Antifibrotic activity of intranasally administered conjugates of pluronics L31 and F68 with hyaluronate-endo-β-N-acetylhexosaminidase was studied in C57Bl/6 mice under conditions of single and repeated bleomycin-induced injury to the alveolar epithelium. Conjugates were prepared using the technique of protein immobilization with ionizing radiation. We demonstrate that in cases of single and repeated injuries to the alveolar epithelium, the conjugates administered during phases of inflammation or deposition of fibrotic masses prevent the development of pulmonary fibrosis. The conjugates demonstrated more pronounced antifibrotic activity than hyaluronate-endo-β-N-acetylhexosaminidase. The conjugate based on hydrophobic pluronic L31 showed higher effectiveness in comparison with the conjugate based on amphiphilic pluronic F68.

The effect of immobilized hyaluronidase on stem and progenitor cells of the lungs was studied on the model of partially reversible toxic bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. During the inflammation phase, immobilized hyaluronidase reduced infiltration of alveolar interstitium with hemopoietic stem cells Sca-1+, c-Kit+, CD34–, (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)–. Improvement of histological parameters of bleomycin lungs during the phase of collagen fiber deposition after the treatment was accompanied by accumulation of mesenchymal multipotent stromal cells (CD31–, CD34–, CD45–, CD44+, CD73+, CD90+, CD106+decrease in the population of pan-hemopoietic cells (CD45+), accelerated restoration of the content of endothelial cells, and inhibition of clonal activity of fibroblast precursors (CD45–).

We have demonstrated the possibility of stimulation of the function of various types of precursor cells with hyaluronidase modified with chondroitin sulfate. Parenteral administration of modified hyaluronidase increased the number of fibroblast, granulomonocyte, and erythroid CFU in the hemopoietic tissue. The changes in the pool of mesenchymal progenitor cells were more pronounced in comparison with those induced by native enzyme.

Hyaluronidase immobilized on polyethylenoxide obtained by electron bean synthesis was administered intranasally and intravenously to C57Bl/6 mice after intratracheal bleomycin and the enzyme effects on the development of pneumofibrosis in animals were studied. Intranasal immobilized hyaluronidase prevented connective tissue growth in the lungs exposed to bleomycin and virtually did not modulate the infiltration of the alveolar and alveolar duct interstitium by inflammatory cells (lymphocytes, macrophages, neutrophils, plasma cells). The antifibrotic effect developed sooner after intranasal inoculation of immobilized hyaluronidase and was more pronounced than after intranasal native hyaluronidase. Intravenous injection of immobilized hyaluronidase did not modify the inflammatory process and deposition of collagen fibrils in the lung parenchyma in pneumofibrosis.

Using the model of lung fibrosis induced by intratracheal administration of bleomycin we studied anti-fibrotic activity of combined treatment with neuroleptic haloperidol and hyaluronidase immobilized on polyethylene oxide using electron-beam synthesis. It was shown that successive administration of immobilized hyaluronidase and the neuroleptic drug inhibits deposition of collagen fibers in the bleomycin-treated lungs. Combined treatment with the test compounds reduced swelling of the alveolar epithelium, exudation and infiltration of the alveolar interstitium and alveolar passages by inflammatory cells, and desquamation of alveolocytes into alveolar lumen, so that the alveolar-capillary membrane function was preserved.

Pegylated hyaluronate-endo-β-N-acetylhexosaminidase was shown to potentiate significantly the hemostimulatory effect of pegylated erythropoietin. It was found that enhanced production of hemopoietin by adherent and non-adherent cells of the hemopoiesis-inducing microenvironment and elevated serum content of endogenous erythropoietin along with increased susceptibility of erythroid precursors to pegylated erythropoietin underlay this phenomenon.

We studied the effect of hyaluronate-endo-β-N-acetylhexosaminidase on the secretory function of the liver and bone marrow microenvironment cells in chronic hepatitis. Enhanced production of substances stimulating parenchymal tissue-specifi c precursors and stem cell homing factors by liver cells was revealed. At the same time, production of SDF-1 and other chemoattractants and adhesion factors of progenitor cells by bone marrow elements was reduced.

Antifibrotic activity of testicular hyaluronidase, immobilized on polyethylenoxide and obtained by electron beam synthesis, was studied on the model of bleomycin injuries to the alveolar epithelium (irreversible pneumofibrosis) in C57Bl/6 mice and compared to the effect of testicular hyaluronidase. Intranasal therapy with immobilized and testicular hyaluronidases prevented the deposition of fibrotic mass in the parenchyma of "bleomycin" lungs. The effect of immobilized hyaluronidase was more pronounced than that of testicular hyaluronidase. The studied compounds were virtually inessential for infiltration of the alveolar interstitium and alveolar tracts by lymphocytes, macrophages, neutrophils, and plasma cells. Unchanged histoarchitectonics of bleomycin-damaged lungs in immobilized hyaluronidase therapy was due to suppression of the progenitor fibroblast cells (CD45(-)).

The possibility of boosting antifibrotic activity of testicular hyaluronidase immobilized on polyethylene oxide with spiperone was studied on the bleomycin models of a single (partially reversible pneumofibrosis) and repeated (irreversible pneumofi brosis) injuries to the alveolar epithelium in C57Bl/6 mice. The antifibrotic effect was more pronounced after successive treatment with immobilized hyaluronidase and spiperone than after individual treatment with each of the compounds: no collagen deposition in the parenchyma of bleomycin-damaged lungs was found. The decrease in inflammatory cell (lymphocytes, macrophages, neutrophils, plasma cells) infi ltration of the alveoli and alveolar tracts interstitium in mice treated by immobilized hyaluronidase and spiperone did not differ from the anti-inflammatory effect of spiperone monotherapy.

We studied the effect of mobilization of bone marrow multipotent stem cells induced by intragastric administration of pegylated hyaluronate-endo-β-N-acetylhexosaminidase (Peg-HEAHA) on hemopoiesis under conditions of experimental chronic hepatitis. Peg-HEAHA increased the counts of hemopoietic precursors in the hemopoietic tissue against the background of their decelerated maturation. This was paralleled by a short-term decrease in the count of neutrophilic granulocyte in the bone marrow and a slight increase of neutrophil count in the peripheral blood.

Hypoglycemic effect of hyaluronate-endo-β-N-acetylhexosaminidase immobilized by electron-beam synthesis nanotechnology (imHEA-HA) was studied in experimental insulindependent and insulin-independent diabetes mellitus. The drug exhibited a hypoglycemic effect of its own and potentiated the pharmacological effect of exogenous insulin injected in vivo . Studies on liver cell culture demonstrated an increase of cell sensitivity to insulin after treatment with imHEA-HA.

Pegylated hyaluronate-endo-β-N-acetylhexosaminidase considerably potentiates the hemostimulating effects of erythropoietin due to intensifi cation of proliferation and differentiation of erythroid precursors against the background of enhanced secretion of hemopoietins by nonadherent hemopoiesis-inducing environment cells and elevation of serum erythropoietin concentration. The use of the enzyme allows 10-fold reduction of the maximum effective erythropoietin dose.

Pharmacological characteristics of somatotropin pegylated using electron-beam synthesis nanotechnology (PEG-STH) were studied. Oral PEG-STH stimulated the intensity of protein and lipid metabolism and endochondral bone growth without modifying the processes of periosteal and endosteal bone formation. Specific activity of this substance administered orally significantly surpassed that of parenteral non-modified growth hormone.

Работа выполнена при поддержке гранта ФЦП «Развитие фармацевтической и медицинской промышленности Российской Федерации на период до 2020 года и дальнейшую перспективу» (госконтракт № 16.N08.12.1008).

Hemopoiesis-stimulating activity of immobilized oligonucleotide preparation was studied on the model of cytostatic myelosuppression induced by injection of cyclophosphamide and 5-fluorouracil. Immobilized oligonucleotides stimulated regeneration of erythro- and granulocytopoiesis in the bone marrow under conditions of cytostatic treatment. The counts of neutrophilic granulocytes and platelets in the peripheral blood increased. The stimulatory effect of the drug was more manifest in animals with active behavior. The mechanism of immobilized oligonucleotide effect was based on stimulation of functional activity of erythroid and granulocytic macrophage precursors.

High hepatoprotective activity of granulocytic CSF and hyaluronidase immobilized using electron-beam immobilization technology was demonstrated on the model of CCl4-induced hepatitis: the preparations produced anticholestatic, anti-inflammatory, and antisclerotic effects. These effects developed against the background of stimulation of bone marrow multipotent precursor cells and their mobilization into circulation accompanied by an increase in the content of parenchymatous progenitor cells in the liver. The most pronounced positive effect was observed in combined treatment with the test preparations.

In vitro experiments demonstrated increased colony-forming capacity of erythroid, granulomonocytic, and mesenchymal progenitors of the bone marrow and parenchymal progenitor elements of the liver after treatment with immobilized hyaluronidase. Increased sensitivity of these progenitor cells to erythropoietin, granulocyte colony-stimulating factor, fibroblast growth factor, and stem cell factor, respectively, was demonstrated. Immobilized hyaluronidase enhanced the formation of tissue-specific hepatic CFU against the background of reduced yield of stromal precursors in liver tissue culture containing insulin.

We evaluated whether immobilized hyaluronidase can modify the hematotropic effect of immobilized granulocyte CSF (G-CSF). The preparation of immobilized hyaluronidase (50 arb. units per mouse) potentiated the specific effect of immobilized G-CSF on granulomonocytopoiesis. The preparation was shown to facilitate the indirect effect of immobilized G-CSF on hemopoiesis (stimulation of the erythroid and lymphoid hemopoietic stems). These changes were accompanied by an increase in functional activity of hemopoietic precursor cells, secretion of humoral factors by bone marrow myelokaryocytes, and concentration of hemopoietins in the serum.