Regenerative Potential of Granulocyte Colony-Stimulating Factor Immobilized by Using Electron-Beam Synthesis Nanotechnology in an Experimental Model of Ovarian Reserve Depletion.

The pharmacological activity of granulocyte CSF (G-CSF) immobilized using electron-beam synthesis nanotechnology (imG-CSF) was evaluated in an experimental model of ovarian reserve depletion. The effectiveness of the drug was compared with that of its unmodified form. Depletion of the ovarian follicular pool in female Sprague-Dawley rats was caused by a single intravenous injection of the antitumor drug etoposide in the maximum tolerated dose. The effectiveness of the studied drugs was assessed by serum concentration of anti-Mullerian hormone (AMH) measured by ELISA and by the number of primordial, two-layer, multilayer, and atretic follicles counted on serial sections of the ovaries (5-µm thick; through the entire organ) stained with hematoxylin and eosin. It was found that imG-CSF prevents depletion of the ovarian reserve in the model used, which was confirmed by high AMH concentration and higher numbers of primordial, two- and multilayer follicles in comparison with the corresponding parameters in the control (etoposide), and by a decrease in the severity of atretic processes. Unmodified form of the drug demonstrated lower efficiency.

Effect of Granulocyte Colony-Stimulating Factor Immobilized by the Electron-Beam Synthesis Nanotechnology on Reparative Regeneration of Spermatogenous Tissue.

Effectiveness of the granulocyte colony-stimulating factor immobilized by using electronbeam synthesis nanotechnology was investigated on the model of experimental testicular failure caused by the toxic effect on stem spermatogonia. Administration of the drug to experimental paclitaxel-treated animals increased the number of sources of the proliferative pool of spermatogenesis and its productivity. The effectiveness of immobilized granulocyte colony-stimulating factor was based on its ability to stimulate reparative regeneration of the spermatogenic tissue, which manifested in a decrease in spermatogenic layer maturity and increase in the number of microenvironment cells. Effectiveness of the immobilized form of the drug was superior to that of non-immobilized form.

[Efficacy of oral drug Thrombovasim® in therapy of lower extremity deep vein thromboses]. / Éffektivnost' peroral'nogo preparata Trombovazim® v terapii trombozov glubokikh ven nizhnikh konechnostei.

Within the framework of the multicenter randomized placebo-controlled double-blind clinical trial "VETTER-1" the authors carried out assessment of therapeutic efficacy and safety of oral drug Thrombovasim® possessing a thrombolytic effect in comprehensive treatment of lower-extremity deep vein thrombosis (LEDVT). The clinical study comprised a total of 154 patients. All patients received standard therapy accepted in LEDVT. The patients were subdivided into 4 groups. Patients from the three study groups received Thrombovasim® at a daily dose of 1,600, 3,200, and 4,800 IU. The control group patients were given placebo. Efficacy was assessed by the results of ultrasound duplex scanning first performed before treatment commenced and then after it terminated. The relative frequency of positive dynamics according to the findings of instrumental methods of study in patients taking Thrombovasim® amounted to 0.728 and in the group of patients receiving placebo to 0.585, p=0.0031. Comparing the degree of blood flow normalization in the zone of the compromised blood flow revealed a pronounced dose-dependent effect: in patients taking the drug at a daily dose of 1,600 IU, the relative frequency of positive dynamics amounted to 0.707 corresponding to an increase in therapeutic efficacy by 21%, for a dose of 3,200 IU these parameters amounted to 0.0257 and 24% and for 4,800 IU - 0.747 and 28%, respectively. In patients taking Thrombovasim® there were no cases of negative dynamics observed. Of the patients taking Thrombovasim®, none developed undesirable or severe adverse events. Inclusion of Thrombovasim® into the composition of comprehensive therapy for LEDVT increases efficacy of treatment at the expense of a spontaneous thrombolytic effect. The most effective dose amounted to 4,800 IU daily. Thrombovasim® turned out to be an efficient and safe agent in treatment of venous thromboses.

[Experience with clinical administration of new drug Thrombovasim® in vascular surgery].

Recent years have witnessed an increased worldwide interest in medical use of unique naturally occurring enzymes - subtilisins possessing pronounced fibrinolytic and anti-inflammatory properties. The article deals with experience in clinical administration in vascular surgery of new therapeutic agent Thrombovasim® containing pegylated subtilisin as an active substance. Thrombovasim® has a favourable profile of safety, good tolerance and causes no severe haemorrhagic complications. The pharmacological action of Thrombovasim® consists in combination of the targeted effect on the fibrin carcass of the thrombus without participation of the own system of haemostasis and anti-inflammatory effect. In the Russian Federation Thrombovasim® has been used for 6 years predominantly in vascular pathology of lower limbs accompanied by the development of chronic venous insufficiency.

Antifibrotic activity of conjugates based on amphiphilic pluronic F68 and hydrophobic pluronic L31 with hyaluronate-endo-ß-N-acetylhexosaminidase in pulmonary fibrosis.

Antifibrotic activity of intranasally administered conjugates of pluronics L31 and F68 with hyaluronate-endo-ß-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-ß-N-acetylhexosaminidase. The conjugate based on hydrophobic pluronic L31 showed higher effectiveness in comparison with the conjugate based on amphiphilic pluronic F68.

Mechanisms of reparative regeneration of rat testis after injection of paclitaxel.

Population of spermatogonia was reduced in 2, 3, and 6 months after single intravenous injection of antitumor drug paclitaxel in maximum tolerated dose (MTD). The count of Sertoli cell increased in 3 months after the start of the experiment. The maturity of the seminiferous tubule epithelium was lower than in intact rats. Spermatogenesis productivity did not differ from that in intact animals 6 months after start of the experiment. These data indicate that regeneration of the spermatogenous tissue after paclitaxel treatment is realized via renewal of the spermatogenic epithelium, but considering the amount of spermatogonial cell population, the recovery rate would be low.

Specific effects of chondroitin sulfate-modified hyaluronidase on the function of progenitor cells.

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.

Effect of immobilized hyaluronidase on stem and progenitor cells in pulmonary fibrosis.

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(-)).

Effect of pegylated hyaluronate-endo-ß-N-acetylhexosaminidase on humoral mechanisms regulating the functions of progenitor cells during chronic hepatitis.

We studied the effect of hyaluronate-endo-ß-N-acetylhexosaminidase on the secretory function of the liver and bone marrow microenvironment cells in chronic hepatitis. Enhanced production of substances stimulating parenchymal tissue-specific 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 effects of immobilized hyaluronidase in repeated bleomycin-induced lesions of the alveolar epithelium.

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(-)).

Mechanisms underlying modulation of the pharmacological properties of pegylated erythropoietin by pegylated hyaluronate-endo-ß-N-acetylhexosaminidase.

Pegylated hyaluronate-endo-ß-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.

Antifibrotic activity of hyaluronidase immobilized on polyethylenoxide under conditions of bleomycin-induced pneumofibrosis.

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.

Specific activity of electron-beam synthesis immobilized hyaluronidase on G-CSF induced mobilization of bone marrow progenitor cells.

The effects of nanotechnology (electron-beam) -PEGylated (or immobilized; Im) hyaluronidase (HD) on the state of the pool of bone marrow progenitor cells and their mobilization induced by granulocyte colony stimulating factor (G-CSF) were studied. A high specific activity of the drug Im-HD on progenitor cells of different classes was demonstrated using parenteral and enteral administration. An increase in the content of erythroid (E), granulomonocytic (GM), fibroblast (F) colony-forming units (CFU) and mesenchymal stem cells (MSC) in bone marrow was shown, as well as G-CSF-induced stimulation of mobilization of precursors into the peripheral blood under the influence of Im-HD. The detected activity of this novel drug on progenitor cells indicates the potential for a safe and highly effective treatment for hematology practice and regenerative medicine.

Effects and mechanisms of hemopoiesis-stimulating activity of immobilized oligonucleotides under conditions of cytostatic myelosuppression.

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.

Pharmacological properties of granulocytic colony-stimulating factor pegylated using electron beam synthesis nanotechnologies.

Granulocytic CSF pegylated using electron-beam synthesis nanotechnology exhibits pronounced granulomonocytopoiesis-stimulating and SC-mobilizing activity. More potent stimulation of committed precursors against the background of less pronounced activation of polypotent hemopoietic cells is a peculiarity of hemostimulating action of pegylated using electron-beam synthesis nanotechnology granulocytic CSF in comparison with its non-modified analog. The mobilizing effect of pegylated using electron-beam synthesis nanotechnology granulocytic CSF on early progenitor elements surpasses that of non-conjugated cytokine.

Hemostimulating effects of erythropoietin immobilized by the nanotechnology method of electron-beam synthesis.

The hemostimulating effect of erythropoietin immobilized by the nanotechnology method of electron-beam synthesis was studied on the model of carboplatin-induced myelosuppression. Subcutaneous injection or oral administration of immobilized erythropoietin was followed by stimulation of erythropoiesis. The effect was most pronounced after parenteral treatment with this agent. The increase in proliferative activity and maturation of erythroid precursor cells serve as a factor determining acceleration of reparative processes in the hemopoietic tissue.

Mechanisms of hepatoprotective effect of immobilized granulocyte colony-stimulating factor.

The effect of immobilized granulocyte CSF on morphological characteristics and functional state of the liver was studied during chronic toxic hepatitis. The mechanisms of the therapeutic action of this agent were evaluated. The product had a strong hepatoprotective effect and exhibited the antiinflammatory and antisclerotic properties. The mechanism of activation of reserve systems for cell renewal (involved in restoration of the liver tissue) is probably related to an increase in proliferative activity of early precursor cells in the bone marrow, mobilization of these cells into the peripheral circulation, and directed homing into the liver tissue where they activate local regenerative mechanisms and prevent hepatocyte destruction. It should be emphasized that the concentration of SDF-1 increases in the liver tissue, but decreases in the bone marrow. These changes create the concentration gradient, which determines the migration of undifferentiated precursor cells to the liver.

Mechanisms for hepatoprotective effects of hyaluronidase immobilized by the nanotechnology method of electron-beam synthesis.

Immobilized hyaluronidase (nanotechnology method of electron-beam synthesis) exhibited high hepatoprotective activity on the model of Cl4-induced hepatitis. This agent produced anticholestatic, anti-inflammatory, and antisclerotic effects. These effects were shown to accompany stimulation of multipotent bone marrow precursors, mobilization of these cells into the peripheral blood, and cell migration to the target organ increasing the number of parenchymal progenitor cells in the liver. The mechanisms for targeted migration of progenitor cells suggest a decrease in SDF-1 production by bone marrow stromal cells and increase in the synthesis of this factor by microenvironmental cells of the liver tissue.

Effect of hyaluronidase immobilized using electron-beam synthesis nanotechnology on sensitivity of progenitor cells to regulatory factors.

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.

Modulation of the blood-stimulating effect of immobilized granulocyte colony-stimulating factor by immobilized hyaluronidase.

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.