Drug development & registration
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Introduction. Lately, medical services have reported a lot of cases caused by taking Tropicamide alone or with other drugs together. Moreover, it has been declared that the increase in the number of resistance cases to Tropicamide consumption has. Due to those facts, Tropicamide was included in the List of Drugs for Medical Use that should be served by the prescriptions in 2015. However, nowadays in Russia there are many combinations of medicines, for instance, Tropicamide and α-adrenergic agonist (phenylephrine) (Midrimax, Fenikamid, Appamide plus) that are not under that regulation. As a result, those medicines are served in pharmacies without any prescriptions. Thus, method developing for Tropicamide determination in the hair samples to establish his consumption period has become a perspective one.Aim. The research aimed to develop a method for the isolation and determination of Tropicamide in the hair samples.Materials and method. Reference standard of Tropicamide was used in this research. The following enzymes – papain, chymopsin, chymotrypsin, and hyaluronidase – were applied in the experiment. To design the long-term consumption of Tropicamide, laboratory animals (Guinea pigs, average masses about 200 – 250 g) with fair and brown nature colour hair were used in this research. The hair of laboratory animals was dyed by professional hair-dye "Estel Professional De Luxe". The following equipment was applied: balance "Sartorius СР224S", pH-meter " FiveEasy ", ball mill Retsch MM-200. The hair samples extracts were analyzed by gas chromatography with mass selective detection (Gas chromatograph model 7890А with mass selective detector model 5977 and MassHunter GC/MS software by Agilent Technologies).Results and discussion. All developed methods of enzymatic hydrolysis (by papain, chymopsin, chymotrypsin, and hyaluronidase) revealed comparable results for the Tropicamide determination in the hair samples. The research showed that the amount of the analyte isolated from the pigmented hair was a bit higher in comparison with the other hair samples (fair hair), despite the melanin gives chemical steadiness property to hair stuff. Moreover, the amount of Tropicamide extracted from the dyed hair samples increased by 30 %. The degradation products of the analyte of interest were not found in the extracts obtained for the dyed hair samples. Thus, the colorant does not destroy the xenobiotic during the hair dying procedure and does not impact the enzymatic hydrolysis process. The values of the validation parameters (precision and accuracy) met the required criteria for bioanalytical methods. Therefore, the enzymatic hydrolysis method can be recommended for application in laboratory practice.Conclusion. In the course of the study, a method for laboratory diagnostics of non-drug use of tropicamide was developed, the reproducibility of which meets the acceptance criteria for bioanalytical methods, which makes it possible to recommend it for work in laboratory practice.

Introduction. The search for and development of new drugs capable of reducing the severity of neurological deficit in traumatic brain injury are a critical task for investigational pharmacology. Chromone-containing allylmorpholines are a new group of neuroprotective drug candidates that have been shown to inhibit acetylcholinesterase and butyrylcholinesterase, and block N-methyl-D-aspartate receptors in vitro.Aim. This study aimed to evaluate the neuroprotective activity of the allylmorpholine derivative (E)-4-[3-(8-bromo-6-methyl-4-oxo-4H-chromen- 3-yl)-1-cyclohexylallyl]morpholin-4-ium chloride (33b) in vivo using a rat model of traumatic brain injury.Materials and methods. Traumatic brain injury was induced using the controlled cortical impact model. The allylmorpholine derivative was administered intraperitoneally at 1, 10, or 50 mg × kg-1 b.w. at 1 h after trauma induction, and then daily for the next 6 d. The neurological deficit was assessed using the Limb Placing, Open Field, Elevated Plus Maze, Beam Walking, and Cylinder tests.Results and discussion. At all doses administered, the allylmorpholine derivative had no positive effect on the motor function or exploratory behavior following traumatic brain injury. In the Elevated Plus Maze, 10 mg × kg-1 b.w. of the compound further suppressed exploratory behaviour in the injured animals, which appears to be consistent with its sedative properties observed previously in zebrafish.Conclusion. Despite the previously described in vitro affinity of allylmorpholines towards several molecular targets crucial for the pathogenesis of brain trauma and posttraumatic functional recovery, an allylmorpholine derivative had no neuroprotective effect in a rat model of traumatic brain injury in this study. These results further emphasize the importance of in vivo evaluation of potential neuroprotective drug candidates.

Introduction. Alopecia is a polyetiological disorder characterized by hair loss and reducing their number per unit area. Baldness causes psychological and social discomfort to patients, in connection with what an important task is to develop formulations that are more effective than the reference agents.Aim. Investigate the possibility of applying the original substance Y in several dosage forms for the treatment of alopecia in comparison with reference drugs: minoxidil and burdock oil.Materials and methods. The research subject was the original substance Y, for which several dosage forms were made: gel, alcohol and oil compositions. The study on the effectiveness and safety of the developed formulations was carried out on 9 groups of male C57BL/6 mice. Depilation with further assessment of the percentage of hair follicles in the growth and resting phases was tested as a pre-clinical model of alopecia. In the study of the mechanism of action of substance Y, chemiluminescent assay was performed compared with natural antioxidant quercetin in the system luminol – 2,2'-azo-bis(2-amidinopropane)dihydrochloride, in potassium-phosphate buffer medium (pH = 7.4). Statistical processing of the results was carried out using two-way ANOVA using GraphPad Prism 8.0.2, USA software at the level of statistical significance of differences p < 0.05 and p < 0.005.Results and discussion. Based on the results of histological analysis and visual changes, it was found that the effectiveness of the topical forms of substance Y decreases in the following order: gel, alcohol form, oil composition. The use of a combination of the gel base with the test substance Y resulted to the appearance of a larger number of hair follicles in the growth phase than when using the reference preparation – 2 % minoxidil solution (the differences are statistically significant). Chemiluminescent assessment of antioxidant activity showed the lack of antioxidant effect in substance Y.Conclusion. The study combines two pharmaceutical profiles: technological and pharmacological. In the course of the experiments, the prospects of the gel form of the original substance Y for topical therapy of alopecia were shown. In the near future, it is planned to study the mechanism of action of substance Y, as well as registration of patent protection for a new drug.

Introduction. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and non-alcoholic steatohepatitis is the second most common cause of liver transplantation in the adult population. An urgent task is to find and develop an optimal model of NAFLD in laboratory animals, which would reproduce all the features of this disease in the clinic.Aim. Modification of the NAFLD model in laboratory animals (rats), which allows the obtained data to be transmitted to humans as fully as possible.Materials and methods. The study was conducted on 52 outbred white male rats of the same age. As the basis of the model, a hypercaloric high-fat diet was used with the addition of food appeal enhancers (sodium glutamate and liquid shrimp extract) and for the first-time conditions of hypodynamia were used – restriction of the motor activity of animals using specially designed cells, in which an individual 11 × 18 cm cell was allocated for each individual. The duration of the study was 12 months. In the course of the experiment, body weight, physical performance, biochemical parameters of blood serum and urine in dynamics were assessed, and lethality was recorded. After the end of the study, the mass of internal organs, visceral and epididymal fat was analyzed, and a histological examination of the liver was performed.Results and discussion. In the course of the experimental study, the development of NAFLD in rats of the control group of animals was histologically confirmed. A high mortality rate was revealed in the group of animals with pathology. Compared with animals of the intact group, a statistically significant increase in their body weight, liver weight, visceral and epididymal fat, a decrease in physical performance, disturbances in lipid, carbohydrate and protein metabolism were revealed, as well as signs of deterioration of the protein synthesis and excretory functions of the liver.Conclusion. A number of advantages of the NAFLD model with a combination of a hypercaloric diet and hypodynamic conditions were revealed, including the similarity of the conditions for the formation and pathogenesis of the disease in experimental animals and humans, which ensures the adequacy of data translation from preclinical practice to clinical practice.

Introduction. Non-alcoholic fatty liver disease is one of the most common chronic diseases of this parenchymal organ among the adult population. The search and creation of supporting drugs is an urgent task of modern pharmacy. The malonic acid derivative, sodium 4,4'-(propanediamido) dibenzoate, synthesized by the employees of the Department of Organic Chemistry of the SPСPU, has antisteatous activity, is a potential agent for the treatment of liver diseases. Sustained release tablets were prepared based on sodium 4,4'-(propanediamido)dibenzoate. An integral part of the pharmaceutical development of a medicinal product is the development of a method for conducting the Dissolution test and the selection of optimal conditions, which became the purpose of this study.Aim. To develop the "Dissolution" test method for the sustained-release tablets based on sodium 4,4'-(propanediamido)dibenzoate.Materials and methods. The objects of research are the active pharmaceutical ingredient sodium 4,4'-(propanediamido)dibenzoate, as well as sustained-release tablets based on this substance. Equilibrium biopharmaceutical solubility was determined by UV-spectrophotometry. To establish the conditions for the "Dissolution" test, an ERWEKA DT-620 dissolution tester (ERWEKA GmbH, Germany) was used.Results and discussion. The suitability of the UV-spectrophotometry method for the quantitative determination of sodium 4,4'-(propanediamido) dibenzoate in solutions was determined. The established high biopharmaceutical solubility of sodium 4,4'-(propanediamido)dibenzoate in a buffer solution with a pH of 6,8, as well as in a 0,01 M solution of hydrochloric acid with a pH of 2,6, determined the choice of these media for the "Dissolution" test of the dosage form. The apparatus "Rotating basket" (rotation speed of 100 rpm in a dissolution medium with a volume of 1000 ml) was reasonably chosen for the test on the basis of the obtained linear dependence of the rate of release of the substance on time, as well as the best test results by the end of the experiment.Conclusion. A study of the biopharmaceutical properties of the original substance with antisteatous activity has been carried out. High biopharmaceutical solubility was established in media with pH 2,6 and pH 6,8. The conditions of the "Dissolution" test for sustained-release tablets based on the original sodium 4,4'-(propanediamido)dibenzoate were experimentally substantiated.

Introduction. The article presents the results of the detection of pharmacologically active secondary metabolites in black crowberry Empetrum nigrum L. using the method of high performance thin layer chromatography (HPTLC).Aim. To show the efficiency of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites in promising medicinal plant species.Materials and methods. HPTLC analysis was carried out on a CAMAG device (Switzerland), using MERCK HPTLC silica gel 60 F154, 20 × 10 cm plates. For the evaporation of the samples, a Heidolph vacuum rotary evaporator (Germany) was used. The aerial parts (shoots) of Empetrum nigrum were harvested next to St. Petersburg State Chemical and Pharmaceutical University (SPCPU) nursery garden of medicinal plants (Leningrad Region, Vsevolozhsky District, Priozerskoe Highway, 38 km) in August 2019.Results and discussion. In the course of the research, four fractions from the aerial parts of Empetrum nigrum were obtained: hexane, dichloromethane, butanol, and water. Then, these fractions were investigated by HPTLC in two solvent systems – n-butanol : acetic acid : water (BAW) (4 : 1 : 2) and hexane : dichloromethane : methanol (HDM) (1 : 2 : 0.5). After scanning densitometric analysis of the plates eluted in the HDM system, it was revealed, that the hexane and dichloromethane fractions have a similar composition and contain the greatest amount of compounds, compared to the butanol and water fractions, and in the BAW system, it was found, that the butanol fraction contains the greatest variety of metabolites. As a result of UV spectroscopy, it was found, that the main groups of compounds contained in the hexane and dichloromethane fractions are derivatives of chalcones, dihydrochalcones, bibenzyls and 9,10-dihydrophenanthrenes. While in the butanol fraction, the main groups of secondary metabolites were derivatives of flavonoids and tanninsConclusion. The data obtained allow us to note the efficiency, speed and simplicity of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites of promising medicinal plant species.

Introduction. A number of studies have shown that various genetic and environmental factors can affect the biosynthesis and accumulation of secondary metabolites. In particular, it is known that the local geoclimate, seasonal changes, external conditions such as light, temperature, moisture and soil fertility can affect the chemical composition and, as a result, the therapeutic properties of plants used in the pharmaceutical and food industries. Empetrum nigrum L. is a rich source of various pharmacologically active secondary metabolites – chalcones, dihydrochalcones, bibenzyls, 9,10-dihydrophenanthrenes, flavonoids, and proanthocyanidins. In the scientific literature, there is no data on the variation in the chemical composition of E. nigrum depending on the growing area. The obtained data are necessary for a reasonable choice of the collecting location for the plant, with the aim of its further chemical and pharmacological research for the development of promising drug candidates.Aim. To carry out a comparative analysis of secondary metabolites composition in the aerial parts of Empetrum nigrum growing in different regions of the Russian Federation.Materials and methods. Samples collected in three different areas were used to compare HPLC profiles: sample 1 was collected next to SPCPU nursery garden of medicinal plants (Leningrad region, Vsevolozhsky district, Priozerskoe highway, 38 km) in July 2020, sample 2 was collected on the Kola Peninsula, around the Khibiny mountains in July 2020, sample 3 was collected on the Kamchatka Peninsula, next to Khalaktyrsky beach (Pacific Ocean coast) in July 2020. Extracts were analyzed by analytical high performance liquid chromatography (HPLC) using a Prominence LC-20 device (Shimadzu, Japan) equipped with a diode array detector.Results and discussion. As a result of the research, for the first time, a significant variation in the qualitative chemical composition in the aerial parts of Empetrum nigrum growing in different regions of Russian Federation was established. Sample 3, collected on the Kamchatka Peninsula, in comparison with samples 1 and 2, contain the greatest variety of polar secondary metabolites belonging to the classes of flavonoids, tannins, and phenol carboxylic acids, while in the shoots collected in the Leningrad region, the major metabolites were weakly polar compounds belonging to the classes of chalcones, dihydrochalcones, bibenzyls, and 9,10-dihydrophenanthrenes, and in sample 2, collected in the Khibiny mountains, the lowest qualitative content of secondary metabolites was found. This variation may be caused by various environmental factors (biotic and abiotic).Conclusion. For the first time, the comparison of HPLC profiles of aerial part samples of E. nigrum, collected in different regions of the Russia Federation has been carried out. As a result, significant variations in the secondary metabolites composition of the studied samples were established, depending on the regions and growing conditions of the plants. The data obtained can be used for a reasonable choice of the collection location for the plant, with the aim of its further chemical and pharmacological research for the development of promising drug candidates.

Introduction. Type 2 diabetes mellitus is currently considered one of the most common non-communicable diseases. For the prevention and concomitant treatment of this pathology, various herbal remedies are successfully used, such as, for example, blueberry shoots. The plant contains phenolic compounds (anthocyanins, flavonoids, phenolcarboxylic and organic acids), which have antioxidant and hypoglycemic effects, and also accumulates macro- and microelements (Ca, Mg, Zn, Mn), which in turn can affect the course of diabetes mellitus. Complexes of elements with phenolic biological active substances (BAS) can affect the formation of a pharmacological response or change its severity. Therefore, it is possible to put forward a hypothesis about the potentiation of the antidiabetic action of phenolic compounds when they exist in the form of mineral complexes.Aim. To carry out a comparative assessment of the antidiabetic activity of the mineral complex rutin with zinc in comparison with precursor substances and extraction from blueberry shoots to predict the effect of elements on the course of this pathology.Materials and methods. The objects of the study were an aqueous solution (C = 0.18 mg/ml) of a model complex of rutin with zinc with a molar ratio of components of 1 : 1 and blueberry shoots purchased from a pharmacy in St. Petersburg. According to the information on the packaging, the region of raw material procurement is Altai Territory, Barnaul, the period for harvesting blueberries is July 2020. The complex of rutin with zinc was obtained according to the method described in the literature from the pharmaceutical substance rutin (Rutin, batch 332, valid until 26.03.2023, Sichuan Guangsong Pharmaceutical Co., Ltd., China, FS 000569-060514) and an aqueous solution (С = 0.13 mg/ml) zinc chloride (Zinc chloride, batch 39/G 4, valid until 09.10.2021, Neva Reaktiv, Russia, STP TU COMP 1-533-2012). The optimal ratio of components 1 : 1 for the formation of a mineral complex was established by us earlier experimentally using the Job's method. The mass of zinc chloride, which must be added to the extraction, and the mass of the complex for the preparation of its aqueous solution were calculated on the basis of the quantitative content of biologically active substances in blueberry shoots and the molar ratio of the components involved in the formation of the complex compound determined by the spectral method. The quantitative content of the main groups of biologically active substances (flavonoids, hydroxycinnamic acids, organic acids) was determined spectrophotometrically on SF-2000 instrument (Russia) and titrimetrically using the methods presented in Russian Pharmacopoeia XIV FS.2.5.0093.18 and FS.2.5.0012.15. The antidiabetic effect of the complex of rutin with zinc was evaluated in comparison with an aqueous extract from the shoots of common blueberries (the ratio of raw materials: extractant – 1 : 80), an aqueous solution of zinc chloride (concentration – 0.36 mg/ml) and their mixture (ratio 1 : 1) on a model of dexamethasone-induced type 2 diabetes mellitus in laboratory animals. Determination of blood glucose concentration was carried out using a portable glucometer "AccuChek Active" (Roche Diabetes Care, Germany). The presence of glucosuria and ketonuria was established using Ketoglyuk-1 test strips (Biosensor AN, Russia). Statistical processing of the results was carried out using the Microsoft Excel program according to OFS.1.1.0013.15.Results and discussion. The results of the content of glucose and ketones in urine showed that in the process of modeling diabetes mellitus, glucosuria and ketonuria are characteristic for all groups of animals, which confirmed the formation of a pathological process in rats. A lower content of glucose and ketones in urine was found in a group of animals that received a complex of rutin with zinc (glucose concentration – less than 2.3 mmol/l, ketone concentration – less than 0.2 mmol/l). Measurement of the concentration of glucose in the blood showed that the complex of rutin with zinc has the most pronounced hypoglycemic effect, while the water extract from blueberry shoots and an aqueous solution of zinc chloride have a minimal antidiabetic effect (blood glucose is 6.9 mmol/l versus 8,1 mmol/l and 7.9 mmol/l, respectively).Conclusion. The study of the influence of elements in the composition of phenolic complexes on the course of diabetes mellitus showed that the introduction of minerals has a positive effect on the severity of the pharmacological effect, which can serve as confirmation of the hypothesis about the potentiation of the antidiabetic effect of phenolic compounds when they exist in the form of mineral complexes. Thus, the complex of rutin with zinc showed the maximum activity in comparison with the solution of zinc chloride, extraction from blueberry shoots and their mixture, which suggests that the form of existence of natural phenolic compounds in the composition of mineral complexes is optimal both from the point of view of accumulation and from the side manifestations of a pharmacological response. The results of the study create the preconditions for further study of the effect of various elements in combination with marker phenolic components of antidiabetic herbal preparations on the course of diabetes mellitus, and also make it possible to conclude that natural mineral complexes have a prophylactic effect against this pathology.

Introduction. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. Non-alcoholic steatohepatitis (NASH), a clinically progressive morphological form of NAFLD, ranks second in the list of reasons for liver transplantation in the adult population. In the pathogenesis of this disease, metabolism and distribution of free fatty acids (FFA) play an important role. A large number of studies have established that the level of FFA in peripheral blood directly correlates with the severity of NASH, but it is still unclear what effect fluctuations in the profile of fatty acids (FA) in the liver have in steatohepatitis.Aim. Study of changes in the profile of fatty acids in the liver of laboratory animals with experimental non-alcoholic steatohepatitis.Materials and methods. The study was carried out on 17 white outbred male rats, which were randomized into two groups – intact (n = 6) and control (steatohepatitis) (n = 11). Steatohepatitis was modeled by 12-month use of a hypercaloric high-fat diet against the background of hypodynamia. The content of fatty acids in the liver was determined in the reaction of methanolysis and extraction with a hexane mixture of their methyl esters. The LC was separated by gas chromatography-mass spectrometry. Calibration for quantitative calculation was carried out with deuterated tridecanoic acid. The content of saturated and monounsaturated higher FAs, their aldehydes and hydroxy derivatives, as well as sterols were studied.Results and discussion. A total decrease in the content of FFA in the liver of animals with steatohepatitis was revealed. The most significant decrease occurred mainly in the class of monounsaturated fatty acids and cholesterol. Also, a significant decrease in the activity of Δ9-desaturase, a key enzyme in the synthesis of monounsaturated FAs from their precursor with the same carbon chain length, was revealed, which was manifested by a significant decrease in their amount in the liver. There were no statistically significant changes in the levels of aldehydes and hydroxy acids between the study groups, as well as in the level of sterols (except for cholesterol, the content of which decreased significantly).Conclusion. Thus, in the liver of rats with steatohepatitis caused by a combination of a hypercaloric diet and hypodynamia, statistically significant changes in the profile and concentration of fatty acids were found in comparison with healthy animals. The demonstrated shifts in FA composition may reflect both adaptive and pathological changes in the liver of animals with NAFLD and require further study.

Introduction. Selenopyran is an organic selenium compound with sharply hydrophobic properties. An increase in solubility in water (and as a consequence – and bioavailability) is possible due to the formation of inclusion complexes with cyclodextrins.Aim. The aim of this work was to study the effect of a gel containing a clathrate complex of selenopyran with β-cyclodextrin on the rate of wound healing on a model of a conditionally aseptic full-thickness planar wound in rats.Materials and methods. The object of the study was a gel containing a clathrate complex of selenopyran with β-cyclodextrin (the content of selenopyran in the gel was 0.1 %). A model of a full-thickness planar wound in sexually mature male rats was used. 20 individuals were divided into 2 groups – intact (without treatment) and experimental (received gel treatment). Efficacy was assessed by the change in wound area at 3, 5, 7, 9, 11 and 14 days after application of wound.Results and discussion. The results of the study showed that the relative area of the wounds in the treated animals by the 3rd day of the experiment was less than in the intact ones. On the fifth day of the experiment, the differences were statistically significant (57.49 ± 12.51 % in treated animals versus 85.27 ± 26.61 % in intact animals). By the 14th day of the experiment, there were practically no differences in the groups of animals.Conclusion. The results obtained indicate that when using a gel containing selenopyran in combination with β-cyclodextrin, it accelerates the transition from the inflammation phase to the proliferation phase. This is most likely due to the antioxidant properties of selenopyran. Considering the lower concentration of selenopyran in comparison with the therapeutic concentrations of other antioxidants (taurine, allantoin), it can be considered as a promising wound healing agent for further study.