Hyaluronidase is an enzyme that helps maintain an optimal balance of hyaluronic acid in connective tissue and ensures full tissue regeneration. Currently, hyaluronidase drugs are used in various fields of medicine. Meanwhile, there are no oral preparations based on hyaluronidase on the pharmaceutical market due to its low bioavailability in this method of administration. One of the options to increase enzyme bioavailability is electron-beam pegylation, which involves obtaining drugs with a modified pharmacokinetic profile in the direction of increasing enteral bioavailability, as well as having additional protection from the action of proteolytic enzymes. Aim of the study was using the electron beam pegylation method to obtain a laboratory batch of a preparation based on highly purified hyaluronidase, which can be further used in the development of an oral drug product. Material and methods. Hyaluronidase, which was isolated from bovine testicular hyaluronidase and polyethylene glycols of different molecular weight, was used in the experimental study. Experiments were carried out using a pulsed linear accelerator ILU-10 for enzyme electron beam immobilization on a water-soluble polymer matrix of polyethylene glycol molecules. The formation of hyaluronidase and polyethylene glycols conjugates was confirmed by exclusion chromatography. Results. As a result of accelerated electron irradiation of hyaluronidase and polyethylene glycol of molecular mass 1500 Da conjugates of enzyme and polymer matrix were formed. After electron-beam pegylation, the specific activity of the enzyme was retained. Conclusions. A laboratory batch of prototype pharmaceutical substance based on immobilised hyaluronidase on polyethylene glycol was obtained.

Introduction. The balance of estrogens and progesterone ensures the harmonious development of the uterus during pregnancy: progesterone provides an increase in the number of myocytes, and estrogens – their volume. Hormonal balance achieved through the catabolism of estrogens in the liver plays an important role in postpartum uterine involution. In this regard, liver diseases during pregnancy can disturb the elimination of estrogens, which leads to hyperestrogenism and problems with maintenance of pregnancy, and can also cause impaired uterine postpartum involution. Aim. The study of the expression of estrogen and progesterone receptors in the myometrium of C57Bl/6 mice during pregnancy and the late postpartum period in acute CCl4-induced hepatosis and in its correction with immobilized hyaluronidase (IH). Materials and methods. The experiment was performed on 100 female C57Bl/6 mice. Acute hepatosis was induced on the 13th day of pregnancy by a single intraperitoneal injection of a 50% carbon tetrachloride solution in olive oil at a dose of 0.3 ml/kg. The mice were divided into four groups: group 1 (control) – intact pregnant mice; group 2 – mice with acute toxic hepatosis; group 3 – pregnant mice with induced acute toxic hepatosis and its correction with IH on the 14th day of pregnancy; group 4 – intact pregnant mice receiving IH on the 14th day of pregnancy. In all groups, sampling was carried out on the 18th and 21st days of pregnancy and on the 1st, 10th and 15th days after parturition. The numerical density (Nai) of positively stained nuclei of the receptors in the test area in myocytes was calculated, regardless of the staining intensity. Results. During pregnancy in acute CCl4-induced hepatosis, the expression of progesterone and estrogen receptors in the myometrium does not change and corresponds to that of intact pregnant mice. In animals with acute CCl4-induced hepatosis, in the late postpartum period (up to 15 days), an increased expression of estrogen receptors in the myometrium is noted, with relatively stable expression rates of progesterone. In the correction of acute CCl4-induced hepatosis with IH, the expression of estrogen receptors in the postpartum period decreases and generally corresponds to similar indicators in intact pregnant mice. Conclusion. In acute CCl4-induced hepatosis, the balance of estrogen and progesterone receptors in the myometrium in the postpartum period is disturbed. In the correction of acute CCl4-induced hepatosis with IH, the levels of expression of estrogen and progesterone receptors in the myometrium are normalized which contributes to the completion of postpartum uterine involution.

Introduction. One of the stages in the development of medicinal products (MP) is the study of safety assessment. Determination of possible toxic effects, establishment of the nature and severity of the damaging effect of drugs under development during their administration is carried out, including studying the effect on hematologic parameters and hematopoiesis. Aim. To assess the possibilities of PEG-HYAL impact on hematologic parameters and hematopoiesis when administered per os. Material and methods. Hyaluronidase immobilized by electron beam synthesis on polyethylene oxide (PEG-HYAL). Laboratory animals – white mature conventional outbred rats and chinchilla rabbits. Hematological parameters were determined using an automatic hematological analyzer Abacus (Diatron, Austria) and conventional manual methods of research. The effect of PEG-HYAL on the state of medullary hematopoiesis was determined by counting the total number of myelokaryocytes per femur (106/femur) and myelograms on smears (in rats). The percentage of individual cell forms in rat myelogram counts was converted to absolute numbers, x106 cells per femur. In rabbits, the myelogram indices of bone marrow, taken from the sternum segment, were determined in relative units (%) only. Results. The analysis of the obtained results of the effect of PEG-HYAL administration on hematologic indices in rats (male and female) did not show any pathological changes. The revealed changes in hematologic parameters in rats are neither significant, nor systemic and dose-dependent. PEG-HYAL administration to rabbits has no toxic effect on hematologic parameters with multiple intragastric administration. Administration of PEG-HYAL for 28 days to rats in all studied doses does not produce a toxic effect on the main studied bone marrow parameters. While the revealed changes in the quantitative composition of myelokaryocytes are non-systemic and reversible. PEG-HYAL administration has no toxic effect on bone marrow indices of rabbits when administered intragastrically in the studied doses during 28 days. Conclusion. Interpretation of the obtained results, concerning the effect of the per os PEG-HYAL administration to experimental animals on hematologic parameters and hematopoiesis, can be used as a preclinical dossier of the drug.

Introduction. Testicular hyaluronidase preparations are widely used in medical practice. Modification of native biologically active molecules by electron beam PEGylation allows to improve their pharmacokinetic properties, which determines a subsequent improvement of pharmacodynamic effects. The development of an original oral drug with a pharmacologically active core, which is hyaluronidase, is promising, but requires studying the safety of use at the preclinical stage. Aim. Ultrastructural evaluation of hepatotoxic effects of PEGylated hyaluronidase in vitro. Materials and methods. The object of a study was testicular hyaluronidase PEGylated on polyethylene oxide (PEG-HYAL) using electron beam synthesis. The culture medium is a continous culture of human liver cells Chanq liver. The cytotoxic effect was detected by the MTT assay. Ultrastructural changes were evaluated by electron microscopy. Results. The original drug PEG-HYAL in the studied concentrations of 37, 75 and 150 U/ml has no cytotoxic effect on the human liver cell culture (hepatocytes). Cell viability is virtually at the control level when PEG-HYAL administration at concentrations of 37 and 75 U/ml, and in the maximum concentration of 150 U/ml, cell proliferation is stimulated significantly, as evidenced by an increase in cell viability up to 106%. The results of evaluation of ultrastructural changes in hepatocytes showed that exposure to PEG-HYAL in all concentrations leads to the enhancement of metabolic processes in cells, development of autophagy, which is one of the main homeostatic processes. Conclusion. The drug PEG-HYAL in all studied concentrations has no toxic effect on the human hepatocyte culture. The results obtained can be used in further research in the development of PEG-HYAL.

The widespread use of testicular hyaluronidase preparations determines the particular interest in the development of oral dosage form. The drug under development, which is hyaluronidase pegylated using electron beam immobilization technology (PEG-GIAL), requires an assessment of its safety. Identification of possible neurotoxic action, as well as the influence on cardiac function seems to be very relevant. Aim of the study was to investigate the effect of PEG- GIAL on behavioural reactions and electrical activity of the heart of experimental animals. Material and methods. Eight hundred of white outbred rats (male and female) were used as experimental animals. The effect of PEG-GIAL on the central nervous system was determined using the «open field with holes» test, examining such characteristics as emotional reactions and orientation-research behaviour. The functional state of the cardiovascular system was assessed by electrocardiography data. Results. PEG-GIAL administration in all studied doses does not lead to changes in the functional activity of CNS, does not have a pronounced effect on the indices of orientation-research behaviour and emotional reactions in laboratory animals. No statistically significant changes in the cardiogram of rats were revealed, including no intersex differences. Conclusions. PEG-GIAL administration in all studied doses has no toxic effect on the electrical activity of the heart and has no pronounced effect on changes in the functional activity of the CNS and behavioural reactions.

Introduction. Lambda interferons (IFN-λ), known as type III interferons (type III IFN), demonstrate significant prospects in the field of antivirus defense. IFN-λ are similar in their antiviral activity to type I IFN, but have unique mechanisms of induction and functioning because of the specific location of their receptors. Type I receptors are expressed in most nucleated cells, IFN-λ receptors (IFN-λR1 or IL-28Rα) are mainly found in epithelial cells. IFN-λ are considered as the first line of defense against microbial attacks, therefore, the development of IFN-λ-based drugs seems very promising. A prototype of an IFN-λ-based drug for enteral administration, PEGylated (polyethylene glycol) using electron beam synthesis technology, has already been developed. Aim. To study the common toxic properties of the intragastrically administered IFN-λ1-based drug in experiments in vivo. Materials and methods. The IFN-λ1-based drug was administered daily intragastrically at doses of 2.6, 13.0 and 26.0 µg/kg (1 therapeutic dose (TD), 5 TD and 10 TD, respectively) to rats and 2.6 and 13.0 µg/kg (1 TD and 5 TD) to rabbits once a day for 180 days. The doses of the drug were calculated for each animal individually, based on body weight. Rabbits were followed-up during the drug administration (180 days), and rats – additionally for 30 days after the end of administration (210 days). In animals, vital signs, behavioral reactions, laboratory parameters of cellular and biochemical composition of blood, electrocardiography data, hematopoiesis parameters, pathomorphological signs were assessed. Results. No deaths were observed during the study. The animals showed a stable state of health, the absence of visible changes in an appetite, appearance, and behavior. During a pathomorphological examination, no abnormalities were found that could indicate a negative effect of the substance. A significant decrease in the mass and mass coefficient of the thymus and spleen was revealed in rats receiving the drug at a dose of 26.0 µg/kg after 180 days of administration compared with the corresponding control. The thymus and spleen mass and the mass coefficient of the thymus and spleen normalized 30 days after drug withdrawal in all groups, where abnormalities were observed. A microscopic examination of the internal organs of rats receiving the highest dose of the IFN-λ1-based drug (26.0 µg/kg) for 180 days revealed specific changes in the thymus and spleen, while analysis of the thymus and spleen in rats receiving a lower dose of the drug (13.0 µg/kg) did not reveal similar pathological changes.This indicates the possibility of dose-dependent toxic effect of the drug. 30 days after the IFN-λ1-based drug withdrawal, the mass and mass coefficient of the thymus and spleen of rats receiving the highest dose of the drug returned to the reference values and did not differ from the corresponding control. Both in rats and rabbits, the indicators of body weight gain, peripheral blood and bone marrow, biochemical parameters of the blood serum in both males and females in the experimental and control groups did not differ significantly throughout the study. The pathomorphological examination found no changes in such crucial organs as the brain, heart, lungs, liver, etc., which indicates the absence of negative effect of the drug on the health and condition of the tissues of rabbits. Conclusion. Based on in vivo experiments to study the common toxic properties of IFN-λ1-based drug in the intragastric route of administration, it can be argued that the drug belongs to the 4th hazard class (GOST 12.1.007-76).

Introduction. The prevalence of acute and chronic liver diseases in pregnant women increases every year, which is associated with a high risk of adverse outcome for the mother and fetus. At the same time, the mechanisms that ensure an increase in uterine mass, structural changes in the myometrium before childbirth and the process of postpartum involution of the uterus to the initial mass, the participation of molecular cellular mechanisms in pregnant women with liver pathology, and ways to correct it remain poorly understood. Aim. The aim of the study was to investigate morphological changes in the myometrium, during pregnancy and the late postpartum period in the myometrium of mice with acute CCl4-induced hepatosis and under conditions of its correction with immobilized hyaluronidase. Materials and methods. The experiment was performed on 200 female pregnant mice of the C57B1/6 line at two months of age. Acute toxic hepatosis was modeled by a single injection of tetrachlormethane. Correction of acute hepatosis was performed on the next day after administration of tetrachloromethane and subsequent days of pregnancy with a single injection of immobilized hyaluronidase. The animals were divided into 4 groups: a group with physiological pregnancy, animals with the immobilized hyaluronidase drug injection; animals with acute hepatosis; animals with acute hepatosis treated with immobilized hyaluronidase. Uterine samples were collected on the 18th, 21st days of pregnancy and on the 1st, 5th, 10th, 15th days after delivery. Results. The volume density (Vv) of myocytes in the state of apoptosis, interstitial cytoplasmic conglomerates, which are products of clasmacytosis, and necrotized myocytes were calculated, as well as the numerical density (Nai) of myocytes with positive expression of the p53 protein. Under conditions of acute hepatosis, structural transformations of the myometrium in the form of an increase in clasmacytosis, necrotized myocytes occur already during pregnancy. The main structural mechanism of the process of postpartum involution of the myometrium of mice in conditions of acute hepatosis in the long-term period of postpartum involution was clasmacyto sis and, to a lesser extent, necrosis of myocytes. The processes of myometrial involution in mice under conditions of acute hepatosis slow down and do not complete by the 15th day of the postpartum period. When correcting acute hepatosis with immobilized hyaluronidase, the volume density of cytoplasmic conglomerates, apoptotically altered myocytes and necrotized myocytes decreases from the 1st to the 15th day of the postpartum period, which indicates the completeness of postpartum uterine involution. Conclusion. Liver damage is accompanied by a change in the metabolism of sex hormones, which leads to an abnormality of the mechanisms of postpartum involution of the uterus, as well as its structural changes before childbirth.

Introduction. Anticoagulants are included in the treatment protocols for several cardiovascular diseases. Nonea rossica Steven is of scientific interest as a source of biologically active compounds with the properties of an indirect anticoagulant. Aim. Study of anticoagulant activity of total extracts from Nonea rossica Steven herb. Materials and methods. A dry extract was obtained from the herb using 70% ethanol, then the extractant was removed. The anticoagulant effect was studied in vivo (36 male Wistar rats) using a prothrombin time test. As a reference drug, warfarin was used in doses recommended by the patient information leaflet. Results. The extract studied at a dose of 1.7 g/kg showed anticoagulant activity comparable to the reference drug (prothrombin time is 31.3 ± 1.27 and 33.42 ± 1.33 s, respectively). Conclusion. Nonea rossica Steven is a promising plant for practical as an indirect anticoagulant.

Purpose: to study the regenerative potential of immobilized hyaluronidase and subtilisin enzymes preparations in experimental models of chemical and mechanical corneal trauma.Material and methods. The study involved 28 mongrel rabbits weighing 3.5 to 4 kg, divided into 4 groups for separate studies of repair models according to different types of injury (chemical vs. mechanical), and different drugs and the methods of administration thereof. Hyaluronidase (PEG-hyaluronidase) and subtilisin (PEG-subtilisin) pegylated by electron beam synthesis technology were used. The condition of the cornea was assessed 24 hours after the injury had been inflicted, whereupon the eyes were enucleated and histologically examined.Results. The chemical trauma groups treated by PEG-hyaluronidase revealed a decrease in the wound area by an average of 36.6 mm2, while those given a subconjunctival injection showed a 36.08 mm2 decrease. If PEG-subtilisin was applied, the area of the corneal defect revealed an average increase of 11.63 mm2 (with a case of perforation registered). In subconjunctival injection of PEG-subtilisin, the wound area decreased by 27.42 mm2. In the mechanical trauma groups, a similar pattern was observed: with the instillation of PEG-subtilisin, the wound area averaged 53.63 mm2 and 1 case of perforation was registered, whilst with subconjunctival administration, the wound area decreased to 28.76 mm2. With PEG-hyaluronidase instillations, a significant wound area decrease of 36 mm2 was noted, and with subconjunctival administration, the wound area decreased by 70.3%. A higher corneal transparency in the optical zone and a weaker inflammatory reaction with the use of PEG hyaluronidase were revealed by histological testing.Conclusion. A positive effect of PEG-hyaluronidase, specifically concerning the enhancement of regenerative processes in the wound in chemical and mechanical wounds could be seen. PEG-subtilisin showed no positive effects, which may be associated with high fibrinolytic activity.

Introduction. Under conditions of chemical damage to the cornea, its cellular structure is significantly disrupted, requiring emergency highly differentiated regeneration without an expressed proliferative component of inflammation and expansion of immunocompetent cells to gain an antimicrobial potential. For the purpose of pharmacological initiation of these processes, the study of the topical administration of anti-inflammatory enzyme preparations, such as subtilisin and hyaluronidase, is pathogenetically justified. Aim. To study the effect of hyaluronidase and subtilisin, PEGylated (polyethylene glycol – PEG) using the technology of electron beam synthesis on the number of immunocompetent cells in the area of the corneal chemical injury during their subconjunctival and topical administration. Materials and methods. An experimental study of the effect of PEGylated hyaluronidase and subtilisin enzymes, on the cellular composition of the corneal chemical injury was performed on 28 rabbits. Corneal injury was modeled using the Obenberger alkali burn technique. PEG-subtilisin or PEG-hyaluronidase was applied topically or subconjunctivally into the right eye of the animal, depending on the group, the left eye of the animal was used as a control – it was treated with 0.9% NaCl. After the experiment, enucleation was performed. The biomaterial obtained was used to prepare tissue specimens for morphological examination. Results. The total count of cells in the groups of topical and subconjunctival administration of PEG-subtilisin was 43 (40; 52) and 73 (33; 92), and subconjunctival injection of PEG-hyaluronidase – 46 (37; 61), which was higher than the count of cells when using 0.9% NaCl in these groups (p < 0.01) and higher (p < 0.0001) cell numbers in the group of topical administration of PEG-hyaluronidase. The total count of cells with topical application of PEG-hyaluronidase was 15 (13; 16), with topical application of 0.9% NaCl of this group – also 15 (14; 18) (p = 0.38). The neutrophil count with the use of PEG-subtilisin was 1 (1; 2) with topical and 0 (0; 1) with subconjunctival administration, and with the use of PEG-hyaluronidase – 0 (0; 0) both with topical and subconjunctival administration. Conclusion. The administration of PEG-hyaluronidase subconjunctivally and PEG-subtilisin both topically and subconjunctivally leads to an increased migration of immunocompetent cells to the area of the corneal chemical injury, while the migration of neutrophils is insignificant. It is completely absent when PEG-hyaluronidase is injected subconjunctivally. Topical administration of PEG-hyaluronidase does not induce a pronounced cellular response of immunocompetent cells in the area of the corneal chemical injury, and the effect of the application is comparable to that of 0.9% NaCl.

The development of safe drugs occupies a special place in the pharmaceutical industry. One of the main tasks of its preclinical phase is to evaluate possible toxic effects of the developed drug on the body and on various systems, including the immune system. The aim of our work was to study immunotoxic properties of pegylated hyaluronidase (PEG- HYAL). Material and methods. Mice F1(CBA/C57Bl/6) were divided into subgroups which were intragastric and intraperitoneally administered with PEG-HYAL in different dosages (50, 500, 1250, 2500 and 5000 U/kg). The number of antibody-producing cells in the spleen, the mass and cellularity of central and peripheral immune organs, phagocytic activity of peritoneal macrophages and neutrophils, delayed hypersensitivity reaction (DHR), level of hemagglutinin to sheep erythrocytes, spontaneous and mitogen-induced splenocyte proliferation were determined. Results. PEG-HYAL did not induce DHR, did not suppress phagocyte activity of peritoneal macrophages, at a dose of 50 ED/kg did not significantly affect the hemagglutinin content to erythrocytes, but at a dose of 500 ED/kg did statistically significantly reduce the titer of specific antibodies. When experimental animals were exposed to PEG-HYAL at doses of 50 and 500 U/kg, spontaneous and mitogen-induced proliferation of splenocytes decreased. Conclusions. The PEG-HYAL trial produced results that can be used to substantiate the administration of the PEG-HYAL-based medication.

Introduction. Hyaluronidase is an enzyme preparation widely used in medicine, including ophthalmology. Hyaluronidase, having a protein structure, carries a potential danger in the form of undesirable allergic reactions. To avoid allergenic effects, protein structures are modified by combining with a polymer carrier, for example, by polyethylene glycol (PEG). Aim. To study the possible allergenic potential of hyaluronidase, PEGylated using the technology of electron beam synthesis, with various routes of administration. Materials and methods. The object of the study is PEGylated hyaluronidase (PEG-Hyal). Experimental animals are hybrid mice and guinea pigs. Seven types of experiments were carried out: anaphylactogenic activity; conjunctival challenge test; cutaneous applications; delayed-type hypersensitivity (DTH); active cutaneous anaphylaxis; indirect mast cell degranulation; inflammatory response to concanavalin A (ConA). Results. When studying anaphylactogenic activity, an anaphylactic reaction was not observed in guinea pigs. The conjunctival challenge test also revealed no signs of hypersensitivity to the drug. In the study of sensitization, no development of local allergic reactions was observed. In experiments to the study DTH in mice, there were no significant differences for the DTH index in the experimental and control groups. The assessment of active skin anaphylaxis has been showed that the diameter of the stained spot did not significantly differ in both experimental groups from the control one. In indirect mast cell degranulation experiments, it was found that there was no statistically significant change in the mast cell degranulation index. Evaluation of the allergenic potential of PEG-Hyal in the inflammatory response to ConA showed that there is no statistically significant difference between the experimental and control groups. Conclusion. Based on the results of the experiments, it can be concluded that PEG-Hyal does not have an allergenic potential in various routes of administration, which significantly expands the possibilities of its clinical use.

Introduction. Currently, a new pharmacological technology of thrombolytic therapy using immobilized subtilisins (ISs) has been introduced into clinical practice. Experimental and clinical studies have shown the high efficacy and safety of the treatment of thrombotic diseases using the drug Thrombovasim®, created on the basis of ISs. Although this drug has been widely studied as a thrombolytic, its pluripotent pharmacological properties have not been studied in detail. Aim. To study the performance of an isolated rat heart under the coronary perfusion with ISs. Materials and methods. The Langendorff’s perfused heart model was used in the study. The experiment included 50 Wistar rats. Animals were divided into 5 groups (10 animals each): group 1 – control (hearts perfused with Krebs-Henseleit solution) and 4 experimental groups – hearts perfused with ISs at 4 concentrations of 170, 340, 510 and 1020 IU/l respectively. The performance indicator of an isolated heart was assessed, reflecting the actual work of the heart muscle and expressed as the product of the heart rate and the strength of its contractions in the form of the pressure developed by the left ventricle. Results. No negative ISs effect on the performance of the isolated rat heart was found. A tendency to increase the performance in the first 5 minutes of using ISs was revealed. The achievement of the maximum and the duration of this effect depend on the ISs contents in perfusate. At a concentration of 170 IU/l, an obvious increase in the effect is found from the 10th minute, at higher concentrations – from the 5th minute. The duration of the increasing performance effect when using doses of 170–510 IU/l is maintained up to the 20th minute, when using ISs at a dose of 1020 IU/l – up to the 30th minute. The return of the performance indicators to the baseline values is observed at the 30th minute, at a dose of 340 IU/l – at the 40th minute. When perfused with a solution of ISs at concentrations of 510 and 1020 IU/l, by the 40th minute, the effect of increasing performance weakens, but the parameters still remain at higher levels as compaired to those at baseline. Conclusion. The experimental data obtained show that ISs increase the performance of the heart. This circumstance will allow expanding the use of the drug Thrombovasim® in a catastrophe of the coronary circulation.

The pharmacological experiments on isolated organs (ex vivo) are the preferred method for assessing the primary pharmacodynamics of the studied drugs, since this method is completely excluded the systemic influence of neurohumoral regulation. In the last decade, a new group of thrombolytic drugs based on immobilized subtilisins has been formed. At the stage of registrational preclinical and clinical studies, their pleiotropic pharmacological effects have not been studied. Meanwhile, there is a reason to consider that their pharmacological activity in the bloodstream is not limited to thrombolytic action, but may be extended to a systemic effect on the cardiovascular system. The aim of the study was to investigate the chronotropic effects of an isolated heart during its perfusion with solutions of immobilized subtilisins at different concentrations. Material and methods. The isolated rat heart model according to Langendorff was used in the study. The experiment included 50 Wistar rats, which were divided into 5 groups: isolated hearts perfused only with Krebs – Henseleit solution (control) or with immobilized subtilisins in 4 concentrations (170, 340, 510 и 1020 U/l). Results and discussion. The immobilized subtilisins have a negative chronotropic effect. The onset of the effect depends on the drug concentration in the solution: the higher concentration, the earlier effect. From 5 to 10 minutes of perfusion, a negative chronotropic effect is observed using of immobilized subtilisins at any dose. The duration of its increase is manifested up to 10–20 minutes, depending on the drug concentration in solution. After 20 minutes of perfusion, the achieved negative chronotropic effect remains at a plateau level up to 40 minutes. Conclusion. The immobilized subtilisins have an independent pharmacological effect on heart rate.

Introduction. Immobilized subtilisins are currently used as a fibrinolytic agent in antithrombotic drugs. The effect of immobilized subtilisins (ISs) on the contractile function of the heart has not been previously studied. Aim. To study the effect of coronary perfusion with immobilized subtilisins (ISs) on the contractile function of an isolated rat heart in an experiment. Materials and methods. The study used a Langendorff model of coronary retrograde perfusion of an isolated rat heart with solutions of ISs at different concentrations. The experiment included 50 male rats. The animals were divided into 5 groups: group 1 – control (hearts perfused only with Krebs-Henseleit solution) and 4 experimental groups – hearts perfused with a solution of ISs at concentrations of 170, 340, 510 and 1020 IU/l, respectively. The contractile function (inotropic effect) of the isolated rat heart was assessed in terms of the pressure developed by the left ventricle (LVP). Results. Perfusion of an isolated rat heart with a solution of ISs for 40 min causes a positive inotropic effect in all experimental groups. At a concentration of 170 IU/l, the effect is observed from the 10th minute, at higher concentrations – from the 5th minute. The duration of the effect’s rising when using ISs in the dose range of 170–510 U/l manifests itself up to the 20th minute, and when using a dose of 1020 U/l – up to the 30th minute. Then there is a weakening of the inotropic effect. In groups of animals with the introduction of immobilized subtilisins at a concentration of 340–1020 U/l, there is a decrease of LVP by the 40th minute, but the values of this indicator remain higher compared to the control group. Conclusion. Perfusion of the isolated heart with a solution of ISs increases the contractile activity of the isolated heart. When the heart is perfused with ISs, there is an increase of LVP. The inotropic effect is dose-dependent at various time points.

Резюме: Показано влияние иммобилизированных ферментов гиалуронидазы и субтилизина на клетки конъюнктивы человека.Для исследования были использованы два вида фармацевтических субстанций -иммобилизированная на полиэтиленгликоле посредством радиационного синтеза гиалуронидаза (ИмГд) и субтилизин (ИмСб).Препараты иммобилизированных ферментов гиалуронидазы и субтилизина изменяют внутриклеточный биохимизм клеток эпителия конъюнктивы при контакте с их мембраной.Установлено, что ИмГд не оказывает цитотоксического действия на клетки конъюнктивы человека во всех изучаемых концентрациях.Отмечается статистически достоверное

ЭКСПЕРИМЕНТАЛЬНОЕ ОБОСНОВАНИЕ ПРИМЕНЕНИЯ ПЕРОРАЛЬНОГО ТРОМБОЛИТИКА НА ОСНОВЕ ИММОБИЛИЗИРОВАННЫХ СУБТИЛИЗИНОВ И АНТИКОАГУЛЯНТА НА ОСНОВЕ НИЗКОМОЛЕКУЛЯРНОГО ГЕПАРИНА ДЛЯ ЛЕЧЕНИЯ ТРОМБОЗА СОСУДОВ ЛЕГКИХ

Введение. Тромболитическая терапия с применением активаторов плазминогена достаточно эффективна, но сопряжена с риском возникновения геморрагических осложнений и реализуется только внутривенным путём введения. Существует необходимость в пероральной тромболитической терапии. Среди возможных кандидатов для эффективного перорального фибринолиза/тромболизиса следует обратить внимание на сериновые протеиназы, не вызывающие сайт-специфического гидролиза факторов свёртывания крови. Необходимо изучить их энтеральную биодоступность и тромболитическую активность. Цель исследования: изучить путь поступления сериновой протеиназы субтилизина в системный кровоток при пероральном применении, а также сравнить тромболитическую активность протеолитических ферментов трипсина, химотрипсина и субтилизина. Материалы и методы. Проведено открытое сравнительное экспериментальное исследование в параллельных группах крыс по определению тромболитической активности изучаемых препаратов in vitro. Сформировано 4 группы животных: 3 для исследования лекарственных препаратов субтилизина, трипсина, химотрипсина и 1 контрольная. В каждой группе происходила инкубация фрагментированных тромбов с предполагаемым тромболитическим агентом. Для исследования всасывания иммобилизированного субтилизина (ИС) 30 крысам после срединной лапаротомии интубировали фрагмент тощей кишки для инфузии растворов. Для ИС, меченного флуоресцеин‑5‑изотиоцианатом (ФИТЦ), определены особенности всасывания из тощей кишки в систему кровь–лимфа. Меченный ИС экстрагировали из прилегающей к области интубации интерстициальной жидкости кишечника, слизистый слой которого был удален, и из паренхимы лимфатических узлов (I и II порядка). Для визуальной детекции абсорбции ИС на эпителии тощей кишки применили метод конфокальной микроскопии. Тромболитическая активность ИС, трипсина и химотрипсина исследована на фрагментированном тромбе. Результаты. In situ установлено, что ИС через 60 мин после начала инкубации в тощей кишке детектируется и в плазме крови и в лимфе (p < 0,01). Концентрация препарата в плазме крови из v. mesenterica (223,8 ± 39,8 мЕд/мл) более чем в 10 раз выше, чем в лимфе из цистерны грудного протока (19,27 ± 2,33 мЕд/мл). Снижение концентрации препарата в v. cauda (143,7 ± 24,4 мЕд/мл) по сравнению с v. mesenterica (p < 0,01) объясняется первичным метаболизмом в печени и последующим почечным клиренсом. При конфокальной микроскопии хорошо видно, что ИС активно абсорбируется на слизистой тонкого кишечника с накоплением в толще крипт и поступлением в сосудистую сеть. В экспериментах in vitro показано, что в условиях нормального или компенсированного кислотно-основного состояния крови достоверно наибольшую тромболитическую активность имеет препарат ИС (p < 0,05 по сравнению с контролем, трипсином и химотрипсином). За 30 мин инкубации ИС уменьшает площадь тромба более чем на 20%. Заключение. Энтеральная биодоступность ИС обеспечивается за счёт активной абсорбции на слизистой тонкого кишечника, прямого поступления в сосуды портальной системы и накопления в лимфатическом русле с дальнейшим дренажем в системный кровоток. Тромболитическое действие ИС существенно превосходит трипсин и химотрипсин, что позволяет считать малоперспективным разработку пероральных тромболитиков на основе трипсина и химотрипсина. Background. Thrombolytic therapy with plasminogen activators is quite effective, but it is associated with the risk of hemorrhagic complications and is implemented only by intravenous administration. Need for oral thrombolytic therapy exists. Among the possible options for effective oral fibrinolysis/thrombolysis, attention should be paid to serine proteinases that do notcause site-specific hydrolysis of blood coagulation factors. It is necessary to study their enteral bioavailability and thrombolytic activity. Objectives: to study the route of entry of serine proteinase subtilisin into the systemic circulation after oral administration, and also to compare the thrombolytic activity of the proteolytic enzymes trypsin, chymotrypsin, subtilisin. Materials/Methods. We carried out an open comparative experimental study in parallel groups of rats to determine the thrombolytic activity of the studied drugs in vitro. Four groups were formed: 3 groups to study subtilisin, trypsin, chymotrypsin and 1 control group. In each group, fragmented thrombi were incubated with the putative thrombolytic agent. To study the absorption of immobilized subtilisin (IS), 30 rats after midline laparotomy were intubated with a jejunum fragment for infusion of solutions. For IS labelled with fluorescein‑5‑isothiocyanate (FITC), the features of absorption from the jejunum into the blood-lymph system were determined. The labelled IS was extracted from the interstitial fluid adjacent to the intubation area, the mucous layer of which was removed, and from the parenchyma of the lymph nodes (sentinel and second-tier nodes). Confocal microscopy was used to detect visually the IS absorption on the jejunal epithelium. The thrombolytic activity of IS, trypsin, and chymotrypsin was studied on a fragmented thrombus. Results. It was found in situ that IS 60 min after the start of incubation in the jejunum was detected in both blood plasma and lymph (p < 0.01). Plasma drug concentration from the mesenteric vein (223.8 8 ± 39.8 mU/ml) is more than 10 times higher than that in the lymph from the thoracic duct cistern (19.27 ± 2.33 mU/ml). Reducing drug concentration in the caudal vena (143.7 ± 24.4 mU/ml) compared to the mesenteric vein (p < 0.01) is explained by primary hepatic metabolism and subsequent renal clearance. Confocal microscopy clearly shows that IS is actively absorbed at the mucous membrane of the small intestine with accumulation in the crypt and entering the vasculature. In vitro experiments have shown that under conditions of the normal or compensated acid-base blood state, the IS has significantly the highest thrombolytic activity (p < 0.05 compared to control, trypsin and chymotrypsin). After 30 minutes of incubation, IS reduces the thrombus area by more than 20%. Conclusions. IS enteral bioavailability is provided due to active absorption at the mucous membrane of the small intestine, direct entry into the vessels of the portal system and accumulation in the lymphatic bed with further drainage into the systemic circulation. IS thrombolytic effect is significantly superior to trypsin and chymotrypsin, which makes the development of oral thrombolytics based on trypsin and chymotrypsin unpromising.

Introduction. In clinical medicine, some phenomenon has been noted: in many patients with anginal attacks during coronary angiography, significant obstructive processes in the coronary arteries are not detected. There is impaired coronary microcirculation due to dysfunction of endothelial and smooth muscle cells. This circumstance initiates the search for new drugs for pharmacological correction of the vasomotor status of coronary vessels in order to eliminate myocardial ischemia. Aim. To study the parameters of the coronary blood flow during the perfusion of an isolated heart with immobilized subtilisins (IS) in different dosages. Materials and methods. The effect of IS on coronary blood flow was studied using the Langendorff-perfused rat heart. The experiments were carried out on 50 male Wistar rats. Animals were divided into 5 groups: control (hearts perfused only with Krebs-Henseleit solution) and 4 experimental (1st — perfusion with IS at a concentration of 170 U/l; 2nd — perfusion with IS at a concentration of 340 U/l; 3rd — perfusion with IS at a concentration of 500 U/l; 4th — perfusion with IS at a concentration of 1000 U/l). Results. An increase in coronary blood flow was observed during perfusion with IS at concentrations of 170 and 340 U/l. With perfusion of IS at a concentration of 340 U/l, the vasodilating effect was significantly expressed. With perfusion at a concentration of 500 and 1000 U/l, a decrease in coronary flow was noted, which is most likely caused by vasoconstriction. Conclusion. When the heart is perfused with IS solution, the phenomenon of vasomotor activity of the coronary vessels is observed. The vasomotor effect caused by IS has a dose-dependent and multidirectional effect and is realized, most likely, through an allosteric effect on the natural mechanism of regulation of the coronary vessels tone.

For the treatment of bipolar affective disorders, lithium preparations are the most famous and effective. But the main problem with the use of lithium preparations is the narrow «therapeutic corridor». An urgent task is the creation of dosage forms of lithium with a slow release and a wide therapeutic range. The study object was a new normotymic drug based on lithium, aluminum oxide and polymethylsiloxane. Due to the new carrier matrix lithium is released from its porous structure gradually providing a prolonged effect and maintaining an optimal concentration in the blood which also helps to minimize side effects. The purpose of the study was to explore the pharmacokinetic parameters of a normotymic drug based on a complex lithium citrate, aluminum oxide and polymethylsiloxane (LOAP).Material and methods: for the assessment of pharmacokinetic parameters the method of atomic emission spectrometry with inductively coupled plasma and two-chamber modeling were used.Results and discussion. The pharmacokinetic data showed a linear nature of pharmacokinetics of the drug based on LOAP as the foundation of data of the lithium’s amount in the blood plasma of rabbits after intragastric administration at doses of 200, 400 and 800 mg/kg. The drug with intragastric administration at a dose of 800 mg/kg is well absorbed from the gastrointestinal tract, with bioavailability (F) 74 %. This dose shows the maximum increase of the area under the pharmacokinetic curve (AUC - 32787.1 (ng x h)/ ml), and indicators of elimination constant (Kel - 0.062 h-1), clearance (Cl - 0.09 l/(kg x h)), elimination half-life (T1/2p - 11.436 h) in comparison with other doses remain unchanged.