, (X ± m) Groups Intact Diabetes (before the introduction of hyaluron
Dazi) (7 ⁴⁵ ) Before administration of insulin preparations (in 3 groups after administration of hyaluron
Dazi) (10 ⁰⁰ ) 2 hours after administration of insulin preparations (12 ⁰⁰ ) 4 hours after administration of insulin preparations (14 ⁰⁰ ) 6 hours after administration of insulin preparations (16 ⁰⁰ ) 24 hours after administration of insulin preparations (10 ⁰⁰ ) Control (DM) one 7.78 ± 0.23 28.54 ± 1.07 * 29.66 ± 1.57 * 24.36 ± 2.51 * 21.1 ± 1.13 * 32.88 ± 0.42 * 30.48 ± 1.37 * SD + DG 2 16.78 ± 2.74 * # 12.02 ± 0.7 # 15.42 ± 0.43 * # 31.2 ± 1.08 * 13.02 ± 1.07 * # Diabetes + insulin 3 26.64 ± 1.88 * 4.91 ± 1.25 * # 3.96 ± 0.66 * # 17.71 ± 3.97 * # 17.64 ± 1.94 * # DM + insulin + HD four 19.44 ± 2.71 * # & 2.08 ± 0.12 * # & 1.94 ± 0.16 * # & 3.9 ± 0.68 * # & 9.17 ± 0.43 * # & SD + glucobai 5 29.53 ± 2.24 * 21.08 ± 1.38 * 16.8 ± 4.04 * 26.18 ± 2.82 * # 20.72 ± 2.33 * # SD + glucobay + GD 6 17.63 ± 1.93 * # & 8.48 ± 0.2 * # & 12.9 ± 0.33 * # & 26.04 ± 1.73 * # 16.62 ± 1.57 * # * - the significance of the difference of the indicator from its value was noted in intact animals at ≤0.05; # - the significance of the difference of the indicator from its value in control animals (DM) was noted at p≤0.05; & - the significance of the difference of the indicator from its value was noted in animals treated with the insulin or glucobai preparation together with hyauronidase, at p≤0.05. Note: Scheme of drug administration and registration of blood glucose levels by time and groups: 7 ⁴⁵ - determination of glucose in all groups (DM); 8 ⁰⁰ - the introduction of imGD in 2, 4, 6 groups; 10 ⁰⁰ - determination of glucose in all groups; 10 ⁰⁰ - the introduction of insulin preparations in 3, 4, 5, 6 groups; 12 ⁰⁰ , 14 ⁰⁰ , 16 ⁰⁰ , 10 ⁰⁰ - determination of glucose in all groups.

In general, the obtained results indicate a significant potentiating ability of immobilized hyaluronidase with respect to the specific hypoglycemic activity of both endogenous (independent hypoglycemic activity of the drug) and insulin and oral hypoglycemic drug administered from the outside.

Example 3

Study of the potentiating effect of immobilized hyaluronidase on the erythropoiesis-stimulating effect of erythropoietin

The experimental model was cytostatic myelosuppression in mice caused by the administration of carboplatin at a dose of 100 mg / kg (maximum tolerated dose) once intraperitoneally.

The erythropoietin preparation (Recormon, Hoffman-la-Roche, Switzerland) was administered for 5 days, starting from the day after the administration of carboplatin at a dose of 5 units / mouse sc. The specified dose in preliminary experiments was determined as the most effective therapeutic dose (its further increase is not accompanied by an increase in the effectiveness of therapy). Immobilized hyaluronidase was started at the same time as Recormon at a dose of 5 U / kg once a day for 2 days, intraperitoneally. The drug Longidazu (NPO Petrovaks Pharm. Russia), which is a chemical conjugate of hyaluronidase ("Lidase") with polyoxidonium, which was administered in a similar mode and equivalent dose, was used as a comparison drug.

The state of the blood system was studied using standard and cultural hematological methods for the content of erythroid precursors (CFU-E) and erythrokaryocytes in the bone marrow, as well as reticulocytes and erythrocytes in the peripheral blood [8].

Table 3 Dynamics of indicators of the erythroid hematopoietic germ in CBA / CaLac mice treated with carboplatin (1), carboplatin and erythropoietin (2), carboplatin, erythropoietin and longidase (3), carboplatin, erythropoietin, and immobilized hyaluronidase preparation (4) ) Study time, days CFU-E in the bone marrow, for 10 ⁵ myelocaryocytes Erythrokaryocytes in the bone marrow, × 10 ⁶ Reticulocytes, ppm Red blood cells, T / L background 14.45 ± 1.22 2.13 ± 0.2 12.4 ± 0.7 9.04 ± 0.12 3rd one 5.4 ± 0.3 * 0.89 ± 0.03 * 2.3 ± 0.42 * 9.01 ± 0.31 2 7.5 ± 0.2 * # 0.94 ± 0.02 * 4.1 ± 0.3 * # 9.1 ± 0.24 3 7.54 ± 0.36 * # 0.97 ± 0.03 * 3.87 ± 0.5 * # 9.2 ± 0.58 four 9.87 ± 0.47 * # & 1.3 ± 0.04 * # & 5.7 ± 0.4 * # & 9.14 ± 0.36 5th one 8.8 ± 0.9 * 1.1 ± 0.02 * 9.7 ± 0.56 * 8.74 ± 0.2 2 14.6 ± 0.5 # 1.5 ± 0.03 * # 18.9 ± 0.5 * # 8.81 ± 0.12 3 13.7 ± 0.64 # 1.4 ± 0.02 * # 19.7 ± 0.97 * # 8.8 ± 0.1 four 18.5 ± 0.71 * # & 1.8 ± 0.01 * # & 25.6 ± 0.7 * # & 8.4 ± 0.3 7th one 21.3 ± 1.4 * 1.07 ± 0.01 * 19.8 ± 0.3 * 7.52 ± 0.19 * 2 24.3 ± 2.3 * 1.57 ± 0.02 * # 29.3 ± 1.5 * # 8.4 ± 0.1 * # 3 23.7 ± 1.7 * 1.56 ± 0.01 * # 28.7 ± 1.67 * # 8.36 ± 0.12 # * four 29.81 ± 1.32 * # & 2.4 ± 0.03 # & 34.1 ± 0.78 * # & 8.6 ± 0.17 * # 10th one 7.9 ± 0.56 * 1.89 ± 0.02 * 22.1 ± 0.65 * 8.7 ± 0.14 2 8.97 ± 0.34 * 2.3 ± 0.04 # 39.7 ± 0.44 * # 9.2 ± 0.09 # 3 7.98 ± 0.46 * 2.4 ± 0.1 # 40.1 ± 1.3 * # 9.13 ± 0.08 # four 11.3 ± 0.55 # & 2.78 ± 0.01 * # & 44.6 ± 0.82 * # & 11.2 ± 0.05 * # & 14th one 12.3 ± 1.4 2.4 ± 0.1 13.2 ± 1.3 7.6 ± 0.16 * 2 13.5 ± 0.98 2.89 ± 0.06 * # 16.2 ± 0.52 * # 10.4 ± 0.3 * # 3 14.6 ± 0.75 2.67 ± 0.1 * # 15.8 ± 0.63 * # 9.89 ± 0.34 * # four 12.9 ± 0.61 3.4 ± 0.7 * # & 18.1 ± 1.1 * # & 12.5 ± 0.2 * # & * - the significance of the difference of the indicator from its value in intact animals was noted at p≤0.05; # - the significance of the difference of the indicator from its value was noted in control animals (carboplatin) at p≤0.05; & - the significance of the difference of the indicator from its value in animals treated with the erythropoietin preparation was noted at p≤0.05.

During the experiment, the administration of carboplatin led to a pronounced depression of the erythroid germ of hematopoiesis (Table 3). In this case, the introduction of erythropoietin was accompanied by a regular acceleration of hematopoietic tissue regeneration, manifested in an earlier and more pronounced increase in the number of erythroid CFU and erythrokaryocytes in the bone marrow, as well as reticulocytes and erythrocytes in the peripheral blood. Moreover, the additional administration of longidase did not lead to an increase in the action of the erythropoietin drug.

In contrast to the existing hyaluronidase preparation, the use of the claimed agent based on this enzyme (hyaluronidase immobilized using electron beam synthesis technology) led to an even sharper increase in the number of CFU-E in the hematopoietic tissue (5th day) and erythrokaryocytes (5, 7 10th day). The indicated changes in bone marrow hematopoiesis were accompanied by regularly higher values of circulating mature red blood cells (days 7, 10, and 14) compared with the group of animals that received erythropoietin alone (Table 3).

Thus, hyaluronidase immobilized by electron beam synthesis exerted a pronounced potentiating effect with respect to the erythropoestimulating action of the recombinant erythropoietin at the most effective dose.

Example 4

Investigation of the effect of immobilized hyaluronidase on hematopoietic stimulating activity of granulocyte colony stimulating factor

The modeling of cytostatic myelosuppression was carried out by a single intraperitoneal injection of cyclophosphamide in the maximum tolerated dose (250 mg / kg) in 0.3 ml of physiological saline.

Against the background of myelosuppression modeling, experimental animals were injected subcutaneously with G-CSF (Neupogen, Hoffman-la-Roche, Switzerland) at doses of 2 and 125 μg / kg once a day for 5 days. The dose of 125 μg / kg in preliminary experiments was determined as the most effective, and 2 μg / kg as an ineffective dose.

Immobilized hyaluronidase was administered orally, starting simultaneously with the G-CSF preparation at a dose of 20 U / kg once a day for 7 days.

The state of the blood system was studied using standard and cultural hematological methods for the content of granulomonocytic precursors (CFU-GM), immature and mature neutrophilic granulocytes in the bone marrow, and neutrophils in peripheral blood [8].

The introduction of cyclophosphamide naturally led to severe depression of bone marrow hematopoiesis. A decrease in the content of immature and mature neutrophilic granulocytes in the bone marrow was noted. Moreover, the indicated dynamics was replaced by a normalization of the index of mature neutrophilic granulocytes and an increase in the number of red blood cells and immature neutrophilic granulocytes on the 10th, 14th day relative to the background values, indicating a high intensity of regenerative processes in hematopoietic tissue. A reflection of the state of bone marrow hematopoiesis was a picture of peripheral blood (Table 4).

The preparation G-CSF significantly stimulated the regeneration of the hematopoietic granulocyte germ, but only if it was used at a dose of 125 μg / kg. At the same time, a significant increase in the content of hematopoietic precursors in hematopoietic tissue relative to the cytostatic control was noted in almost all periods of the experiment.

Changes in the state of the pool of primordial cells naturally led to a more rapid and significant increase in the number of neutrophilic granulocytes in the bone marrow and neutrophils in the peripheral blood (Table 4).

Table 4 Dynamics of granulomonocytopoiesis indices in CBA / CaLac mice receiving a single cyclophosphamide (1), cyclophosphamide and G-CSF at a dose of 2 μg / kg (2), cyclophosphamide and G-CSF at a dose of 125 μg / kg (3), cyclophosphamide, G -CSF at a dose of 2 μg / kg and imGD (4), cyclophosphamide, G-CSF at a dose of 125 μg / kg and imGD (5), (X ± m) Terms of study
Niya, day CFU-GM in the bone marrow, for 10 ⁵ myelocaryocytes Immature neutrophilic granulocytes in the bone marrow Mature neutrophilic granulocytes in the bone marrow Band neutrophils of peripheral blood, g / l Segmented neutrophils of peripheral blood, g / l background 5.6 ± 0.37 1.08 ± 0.03 4.68 ± 0.22 0.2 ± 0.01 2.79 ± 0.09 3rd one 3.4 ± 0.21 * 0 0 0 2.6 ± 0.04 2 3.42 ± 0.3 * 0 0 0 2.67 ± 0.03 3 9.4 ± 0.37 * # 0.07 ± 0.01 * 0 0.01 ± 0.01 * 2.97 ± 0.04 four 5.7 ± 0.23 # & 0.06 ± 0.01 * 0 0.01 ± 0.01 * 2.98 ± 0.05 5 17.3 ± 0.78 * # & 0.07 ± 0.01 * 0 0.01 ± 0.01 * 2.78 ± 0.06 5th one 8.9 ± 0.27 * 0.05 ± 0.01 * 1.76 ± 0.09 * 0.24 ± 0.02 * 3.64 ± 0.1 * 2 9.4 ± 0.56 * 0.04 ± 0.01 * 1.89 ± 0.24 * 0.23 ± 0.01 * 3.67 ± 0.15 * 3 24.6 ± 1.7 * # 0.24 ± 0.01 * # 2.59 ± 0.6 * # 0.67 ± 0.01 * # 6.4 ± 0.22 * # four 18.4 ± 0.6 * # & 0.26 ± 0.01 * # & 2.47 ± 0.53 * # & 0.56 ± 0.02 * # & 6.37 ± 0.34 * # & 5 32.7 ± 0.81 * # & 0.45 ± 0.01 * # & 3.67 ± 0.54 # & 0.89 ± 0.02 * # & 7.8 ± 0.1 * # & 7th one 6.5 ± 0.23 * 0.87 ± 0.02 * 3.98 ± 0.2 0.37 ± 0.01 * 2.8 ± 0.1 2 7.1 ± 0.45 * 0.92 ± 0.04 * 4.1 ± 0.34 0.41 ± 0.02 * 2.86 ± 0.4 3 14.2 ± 0.63 * # 1.97 ± 0.03 * # 6.89 ± 0.8 * # 0.98 ± 0.02 * # 6.78 ± 0.3 * # four 18.7 ± 0.5 * # & 2.14 ± 0.02 * # & 6.91 ± 0.74 * # & 0.97 ± 0.02 * # & 6.65 ± 0.33 * # & 5 25.7 ± 0.3 * # & 2.87 ± 0.04 * # & 8.1 ± 0.9 * # & 1.2 ± 0.03 * # & 9.64 ± 0.17 * # & * - The significance of the difference of the indicator from its value in intact animals was noted at p≤0.05; # - The significance of the difference of the indicator from its value in control animals (cyclophosphamide) was noted at p≤0.05; & - The significance of the difference in the indicator in animals that received G-CSF and imGD together with the values of similar indicators in mice that were administered only G-CSF in the appropriate dose at p≤0.05 was noted.

The combined use of immobilized hyaluronidase and G-CSF at a dose of 125 μg / kg led to significantly more pronounced changes on the part of granulomonocytopoiesis. IMHD enhanced the specific action of G-CSF on CFU-GM, which led to a more significant increase in the number of precursors and morphologically recognizable elements of the granulocyte series in the bone marrow: immature neutrophilic granulocytes by 87.5%, and mature neutrophilic granulocytes by 41.7% by 5 day of experiment with respect to the group of animals that received only 125 μg / kg G-CSF. Moreover, these changes led to a more significant increase in the number of neutrophils in peripheral blood.

In addition, the additional use of imGD was accompanied by a significant activation of granulomonocytopoiesis in the case of using G-CSF at a dose of 2 μg / kg. At the same time, a statistically significant increase in the values of all the studied parameters was observed compared with both the cytostatic control and the corresponding parameters in mice that were injected with only G-CSF at a dose of 2 μg / kg (Table 4).

The results indicate a significant increase in granulocyte-stimulating activity in the preparation of granulocyte colony-stimulating factor under the action of imGD. Moreover, the experimental data indicate the possibility of using imHD as a means to reduce the effective therapeutic dose of drugs.

Example 5

The study of the ability of immobilized hyaluronidase to enhance the therapeutic activity of anticoagulants

The experiments were performed on intact mice of the CBA / CaLac line.

Experimental animals were once injected intravenously with heparin (Sintez OJSC) at a dose of 200 PIECES / kg, or heparin together with Lidaza (FSUE NPO Microgen of the Ministry of Health of the Russian Federation) (intraperitoneally) at a dose of 10 PIECES / kg, or heparin together with immobilized hyaluronidase (intraperitoneally) at a dose of 1 U / kg. After 1 hour, the activated partial thromboplastin time (APTT) and thrombin time (TB) were studied. In preliminary experiments, the dose of heparin was chosen as the dose at which the methods used are considered insensitive (APTT is increased by less than 2, and TB less than 4 times [10]).

During the experiment, the administration of a low dose of heparin did not lead to significant changes in the plasma coagulation time in the APTT and TB test (Table 5) and amounted to 45.7 ± 1.4 and 34.2 ± 0.87 sec, respectively. Significant changes were also not recorded when using the indicated dose (1 UNITS / kg) of imGD and the combined use of heparin with the standard preparation of hyaluronidase ("Lidase").

Table 5 Hemostasis in animals treated with heparin (1), immobilized hyaluronidase (2), heparin together with lidase (3) and heparin together with immobilized hyaluronidase (4), (X ± m) Study time, days APTT, sec TV, sec background 28.65 ± 1.23 19.4 ± 0.7 1 hour one 45.7 ± 1.4 34.2 ± 0.87 2 31.57 ± 0.98 25.4 ± 0.3 3 46.1 ± 1.73 33.6 ± 0.54 four 81.7 ± 0.69 56.9 ± 0.48

At the same time, in animals to which, in addition to heparin, they were given imGD, there was a significant increase in both indicators, reflecting the state of the hemostasis system (Table 5). In this case, the APTT was 81.7 ± 0.69, and the TV 56.9 ± 0.48.

Based on the results of the study, it follows that the additional use of the drug imGD against the background of the introduction of low doses of heparin leads to a significant increase in the degree of its anticoagulant activity.

Example 6

Study of the effect of immobilized hyaluronidase on the specific activity of immunomodulators

The study used 240 male CBA / CaLac mice. Experimental animals were administered reaferon-EC-lipint (CJSC "VECTOR-MEDICA") intraperitoneally for 5 days at a dose (185715 IU / kg) in a volume of 0.2 ml of sterile saline, or reaferon-EC-lipint (ZAO " VECTOR-MEDICA ”) together with immobilized hyaluronidase (intraperitoneally) at a dose of 10 PIECES / kg, control animals received an equivalent volume of solvent (physiological saline). In this case, the used dose of reaferon-EC-lipint in preliminary experiments was determined as the most effective.

We studied the effect of drugs on the phagocytic activity of peritoneal macrophages, the total number of splenocytes, antibody-forming cells, the content of IgM, IgG. Moreover, in the study of the phagocytic activity of peritoneal macrophages, intact animals of the corresponding sex and age were used as the background. In the study of the effect of drugs on the humoral immune response (on the 4th day), mice that received the antigen but without a course of physiological saline were used as the background [10].

During the experiment, it was found that the use of imGD significantly increased the cellular immune response: the phagocytic activity of the peritoneal macrophages of experimental animals (PS) compared with the control (reaferon-EC-lipint). In mice treated with both drugs, there was not only a significant increase in the average number of bacteria absorbed by one cell, but also an increase in the percentage of active peritoneal macrophages (PI) (Table 6).

Immobilized hyaluronidase also enhanced the effect of the immunomodulator on the humoral immune response. In this group of animals, there was also a more significant increase in the number of splenocytes after immunization, as well as the relative and absolute number of antibody-forming cells in the spleens. In addition, in this group there was a significant increase in titers of IgM, IgG and total antibodies in blood serum compared with the group of animals using reaferon-EC-lipint (Table 7).

Thus, the results of the studies indicate that the additional use of immobilized hyaluronidase can significantly enhance the stimulation of the cellular and humoral immune responses of immunotropic drugs (in particular, reaferon-EC-lipint).

Table 6

The effect of drugs on the phagocytic activity of peritoneal macrophages of CBA / CaLac mice

Indicators Background The control RFN-EU-l RFN-EU-l + imGD FI,% 22.50 ± 0.92 25.0 ± 1.37 21.83 ± 0.87 73.50 ± 0.96 * # Ff 2.54 ± 0.26 5.15 ± 0.44 * 4.53 ± 0.25 * 20.82 ± 1.38 * # * The significance of the difference between the indicator and its value in intact animals was noted at p≤0.05; # The significance of the difference of the indicator from its value in animals receiving only reaferon-EC-lipint at p≤0.05 was noted.

Table 7 The effect of drugs on the humoral immune response of CBA / CaLac mice after immunization, (X ± m) Indicators Background The control RFN-EU-l RFN-EU-l + imGD The total number of splenocytes, × 10 ⁶ 193.40 ± 2.89 204.0 ± 3.05 * 213.0 ± 5.44 * 344.83 ± 2.11 * # Antibody cells,% 11.92 ± 0.28 15.30 ± 0.30 * 14.23 ± 0.34 * 35.80 ± 0.87 * # Antibody cells × 10 ⁶ 23.08 ± 0.88 32.86 ± 1.52 * 30.36 ± 1.28 * 48.77 ± 3.02 * # IgM, log ₂ T 2.80 ± 0.37 2.80 ± 0.37 4.60 ± 0.40 * 12.0 ± 0.71 * # IgG, log ₂ T 7.80 ± 0.58 7.0 ± 0.55 5.20 ± 0.20 * 11.20 ± 0.58 # IgM + IgG, log ₂ T 10.60 ± 0.60 9.80 ± 0.73 9.80 ± 0.37 17.20 ± 0.20 # * The significance of the difference between the indicator and its value in intact animals was noted at p≤0.05; # The significance of the difference of the indicator from its value in animals receiving only reaferon-EC-lipint at p≤0.05 was noted.

In general, the obtained data indicate a pronounced potentiating ability of immobilized hyaluronidase with respect to the action of various biologically active substances (hormones, growth factors, cytokines, etc.), including endogenous and also drugs of different pharmacological groups, which can significantly reduce their effective therapeutic doses. In this case, the mechanism of enhancing the effects of biologically active substances and pharmacological agents is the direct effect of the drug on cell structures (receptors, biological membranes, etc.) or humoral regulation factors responsible for the performance of certain functions. Moreover, the ability of IMHD to potentiate the action of endogenous regulators of physiological functions can obviously be considered as an increase under its influence the effectiveness of the regulatory effect of biologically active substances and the optimization of their metabolism.

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Claims (1)

The use of hyaluronidase immobilized by electron beam synthesis as a means of enhancing the action of biologically active substances and drugs of various pharmacological groups, which can significantly reduce their effective therapeutic doses.