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Vol 23, No 9 (2020)


Development of standardization approaches for recombinant endolysin LYSECD7-based pharmaceutical substance

Antonova N.P., Vasina D.V., Balabanyan V.U., Gushchin V.A.


Relevance. Lytic bacteriophages endolysins represent one of the most promising classes of antibacterial agents in the post-antibiotic era. Since this class of antibacterials is quite new there is an urgent need for development of approaches to the endolysins pharmaceutical substances standardization. Objective of the study was to develop the approaches to the recombinant endolysin LysECD7 pharmaceutical substance standardization. Materials and methods. The identity and purity of the endolysin pharmaceutical substance was assessed by SDS-PAGE in 16% polyacrylamide gel. The specific antibacterial activity of the LysECD7 pharmaceutical substance was determined using a novel microbiological method counting the CFU reduction of the Acinetobacter baumannii Ts 50-16 test strain after the endolysin exposure. The sterility of the LysECD7 pharmaceutical substance was determined by the membrane filtration method. The bacterial endotoxins level was determined by a chromogenic endpoint test. The quantity of the protein in the pharmaceutical substance was determined by the colorimetric method with bicinchoninic acid. Results. During the work the approaches to the standardization of the endolysin LysECD7 pharmaceutical substance were developed including the basic quality attributes such as "Electrophoresis in polyacrylamide gel" and "Specific activity" to confirm the identity, "Sterility", "Bacterial endotoxins" and "Quantification". Conclusions. Approaches to the standardization of the LysECD7 endolysin pharmaceutical substance were developed including such quality attributes as "Identity", "Purity" and "Quantification", which allow to reach the stable efficiency and safety of the pharmaceutical substance aiming to develop innovative effective antibacterial drugs.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):3-8
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A comparative analysis of the content of sugars and flavonoids in the hairy roots of sunflower Heliantus annuus L

Yakupova A.B., Musin K.G., Kuluev B.R.


Relevance. Sunflower Heliantus annuus L. roots are used in the treatment of urolithiasis and cholelithiasis. Harvesting of sunflower roots for medical purposes is fraught with a number of problems, of which the impossibility of year-round cultivation, the absence of devices and machines for collecting roots, and contamination of roots with pesticides are most relevant. In this regard, it is of great interest to obtain hairy roots of sunflower that can grow on hormone-free nutrient media in isolated cultures. The aim of our work was to create hairy roots of sunflower using strains A4 and 15834 of Agrobacterium rhizogenes, evaluate their growth parameters when grown on solid media, and analyze their content of water-soluble sugars and flavonoids. Material and methods. The study revealed greater efficiency of strain A4 than strain 15834 for Agrobacterium-mediated transformation of sunflower cotyledon explants. The content of sugars and flavonoids was measured spectrophotometrically using alcohol extracts. Results. The content of water-soluble sugars in the hairy root culture obtained using strain 15834 of A. rhizogenes was more than 2 times higher than that in the hairy roots obtained using strain A4 of A. rhizogenes. Naturally grown sunflower roots contained fewer water-soluble sugars than hairy roots. The total content of flavonoids in sunflower hairy root cultures obtained using strains A4 and 15834 of A. rhizogenes did not differ significantly, but on average it was 2.7 times higher than in native sunflower roots. Conclusion. The hairy roots of sunflower were obtained by agrobacterial transformation of cotyledon explants in vitro. The resulting hairy roots are characterized by unrestricted growth on hormone-free media and a higher water-soluble sugar content than native plant roots.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):9-18
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Comparative study of Pinus pumila (Pall.) Regel needles essential oils from Baikal and Yakut populations

Erdyneeva S.A., Shiretorova V.G., Taraskin V.V., Radnaeva L.D.


Aim of the research. Comparative study of the composition of essential oil obtained from Pinus pumila (Pall.) Regel needles from the Pribaikalsky and Yakutsky regions populations. Materials and methods. The objects of studying were Pinus pumila (Pall.) Regel needles growing within Buryatia and Sacha (Yakutia) territory, collected during July-August, 2019. Essential oils from the pine needles were obtained using commonly used method - hydrodistillation in Clevenger apparatus. The content of essential oil was determined using volumetric method. The qualitative composition and relative quantitative content of the essential oils components were determined by gas-chromato-mass spectrometric method. Results. The yield of essential oils was 1.95-3.20%, calculated to air-dried raw material. The dominant components of essential oils from Pinus pumila were monoterpenoids: a-Pinene (20,3-42,2%), Camphene (2,2-4,0%), ß-Pinene (1,4-3,1%), ß-Myrcene (1,2-1,8%), 3-Karen (0,7-10,9%), Limonene (1,8-6,9%), ß-Fellandren (3,6-12,3%), Terpinolene (4,7-8,2%), a-Terpinyl acetate (3,6-7,3%); sesquiterpenoids: caryophyllene (2,7-4,3%), Humulene (1,4-2,5%), Germacrene D (0,7-2,1%), Y-Cadinene (0,6-2,2%), б-Cadinene (2,0-6,3%), Т-Muurolol (1,2-4,4%), a-Cadinol (1,5-4,7); diterpenoid cembrene (0,1-0,6%). Conclusion. The chemical composition of Pinus pumila (Pall.) Regel needles essential oilsgrowingon Baikal and Yakutiya territories was studied. A comparative analysis with literature data for populations of the southern boundary of the range is carried out, the influence of climatic factors on the content of dominant components is shown. Essential oil of cedar dwarf with a high content of a-Pinene is of practical interest for use in therapeutic and prophylactic purposes.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):19-25
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Influence of the process of grinding and anti-friction agents on the technological characteristics of the pharmaceutical substance of an antihelmint appointment based on the vinyl pyridine series copolymer

Bogunova I.V., Raspopina A.A., Panov A.V., Kedik S.A.


Relevance. The search for new anthelmintic agents with low toxicity and the absence of serious side effects is an urgent problem today. The vinylpyridine copolymer belongs to the V class of «Almost non-toxic» drugs according to the Hodge and Sterner toxicity scale, and to the results of studies it exerts a high anthelmintic activity. The identified unsatisfactory technological characteristics are a significant but solvable problem for the development of solid dosage forms based on a vinyl pyridine copolymer. Purpose. To study the effect of grinding and mixing processes, using special laboratory equipment and the influence of antifriction agents on the technological characteristics of the copolymer. Materials and methods. The substance is avinylpyridine copolymer. The copolymer was milled using a laboratory mill-mixer, ball and disk mills. Mixing was carried out using a V-shaped mixer and a cyclic laboratory mill. Particle size range and fractional composition were determined according to the methods described in Russia State Pharmacopoeia XIV edition (GMP., GMP. 1.1.0015.15). The equipment used: MICROMED 1 VAR 3-20microscope, a digital camera with a «TOUPCAM» 9.0MP video eyepiece and «Toup View» control software, a set of sieves with mesh sizes 1.0; 0.3 and 0.2 mm respectfully. The effect of antifriction substances on the copolymer was studied using magnesium stearate, stearic acid, sodium stearyl fumarate, talc and colloidal silicon dioxide. Assessment of the technological characteristics of the mixtures was carried out according to standard methods described in Russia State Pharmacopoeia XIV edition (GMP. The «ERWEKA GTL» powder flow tester was used, the angle of repose was measured using a goniometer, and the bulk density before and after compaction was determined using the «ERWEKA SWM» tester and the «CASIO FX-82ES PLUS» electronic calculator. Results. The usage of laboratory shredders and mixers independently did not give satisfactory results, as did the introduction of individual antifriction agents into the composition of the mixtures. Introduction of a combination of antifriction agents (talc and stearyl fumarate) into the experimental mixture as well as carrying out the grinding and mixing processes in a cyclic laboratory mill optimized the technological properties of the copolymer. Conclusions. The influence of grinding and mixing processes, using special laboratory equipment, and of antifriction agents on the technological characteristics of the copolymer was studied. Antifriction substances and equipment were selected and the technology of mixing and grinding processes for the further development of solid dosage forms was determined.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):26-31
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Pharmacokinetic modeling in dynamic scintigraphy of the hepatobiliary system

Matveev A.V.


Relevance. Pharmacokinetic modeling is one of the main methods for evaluating the results of radionuclide studies. It can be used to track changes in scintigraphic images over time to evaluate the functions of the hepatobiliary system, as well as to calculate the corresponding quantitative indicators that characterize the various functions of the organs and tissues under study. The complexity of such modeling consists in two aspects. On the one hand, the excessive simplification of anatomical and physiological features of the body when dividing it into kinetic compartments, which can lead to the loss or distortion of important diagnostic information, on the other -in excessive consideration of all possible relationships of the functioning of organs and systems, which, on the contrary, will lead to the appearance of an excessive amount of absolutely useless for clinical interpretation of mathematical data, or the model becomes completely unsolvable. Purpose of work. To develop the compartmental model of kinetics of radiopharmaceutical in the human body in the study of the hepatobiliary system using the dynamic scintigraphy method. Material and methods. The work is based on the principles and methods of pharmacokinetics of drugs (compartmental modeling). To identify the parameters of the pharmacokinetic model, we used the results of dynamic scintigraphy of the hepatobiliary system of a patient with choledocholithiasis before and after endoscopic papillosphincterotomy. Results. Various methods of identification of model parameters based on quantitative data of hepatobiliscintigraphy are proposed. The results of pharmacokinetic modeling for dynamic scintigraphy of the hepatobiliary system in cases of non-visualizing gallbladder (four-compartment model) and visualizing gallbladder with stimulation of its emptying (five-compartment model) are presented and analyzed. Conclusions. From the comparative analysis of model curves "Activity-time" for different zones of interest the time of stimulation of gallbladder emptying is justified, which normally should be 35-40 minutes from the beginning of the study.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):32-41
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Study of the involvement of AMPA receptors in the antihypoxic effect of the neuropeptide cyclo-prolyl-glycine

Koliasnikova K.N., Alyaeva A.G., Vorontsova O.N., Gudasheva T.A.


Relevance. Cyclo-prolyl-glycine (CPG) was designed and synthesized at the Zakusov Research Institute of Pharmacology as a topological analogue of the nootropic drug piracetam and was later discovered as an endogenous compound in the rat brain. It had a spectrum of pharmacological characteristics of piracetam, including antihypoxic activity. However, there is still no evident data of the mechanism of action of this neuropeptide. Therefore, clarification of the mechanism of action of CPG is relevant. Objective. Purpose of the study is to reveal the involvement of AMPA receptors in the antihypoxic effect of the neuropeptide CPG. Material and methods. The method of inhibitory pharmacological analysis using a selective antagonist of AMPA receptors of the compound DNQX was used. Antihypoxic activity was evaluated on a normobaric hypoxia model with hypercapnia in BALB/c mice. Results. The antihypoxic effect of CPG at the dose of 1 mg/kg intraperitoneally was completely removed by a specific blocker of AMPA receptors, compound DNQX, at the dose of 10 mg/kg intraperitoneally. Conclusions. For the first time it was shown that AMPA receptors are involved in the antihypoxic effect of the neuropeptide cyclo-prolyl-glycine.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):42-45
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Antioxidant effects of 11-deoxymisoprostol in experimental liver damage with carbon tetrachloride

Kataeva R.M., Agletdinov E.F., Kataev V.A., Gizatullin T.R.


Relevance. The development of new effective antioxidant drugs, the identification and specification of the properties of antioxidants in drugs of other pharmacological groups remains relevant. The substance studied in this work, ethyl ester of (±) -11,15-dideoxy-16-methyl-16-hydroxyprostaglandin E1 (11-deoxymisoprostol), is considered as a promising drug with a broad spectrum of pharmacological activity and antioxidant properties. Objective. Evaluation of the antioxidant activity of 11-deoxymisoprostol in tissues, blood and erythrocytes during the induction of free radical pathology by experimental liver damage with carbon tetrachloride. Material and methods. The study was carried out on 40 male rats. An experimental model of liver damage with carbon tetrachloride (single intraperitoneal administration of CCl4 at a dose of 0.5 ml / kg) was used to induce free radical pathology. Animals of the experimental group and the comparison group in the next 72 hours intragastrically, daily at a dose of 1 mg / kg, received 11-deoxyimisoprostol and the reference drug (misoprostol), respectively. The control animals received equivalent volumes of the solvent at the same rate. The levels of primary, secondary and final products of lipid peroxidation, products of oxidative modification of proteins, activity of glutathione peroxidase, superoxide dismutase, and catalase were determined in blood and liver homogenates. Results. The three-day administration of 11-deoxyimisoprostol, like the reference drug, did not prevent the development of ССЦ-dependent oxidative stress, but significantly limited its manifestations and modified its character, eliminating the phenomena of carbonyl stress. In terms of the severity of the effect, 11-deoxyimisoprostol was superior to the comparison drug (redistribution of the balance of products oxidative modification of proteins with restoration of the control values of the reserve-adaptive potential and preservation of catalase activity in the liver). Conclusions. Based on the results of the study, it is possible to draw unambiguous conclusions that 11- deoxyimisoprostol has antioxidant properties that are not inferior in severity to the reference drug.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):46-52
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Biochemical markers of liver damage with a fructose-induced diet in rats

Gileva O.G.


Relevance. The increased content of fructose as a common source of carbohydrates in foods leads to metabolic disorders in the body, which may be due to biochemical characteristics of fructose metabolism. The result is an increased risk of obesity, diabetes mellitus, cardiovascular diseases, as well as liver diseases induced by the formation of oxidative stress amid changes in lipid and carbohydrate metabolism. The destructive processes in the liver may be indicated by an increase in the content of the main markers of hepatocyte cytolysis: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH). Objective. To create an experimental model of the development of inflammatory processes in the liver under the influence of a fructose-induced diet. Material and methods. The study was conducted on 50 male rats with a body weight of 250-300 g. Animals were divided into experimental and control groups of 25 animals each. Rats of the control group received a balanced diet of vivarium, animals of the experimental group received a diet containing 60% fructose of daily calories. On the 60th day, the formation of metabolic deviations in the long-term was evaluated. In the obtained serum, the content of glucose, cholesterol (cholesterol), low-density lipoproteins (LDL), triglycerides (TG), insulin, the activity of ALT, AST, alkaline phosphatase, LDH were studied. Biochemical studies were carried out on an automatic analyzer AU-480 (Beckman Coulter, USA) using reagents of the same manufacturer. The insulin content was determined on an enzyme-linked immunosorbent analyzer StatFax-2100 (Awareness Technology, USA) using a set of reagents LLC Vector-best, Russia. Indices of insulin resistance were calculated. Results. During the study, there was a tendency to an increase in glucose concentration with a significant increase on the 21st and 60th day of the experiment by 1.18 and 1.21 times, respectively, and cholesterol on the 35th day of the experiment by 1.59 times relative to the control in rat blood serum. The insulin concentration significantly increased throughout the experiment, exceeding the control values by 1.5, 1.7, and 1.9 times on the 21st, 35th, and 60th days of the experiment. The insulin resistance indices also differed from the control values, which may indicate the formation of insulin resistance. The LDL content was statistically significantly increased throughout the experiment. During the experiment, there was an increase in the activity of hepatocyte cytolysis markers with a significant increase compared with the control. A decrease in the de Ritis coefficient confirms the activation of cytolytic processes in the liver. Conclusion. Thus, a fructose-induced diet increases the content of specific rat liver enzymes (ALT, AST, ALP, LDH) against the background of disturbances in lipid and carbohydrate metabolism with increased concentrations of cholesterol, triglycerides, LDL, as well as glucose and insulin, the formation of insulin resistance. The reason for the initiation of cytolytic processes in hepatocytes may be free-radical oxidation of protein-lipid complexes and activation of inflammatory reactions as a result of increased consumption of fructose.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(9):53-59
pages 53-59 views

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