The aim of the work is to evaluate the hemostimulating activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) conjugates with alendronic acid (ALN) in the model of cytostatic myelosuppression and the dynamics of rhGM-CSF accumulation as a part of the conjugate in the bone tissue and bone marrow of mice.Materials and methods. The conjugates obtained by a solid-phase synthesis using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide or periodate oxidation, were used. A hemostimulating activity was evaluated in a model of a cytostatic myelosuppression induced by the administration of cyclophosphamide to CBA/Calac mice. RhGM-CSF preparations were injected subcutaneously for 4-5 days at the dose of 90 µg/kg. After the injections cycle had been completed, the total leukocyte and segmented neutrophil counts were carried out in the blood samples, and the total karyocyte count was carried out in the bone marrow samples.The tissue distribution of rhGM-CSF preparations was assessed in outbred CD-1 mice after a single intravenous administration at the effective dose. The content of rhGM-CSF in blood, femoral tissue and bone marrow was determined by enzyme immunoassay.Results. RhGM-CSF conjugates with ALN have been shown to retain the ability of the original protein to increase the number of leukocytes, segmented blood neutrophils, and bone marrow karyocytes under the action of conjugates. The stimulation of the neutrophil production used to be observed at earlier times than in the case of rhGM-CSF. The increase in the total number of bone marrow cells after the introduction of all three conjugates was more pronounced compared to the original protein (by 34%). The increased hemostimulatory effect of the AEG conjugate was accompanied by a more intense accumulation of rhGM-CSF in the bone tissue and bone marrow of mice. The rhGM-CSF introduced into the conjugate was detected in the bone tissue for 24 h and it circulated in the bloodstream for a longer time compared to the original protein.Conclusion. The data obtained make it possible to conclude that further work on the development of effective hemostimulating drugs based on rhGM-CSF conjugates with ALN, is promising.

Influence of double-stranded RNA (dsRNA) from Saccharomyces cerevisiae yeast upon expression levels of the macrophage genes encoding TLR3 receptor, interferons alpha and beta (IFNα, IFNβ), 2’,5’-oligoadenylate synthetase (OAS) and protein kinase R (PKR) enzymes has been studied in the J774 mouse histiocytic cell culture and in vivo in Balb/c mice. It has been shown that dsRNA exerts a selective activating effect on genes of TLR3 receptor, antiviral proteins IFNα, IFNβ, and OAS, both in vitro and in vivo. With J774 cell culture, the highest induction capacity was observed for the IFNβ gene: 365 to 802-fold. The stimulatory effect was dependent on the dose of dsRNA in the range of 16.9 to 125 μg/ml. The preparation enhanced IFNα gene activity to lesser degree (more than 10-fold), TLR3 and OAS (3 to 4-fold), while the expression levels for these genes were not significantly dependent on the dose of dsRNA. The stimulating effect of dsRNA was dosedependent in murine peritoneal macrophages. The maximum activating effect of the preparation was shown upon administration of the effective antiviral dose (0.5 mg of dsRNA/kg). Five hours after intraperitoneal injection of dsRNA, the highest level of mRNA synthesis was observed for IFNα (54-fold), OAS (43-fold) and TLR3 (28-fold) genes. Expression of the IFNβ gene increased to a lesser degree (9-fold). An increase in the dose of preparation to 1.5 mg/kg led to decrease of the stimulatory effect. Expression levels of the IFNα, TLR3, and OAS genes in that case decreased by 2-4-fold as compared to a lower dose, and the PKR gene expression was 5-fold lower compared to the control. One day after dsRNA administration, a tendency was observed for both experimental groups towards a decreased transcription of macrophage genes, if compared with the 5-hour term. The weakening of gene activity was less pronounced in animals treated with dsRNA at the dose of 1.5 mg/kg. The transcription indices for IFNβ, OAS, and TLR3 genes were much higher during this period (5-10-fold higher than the control values). The dynamics of PKR gene transcription in both experimental systems was significantly different from the expression of other studied genes. The dsRNA preparation at this dose range did not have a pronounced stimulatory effect upon expression of this gene. A moderate increase in PKR gene activity in macrophages of mice was observed only a day following intraperitoneal administration of dsRNA. Concentrations and length of dsRNA molecules are known to be critical factors to the PKR gene activation. An ability to increase the expression of the gene is shown at low dsRNA concentrations (10-7 g/ml and below), while highly polymeric dsRNAs weaken the gene activity. Since the doses and concentrations of dsRNA used in our experiments were significantly different from those mentioned above, it could, in general, affect regulation of PKR gene transcription towards reduction of the stimulatory effect.

In experiments on mice, the level of expression of antiviral-response-mediating proteins was studied in peritoneal macrophages, in reaction to the intraperitoneal administration of double-stranded RNA (dsRNA) from the yeast Saccharomyces cerevisiae. Dose (0.5 and 1.5 mg/kg) and time (5 and 24 hours) dependencies of the eff ect of dsRNA on the activity of genes encoding Toll-like receptor 3 (TLR3), IFN-alpha, IFN-beta, 2’,5’-oligoadenylatesynthetase (OAS) and proteinkinase R were determined. The obtained results indicate the increase in the level of transcriptional activity in the cells, most pronounced for TLR3, IFN-alpha and OAS genes. The maximum effect was observed 5 hours after the administration of dsRNA in the effective antiviral dose (0.5 mg / kg).

The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain.The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration.Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software.Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg.Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems.

The main problems of using TNF-alpha in antitumor therapy are its rapid degradation in the bloodstream and the limited selectivity of accumulation in the tumor tissue. The SRC VB «Vector» developed a biodegradable molecular construct that provides protection against proteases and ensures targeted delivery of proteins to the tumor tissue. This construct was used to create an antitumor drug containing recombinant human TNF-alpha (rhTNF-alpha). The aim of the study was to analyse rhTNF-alpha pharmacokinetics in the delivery system after a single administration. Materials and methods: the rhTNF-alpha drug carried by the delivery system was intravenously administered to female outbred ICR (СD-1) mice only once at two effective antitumor doses, 2.55 μg and 5.1 μg / 20 g of body weight. The concentration of TNF-alpha in the serum and supernatants of organ homogenates, obtained at different time points after administration, was analysed by immunoenzyme assay. Results: the obtained curves of TNF-alpha concentration in the blood were satisfactorily described by the equation for the twocompartment model without absorption. The rapid phase of elimination from the blood took 0–4 h, the slow one — 4–24 h. The highest specific content of protein was observed in the skin, spleen, and kidneys tissue. The calculation of pharmacokinetic parameters demonstrated that the highest values of tissue availability f T were obtained for the kidneys and skin; the drug was retained for longer periods of time in the kidneys, liver and skin (according to the MRT data). As a rule, complete elimination of the drug was observed by the end of the first day after administration. Conclusions: rhTNF-alpha carried by the delivery system was quickly eliminated from the blood and distributed in the internal organ tissues after a single intravenous administration to mice in the effective doses range. The main organs in which rhTNF-alpha was distributed were skin, kidneys, and spleen. The elimination of the drug from the blood was a two-phase process which was generally over by the end of the first day.

The main problems of using TNF-alpha in antitumor therapy are its rapid degradation in the bloodstream and the limited selectivity of accumulation in the tumor tissue. The SRC VB «Vector» developed a biodegradable molecular construct that provides protection against proteases and ensures targeted delivery of proteins to the tumor tissue. This construct was used to create an antitumor drug containing recombinant human TNF-alpha (rhTNF-alpha).The aim of the study was to analyse rhTNF-alpha pharmacokinetics in the delivery system after a single administration.Materials and methods: the rhTNF-alpha drug carried by the delivery system was intravenously administered to female outbred ICR (СD-1) mice only once at two effective antitumor doses, 2.55 μg and 5.1 μg / 20 g of body weight. The concentration of TNF-alpha in the serum and supernatants of organ homogenates, obtained at different time points after administration, was analysed by immunoenzyme assay.Results: the obtained curves of TNF-alpha concentration in the blood were satisfactorily described by the equation for the twocompartment model without absorption. The rapid phase of elimination from the blood took 0–4 h, the slow one — 4–24 h. The highest specific content of protein was observed in the skin, spleen, and kidneys tissue. The calculation of pharmacokinetic parameters demonstrated that the highest values of tissue availability fT were obtained for the kidneys and skin; the drug was retained for longer periods of time in the kidneys, liver and skin (according to the MRT data). As a rule, complete elimination of the drug was observed by the end of the first day after administration.Conclusions: rhTNF-alpha carried by the delivery system was quickly eliminated from the blood and distributed in the internal organ tissues after a single intravenous administration to mice in the effective doses range. The main organs in which rhTNF-alpha was distributed were skin, kidneys, and spleen. The elimination of the drug from the blood was a two-phase process which was generally over by the end of the first day.

It is shown in experiments in mice that the double-stranded RNAs (dsRNA) of bacteriophage ф6 has pronounced interferon-inducing properties and the ability to enhance the functional activity of phagocytes. The maximum level of IFN-а in serum of outbred ICR mice was observed 5 h after the i.p. administration of the phage dsRNA at doses of 0,8 and 8 mg/kg, and the number of NBT-positive peritoneal macrophages increased 5 – 24 h after the administration. DsRNA of bacteriophage ф6 under conditions of three-fold administration (3 h before, 1 and 3 days after infection) increased the average life span and the number of surviving mice infected with influenza virus (IV) A/Chicken/Kurgan/05/2005 (H5N1). The IV titers in lung homogenates of dsRNA-administered mice were significantly lower on 4th and 6th days after the infection with IV A/Bishkek/03/2009 (H1N1)pdm09 than in untreated animals. The antiviral effects of the phage dsRNA were comparable to those of Tamiflu. These results give the future outlook for the development a new drug based on the dsRNA of bacteriophage ф6 as a preventive and therapeutic medicine for viral infections.

The method of obtaining and purification of the double-stranded RNA of bacteriophage p6 was developed. It was shown that the created method makes it possible to obtain high purity dsRNA preparations possessing interferon-inducing and phagocyte-stimulating activity.

Based on the technology for producting virus-like particles of a killer strain of Saccharomyces cerevisiae Y-448 yeast, which includes stages of extraction, concentration, and chromatographic purification, experimental preparation samples were obtained and their properties were researched. The particle size was detected by electron microscopy. It was identified that dsRNA, which is in the composition of particles, is notable for its higher enzymatic stability as compared to loose dsRNA. It was shown in in vivo experiments that the obtained VLP preparations increase the phagocytic activity of macrophages and the interferon level in mouse blood serum to a greater extent than the drug of comparison, Rhidostin.

Composite preparation of tumor necrosis factor alpha and rheopolyglukin and polyethylene glycol (TNF-α+PG+PEG) was obtained. The specific activity of the samples was 4,13 х 107 IU/mg. The cytolytic activity of drugs TNF-α+PG+PEG and rhTNF-α did not change after 4 months when stored at 6 °С. Preparation TNF-α+PG+PEG provided a moderately prolonged elevation of TNF-alpha in blood of laboratory mice in contrast to TNF-α when they applied to the skin. The composite preparation did not have toxic, allergic and locally irritating action in experiments on laboratory animals.

AIM:Evaluation of composite formulation of yeast double stranded RNA with polyglucinum (dsRNA-PG) effect on non-specific antiviral resistance factors in mice in comparison with commercial formulation Ridostin.MATERIALS AND METHODS:dsRNA and Ridostin formulations were injected intramuscularly once at the dose of 5 mg/ml, polyglucinum--at the dose of 3.75 mg/ml. 3, 5, 24, 48 and 72 hours after the injection serum interferon levels, neutrophil oxidation-reduction activity parameters, peritoneal macrophage phagocyte activity levels were analyzed in mice blood samples.RESULTS:New dsRNA and polyglucinum containing composite formulation is a non-specific resistance system stimulator. dsRNA-PG effect on interferon synthesis and mice phagocyte activity was higher than with Ridostin and developed earlier. Neutrophil function activation by the formulation had a prolonged effect. A possible explanation for increased activity of dsRNA and polyglucinum composite formulation is a modulating effect by the polysaccharide component.CONCLUSION:The new formulation may have a more intensive and prolonged protective effect against influenza virus in comparison with Ridostin.

К числу перспективных противоопухолевых препаратов относятся представители семейства факторов некроза опухоли, в частности фактор некроза опухоли альфа (ФНО-α), который явля-ется противоопухолевым агентом, подавляя рост злокачественных новообразований и вызывая геморрагический некроз опухолей [5, 15]. В то же время результаты клинических испытаний препаратов рекомбинантного ФНО-α человека свидетельствуют о широком спектре его по-бочных эффектов. Этот факт, а также незна-УДК: 615.36:612.1]:616–006.04+57.084.1

Antitumor activity of TNF-α incorporated in nanoparticles (VLP-TNF-α) and dynamics of its accumulation and elimination from the blood and tumor tissue were studied in ICR mice. The VLP-TNF-α preparation exhibited higher antitumor activity compared to free TNF-α, presumably due to longer circulation of the cytokine in the blood and its more intensive accumulation by tumor tissue.

Cytotoxic properties of a liposomal form of the HLDF6 hexapeptide, representing an HL-60 cell differentiation factor fragment, have been studied on a murine primary lymphosarcoma cell culture. It is established that the liposomal HLDF6 peptide is capable of inhibiting proliferation and enhancing death of the cells of both LS and RLS lymphosarcoma strains distinguished by their sensitivity to cytostatic agents. The effect of the preparation is determined by its antiproliferative and apoptogenic actions on the cells. Free HLDF6 peptide showed a lower cytotoxic activity with respect to the tumor cells as compared to the liposomal preparation.

The recombinant attenuated strain Salmonella enteritidis E23/ pcDNA-TCI which carrying DNAvaccine containing gene of CTL-polyepitope HIV-1 immunogen under the control of CMV promoter was constructed. The strain was used for perrectal immunizations of BALB/c mice. Detected after immunization in sera of immunized mice HIV-specific antibodies were demonstrated to recognize both recombinant and native HIV-1 antigens. Double immunization was more effective compared to unitary injection. The results obtained showed that recombinant attenuated Salmonella enteritidis E23/pcDNA-TCI could be perspective for delivery the candidate HIV-1 DNA vaccine.

Studying pharmacokinetics of virus-like particle (VLP) marked by [gamma-32Р] was performed in non-inbred mice having transplanted solid Erlich carcinoma. VLP preparation was established to quickly eliminate from the blood and accumulate mainly in kidneys, spleen and intestine. The degree of accumulation of VLP in tumor tissue was higher than the preparation of comparison, double-stranded RNA. Preparation was eliminated from the organism of laboratory animals mostly via kidneys and completed by the end of the second day after the administration.