The development of a rational dosage form for an API is one of the main links in the chain of comprehensive research to create a drug. The purpose of this study was to use physicochemical and technological methods to create various parenteral forms based on the practically water-insoluble API LCS-1269 and to conduct a comparative study of the developed dosage forms according to the main quality indicators characteristic of injectable dosage forms. Compositions and technologies for producing three model dosage forms based on LCS-1269 were evaluated during the work. Their main advantages and disadvantages were shown.

Разработка рациональной лекарственной формы для активной фармацевтической субстанции (АФС) — одно из основных звеньев в цепи комплексных исследований по созданию лекарственного средства. Цель настоящего исследования — применение физико-химических и технологических приемов для создания различных парентеральных форм на основе практически нерастворимой в воде АФС — ЛХС-1269 и их сравнительное исследование по основным показателям качества, характерным для парентеральных ЛФ. В процессе работы были оценены составы и технологии получения трех моделей ЛФ на основе ЛХС-1269, показаны основные их преимущества и недостатки.

Purpose. Purpose: to study the content of pro-inflammatory cytokines and matrix metalloproteinases in the intraocular fluid in patients with advanced stage of primary open-angle glaucoma. Material and methods. 50 patients were examined with a verified diagnosis of the advanced stage of poag. the control group consisted of 30 patients with a diagnosis of uncomplicated cataract. the concentration of the determined biologically active molecules was carried out by the method of flow fluorimetry on a two-beam laser analyzer -Bio-plex 200, Bio-rad, USA. Results. A statistically significant increase in the concentrations of Il-6, 8, 12, 17, mIp-1β matrix metalloproteinase-2 was found in the intraocular fluid of patients with poag. Conclusion. The data obtained indicate the significance of the local inflammatory process, as well as disturbances in the structure of the extracellular matrix in the mechanisms of poag development. Keywords: primary open-angle glaucoma, intraocular fluid, cytokines, matrix metalloproteinases

The N.N. Blokhin Oncology Research Center synthesized indolo[2,3-a]carbazole N-glycoside, which received the laboratory cipher LСS-1269, which showed a pronounced antitumor effect during research.. During the study of LСS-1269 by HPLC, the presence of two impurities was revealed, called LСS-1269-X and LСS-1269-Y (impurities X and Y). The electronic absorption spectrum (EAS) of the impurity peak X was similar to the EAS of the indolo[2,3-a]carbazole N-glycoside obtained and described earlier under the laboratory cipher LCS-1208, and the EAS of the impurity was the spectrum of LCS–1269, which indicates that the impurities belong to indolo[2,3-a]carbazoles. In this regard, it is suggested about the structure of these impurities: impurity X is a xyloside of aglycone similar in structure to aglycone LCS-1208, which can be formed as a by-product at one of the last stages of the synthesis of LCS-1269, and impurity Y has the structure of triacetyl ester LCS-1269, which is a precursor of LCS-1269 in the synthesis process. The aim of the work was to identify impurities in the substance LHS-1C69, to confirm their structures and belonging to derivatives of indolo[2,3-a]carbazole. The identification of impurities in the substance LCS-1269 was carried out using the HPLC-MSMS method. For this purpose, X and Y impurities having the assumed structures were obtained by directed counter synthesis, which were used as reference samples. Comparison of peak retention times on extracted ion chromatograms (XIC) and MSMS spectra of impurities with XIC and MSMS spectra of samples of comparison of the alleged impurities confirmed their structure. As a result of the work carried out, the structures of impurities X and Y were identified and the affiliation of these impurities to derivatives of indolo[2,3-a]carbazole was confirmed.

Immunohistochemical and ultrastructural analysis revealed signs of structural alterations in neurons and autophagy in all layers of the human retina at the end-stage glaucoma. The most pronounced destructive changes associated with swelling and destruction of mitochondria, endoplasmic reticulum, and Golgi apparatus, as well as structural signs of impaired synaptic activity and apoptosis were noted in ganglion, bipolar, and amacrine neurons. In the structure of photoreceptor cells, alone with destructive processes associated with structural alterations of rods and cones in the outer membrane discs, as well as swelling of organelles, we observed processes aimed at the maintenance of cell homeostasis. Structural signs of autophagy (mainly mitophagy) and changes of the ultrastructural organization in rod neurons were more pronounced than in cones.

Purpose: to study the structural organization of the vascular bed of human retina in the terminal stage of primary open-angle glaucoma (POAG).Material and methods. We performed a comparative immunohistochemical analysis of the content of vessels in the retina of 13 eyes of patients in the terminal stage of POAG, enucleated for medical reasons, and 17 eyes with uveal melanoma, using the markers of blood vessels endothelium CD34. The ultrastructural organization of the interstitium and endothelial cells of retinal microvessels was studied by electron microscopy and morphometry.Results. A significant increase in the volume density of the interstitium and a decrease in the volume density of CD34+-blood vessels in the retina of patients in the terminal stage of POAG, as compared with uveal melanoma, were revealed. An increased volume density of luminal and basal caveolae and the formation of transendothelial channels in the cytoplasm of endotheliocytes of retinal blood capillaries in the terminal stage of POAG were noted.Conclusion. In the terminal stage of POAG, the interstitial spaces of the retina are increased and the volume density of blood vessels is dropping. The increased volume density of luminal and basal caveolae and the formation of transendothelial channels in the cytoplasm of blood capillary endotheliocytes indicate the growth of transcytosis and the permeability of the blood-retinal barrier.

Background: glaucoma is one of the leading causes of blindness worldwide. Diagnosis of glaucoma at an early stage is challenging. Therefore, genetic factors predisposing to the development of primary open-angle glaucoma (POAG) are investigated. One of these predictors is tumor necrosis factor α (TNFα), a multifunctional proinflammatory cytokine involved in glaucoma pathogenesis. Point mutations in the regulatory region of the TNFα gene are hypothesized to be associated with POAG risk. Aim: to analyze the polymorphism of three positions of TNFα gene promoters and their complexes in West Siberian Caucasians with POAG and healthy volunteers. Patients and Methods: the study enrolled 401 individuals, i.e., 99 patients with POAG stage 2 and 302 individuals without POAG (randomized control group). All participants signed an informed consent form. Single nucleotide TNFα gene promoter polymorphism (rs361525, rs1800629, rs1800630) was analyzed. Genotyping was performed by restriction analysis of gene amplification products. Results: the occurrence of minor genotype TNF-308*АА was significantly higher in POAG (odds ratio 11.41, p=0.0011). The occurrence of two other genotypes demonstrated no significant differences between groups. Three complex genotypes were positively associated with POAG (TNF-863*CC:TNF-308*AA, TNF-308*AA:TNF-238*GG и TNF-863*CC:TNF-308*AA:TNF-238*GG). We failed to identify any single nucleotide polymorphism or complexes negatively associated with POAG. Conclusion: minor genotype TNF-308*АА is an essential factor of POAG pathogenesis. Two other polymorphic gene variants were associated with POAG as a part of complex genotypes. These findings demonstrate that potential polymorphic associations should be considered in the case-control analysis. Keywords: primary open-angle glaucoma, polymorphism, TNFα gene, gene promoter, complex genotypes. For citation: Shevchenko A.V., Prokof’ev V.F., Konenkov V.I. et al. Association of TNF-α gene promoter polymorphism with primary open-angle glaucoma. Russian Journal of Clinical Ophthalmology. 2022;22(1):11–15 (in Russ.). DOI: 10.32364/2311-7729-2022-22-1-11-15.

Purpose. To analyze the polymorphism of the regulatory regions VEGFand eNOS genes polymorphism and their combinations in patients with type 2 diabetes with and without non-proliferative DR. Material and methods. The study included 200 patients with type 2 diabetes 111 patients with type 2 diabetes without signs of DR and 89 patients with non-proliferative DR. The polymorphism of the regulatory regions of VEGF(rs699947 and rs3025039) and eNOS(rs2070744) genes was studied using restriction fragment length polymorphism analysis and Real-Time PCR by TaqMan probes. Statistical: used by StatSoft Statistica 10.0, SPSS Statistics 23 and the original programs for volumetric processing of bioinformatics. Results. The polymorphism of the regulatory regions of VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes was studied using restriction fragment length polymorphism analysis and Real-Time PCR by TaqMan. Results: The VEGF-2578heterozygosity and two complex genotypes: VEGF-2578 CA:VEGF+936CC and NOS3-786CT:VEGF-2578CA:VEGF+936CC significantly decreased in patients with DR. Conclusion. The data obtained indicate that the genetic polymorphism of the regulatory regions of the genes analyzed by us can be attributed to risk factors for the development of DR in patients with DM2. A comprehensive analysis of variants of polymorphic gene loci can be used to predict the development of DR. Keywords: non-proliferative retinopathy, VEGF polymorphism, eNOS polymorphism, complex genotypes.

The endothelial NO synthase (eNOS) and vascular endothelial growth factor (VEGF) imbalance and the polymorphism of these genes may be the predisposition for diabetic retinopathy (DR) development and progression.The aim: to analyze VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes polymorphism and their combinations in patients with type 2 diabetes mellitus (DM2) with and without initial non-proliferative DR.Materials and methods. The study included 200 patients with type 2 diabetes (155 women and 45 men, age – 43–70 years): 111 people without and 89 people with DR. The polymorphism of the regulatory regions of VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes was studied using restriction fragment length polymorphism analysis and TaqMan Real-Time PCR by. Statistical processing was carried out using the software packages Statistica 10.0, SPSS Statistics 23 and the package of original programs for volumetric processing of bioinformation.Results. The VEGF-2578 heterozygosity and two complex genotypes – VEGF-2578CA:VEGF+936CC and NOS3-786CT:VEGF-2578CA:VEGF+936CC – signifi cantly decreased in patients with DR. The predisposition to early DR development to minor genotype of eNOS gene in the NOS3-786CC:VEGF+936CT complex and signifi cantly decreased the homozygous wild-type eNOS genotype in DM2 patients with ophthalmopathology were shown. NOS3-86TT:VEGF2578AA genotype signifi cantly decreased in group with retinopathy developing and the glycated hemoglobin high level.Conclusion. Along with the clinical risk factors for the development of DR in DM2, the genetic polymorphism of the regulatory regions of the genes analyzed by us has a signifi cant weight. When analyzing potential genetic markers, it is important to consider possible joint epistatic/hypostatic effects. The complex analysis of polymorphic gene can help early prognosis of the DR development.

Introduction. The search for new antineoplastic agents in a series of indolo[2,3-a]-carbazole derivatives is an urgent and promising direction, since compounds with antitumor activity have been found in this class. In the chemical fusion laboratory, N.N. Blokhin National Medical Research Center оf the Ministry of Health of Russia has developed an original and effective method for the synthesis of glycosides of indolo[2,3-a]-pyrrolo[3,4-c]carbazoles, which makes it possible to synthesize derivatives of N-glycosides of indolo[2,3-a]carbazoles with different substituents in the heterocyclic parts including at the maleimide nitrogen atom and with different carbohydrate residues.The purpose of the study – the primary assessment of the antitumor activity of new derivatives of indolocarbazoles with a carbohydrate residue xylose in models of tumor growth mice.Materials and methods. The compounds studied at transplanted tumors of mice: the Lewis epidermoid carcinoma (LLC), colon cancer ACATOL, cervical cancer RSHM-5, breast adenocarcinoma CA-755. Studies were performed on immunocompetent mice: males and females of BDF1 hybrids (C57Bl/6 × DBA/2), females CBA/Lac and Balb/c. Compound solutions were prepared ex tempore and administered to the mice intraperitoneally at a dose of 60 mg/kg daily for five days. The antitumor effect was evaluated as to of tumor growth inhibition and increase of life span of the treated animals as compared with the control ones.Results. Eight compounds studied, containing D-xylose as a carbohydrate component and various substituents at the maleimide nitrogen atom, showed different degrees of antitumor activity. Two derivatives have been identified: N-[5,7-dioxo-12-(β-D-xylopyranosyl)-indole[2,3-a]pyrrolo[3,4-c]carbazol-6-il]benzamide (compound 4) and N-[5,7-dioxo-12-(β-D-xylopyranosyl)-5,7,12,13-tetrahydro-6H-indole[2,3-a]pyrrolo[3,4-c]carbazole-6-il]pyridin-2-carboxamide (compound 8), which showed high antitumor activity on 4 solid tumors of mice with a duration of effect of 12 days or more. The most pronounced antitumor effect was obtained in compounds 4 and 8 in RSHM-5 and Ca-755, tumor growth inhibition was amounted, respectively: in RSHM-5 – 68–82 % and 80–72 %; for Ca-755 – 57–62 % and 86–68 % (p <0.05).Conclusion. For further research, we chose the compound (N-[5,7-dioxo-12-(β-D-xilopiranosil)-5,7,12,13-tetrahydro-6H-indole[2,3-a] pyrrolo[3,4-c]carbazol-6-il]pyridin-2-carboxamide).

Взаимодействием N-гликозидов индоло[2,3-a]фурано[3,4-c]карбазол-5,7-дионов с гидразингидратом, гидрохлоридом гидроксиламина, формилгидразином получены соответствующие N-6 замещенные гликозиды индолопирролокарбазолов. В качестве углеводных остатков использованы L-арабиноза, D-галактоза, D-ксилоза и D-рибоза. Полученные соединения изучены in vitro и in vivo. Они показали значительную антипролиферативную активность на клетках HCT-116 (IC50 10–6 – 10–7 М) и высокий противоопухолевый эффект на асцитных моделях опухоли Эрлиха и лимфолезкозе Р-388. Увеличение продолжительности жизни животных на первой модели составило от 540 – 495 % до 172 – 132 % и от 80 – 83 % до 120 – 93 % на второй модели.

Reactions of indolo[2,3- a ]furano[3,4- c ]carbazole-5,7-diones with hydrazine hydrate, hydroxylamine hydrochloride, and formyl hydrazine produced the corresponding N -6-substituted indolopyrrolocarbazole glycosides with the carbohydrate residues L-arabinose, D-galactose, D-xylose, and D-ribose. The obtained compounds were studied in vitro and in vivo and showed significant antiproliferative activity against HCT-116 cells (IC 50 = 10 –6 – 10 –7 M) and high antitumor effects on Ehrlich ascites tumor and P-388 lymphocytic leukemia models. The lifespan increase of animals in the first model ranged from 540 – 495% to 172 – 132%; in the second model, from 80 – 83% to 120 – 93%.

Typical blood capillaries and vessels in uveal melanoma were shown and different types of uveal melanoma stromal cells were determined by electron microscopy and immunohistochemical analysis. Macrophages, fibroblasts of varying degrees of differentiation and endothelial-like cells with numerous caveolae in the cytoplasm were found in the channels of the extracellular matrix surrounding accumulations of tumor cells. The presence local structures positively stained for markers of the blood and lymphatic vessels (CD31 and podoplanin) in channels of the extracellular matrix suggests that the described endothelial-like cells can be the structural basis for blood and lymphatic vessels of the tumor.

The aim of this work was a comparative study of the ultrastructural organization of lymphatic structures in the choroid and conjunctiva of the human eye. Material and methods. Samples of the choroid and conjunctiva of the human eye, obtained perioperatively, were studied by immunohistochemical analysis using specific antibodies to endothelial cells markers of blood and lymphatic vessels, and electron microscopic studies, morphometric processing and statistical analysis of the results were performed. Results and discussion. Within simultaneous immunohistochemical staining of conjunctiva and choroid samples using antibodies to markers of lymphatic vessels (LYVE-1, Prox1, podoplanin) the positively stained typical lymphatic vessels have been revealed in the conjunctiva structure and positively stained channels with narrow elongated cells, fibroblasts and pigment cells in choriocapillar layer of the choroid and the transition zone between choroid and sclera. Electron microscopic study revealed that cells expressing lymphatic markers and forming lymphatic channels in the choroid have an ultrastructural organization that is differ from typical lymphatic vascular endothelial cells: they do not have stropic filaments, contain a greater volume density of the granular endoplasmic reticulum membranes, and a lower volume density of mitochondria and micropinocytotic vesicles. The data obtained indicate that the lymphatic pathways of the intraocular fluid outflow in the human eye choroid have organ-specific structural features.

Use of formylindolylacetic acid as a reagent at the stage of preparing the glycosides of bis(indolyl)furan-2,5-diones and dioxane as solvent increased yields from 5 – 10% to 60 – 63%, decreased the duration of the reaction from 6 h to 2 h, and significantly simplified extraction of products adequate for use at the next stage of photochemical oxidation to furancarbazole derivatives. New data are presented on the cytotoxic activity of the indocarbazole N-glycosides synthesized here, covering a wide range of cell structures and giving a more complete view of the antiproliferative properties of the compounds synthesized.

The aim of this work was a comparative study of the ultrastructural organization of lymphatic structures in the choroid and conjunctiva of the human eye. Material and methods. Samples of the choroid and conjunctiva of the human eye, obtained perioperatively, were studied by immunohistochemical analysis using specific antibodies to endothelial cells markers of blood and lymphatic vessels, and electron microscopic studies, morphometric processing and statistical analysis of the results were performed. Results and discussion. Within simultaneous immunohistochemical staining of conjunctiva and choroid samples using antibodies to markers of lymphatic vessels (LYVE-1, Prox1, podoplanin) the positively stained typical lymphatic vessels have been revealed in the conjunctiva structure and positively stained channels with narrow elongated cells, fibroblasts and pigment cells in choriocapillar layer of the choroid and the transition zone between choroid and sclera. Electron microscopic study revealed that cells expressing lymphatic markers and forming lymphatic channels in the choroid have an ultrastructural organization that is differ from typical lymphatic vascular endothelial cells: they do not have stropic filaments, contain a greater volume density of the granular endoplasmic reticulum membranes, and a lower volume density of mitochondria and micropinocytotic vesicles. The data obtained indicate that the lymphatic pathways of the intraocular fluid outflow in the human eye choroid have organ-specific structural features.

Purpose. To study the content of cytokines and growth factors in the intraocular fluid of patients with developed stage of primary open-angle glaucoma (POAG).Materials and methods. 56 patients with a verified diagnosis developed stage of primary open-angle glaucomawere examined. The control group consisted of 30 patients with a diagnosis of uncomplicated cataract. A concentration of 17 cytokinesand 3 isoforms of the transforming growth factor (TGF) β was determined using a Bio-Plex Pro™ Human Cytokine 17-plex Assay and Bio-Plex Pro™ and TGFβ 3-plex Assay kit by flow-through fluorometry on a Bio-Plex 200, Bio-Rad double beam laser analyzer, USA.Results. Astatistically significant increase was shown in the concentrations of cytokines and growth factors (interleukins (IL) 4, 6, 7, 8, 12, 17, TGFβ 1, 2, 3, macrophage inflammatory protein 1 β) in the intraocular fluid of patients with developed stage of the primary open-angle glaucoma in respect to data obtained from the study of the intraocular fluid of the persons with uncomplicated cataract, as well as a statistically significant decrease in the concentrations IL-2, IL-10, granulocyte-macrophage colony-stimulating factor.Conclusion. In the pathogenesis of primary open-angle glaucoma, the activity of the local chronic inflammatory process is determined. This is confirmed by statistically significant changes in the studied cytokines and growth factors. Increase in the concentrations of the studied representatives of the superfamily of transforming growth factors-beta, which have anti-inflammatory activity, the ability to stimulate proliferation, cell growth, synthesis of extracellular matrix proteins, etc., attests to their importance in the mechanisms of primary open-angle glaucoma development. Increase concentrations of IL-7 in the intraocular fluid of patients with primary open-angle glaucoma allows us to assume participation in the pathogenesis of the primary open-angle glaucoma of this autocrine mediator of activation of the growth of lymphatic structures.