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Aging is accompanied by intensification of oxidative degradation of proteins, including lipoproteins, proteolytic system dysfunction and accumulation of oxidized proteins. Similar changes are found in drug addiction. In this regard, we carried out a comparative study of conformational changes of plasma proteins and red blood cells of people of different age groups and people who use drugs. The study was carried out with the use of fluorescence analysis to measure the spectra of fluorescence of amino acid residues in proteins (summary fluorescence and tryptophan). In the analysis of fluorescence spectra it is preferable to use the second derivative of the fluorescence spectra as it is the most informative. The application of this research method was chosen because it has high sensitivity. It provides information on the status of living systems without damaging them; it requires large quantities of biological material; it makes it possible to differentiate between the various stages of the disease. Changes in the general phase and tryptophan fluorescence plasma proteins in older people and drug addicts indicate increase in the availability of tryptophan residues to the aqueous environment as a result of conformational changes of macromolecules and the disruption of the lipid layer due to the intensification of free radical processes.

Цель работы – на основе проведенного эксперимента предложить технологию получения липосомальной формы иринотекана; проанализировать получаемые полупродукты, предложить контрольные точки. Материалы и методы. Для изготовления липосом использовали липиды производства Lipoid, ФРГ. Холестерин, лимонную кислоту моногидрат, растворители использовали производства фирмы Sigma-Aldrich, США. Липидную пленку получали на роторном испарителе Buchi 210 с вакуумным контроллером при остаточном давлении 0,02 атм. рН контролировали на рН-метре Seven Compact (Mettler Toledo, США). Для гомогенизации использовали метод экструзии при высоком давлении, которую проводили на установке Microfluidiser M-110P (Microfluidics, США). Размер липосом определяли при 20 °С на приборе Zetasizer Nano ZS (Malvern Instruments, Великобритания). Технологию «химического градиента» проводили методом ультрафильтрации на опытно-промышленной установке АСФ-018 (Владисарт, РФ). Уровень инкапсуляции иринотекана в липосомы, концентрацию иринотекана, содержание посторонних примесей контролировали методом высокоэффективной жидкостной хроматографии (ВЭЖХ) на хроматографах Shimadzu LC-20 (Япония). Процесс лиофилизации производили на оборудовании Quarco (КНР). Результаты. Технологии получения наноструктурированных препаратов требуют комплексного подхода к разработке. Показан порядок производства липосомальной формы иринотекана, структурирован опыт, полученный при фармацевтической разработке и при внедрении препарата в опытно-промышленное производство. Проанализированы ключевые этапы производства, необходимые точки межоперационного контроля и контроля качества готового лекарственного средства. Рассмотрены отличия лабораторной технологии от технологии, реализуемой в опытно-промышленном масштабе. Реализация представленной технологии заложена на этапе фармацевтической разработки и научно-практически подтверждена рядом проведенных экспериментов. В результате получены серии продукта со стабильными показателями качества при воспроизводимых и контролируемых параметрах технологического процесса. Выводы. Предложена оригинальная опытно-промышленная технология получения липосомальной формы иринотекана. Стадии технологии проанализированы с точки зрения промышленной реализации, предложены контрольные точки. Purpose of the study. Based on the experiment to propose technology for obtaining the liposomal form of irinotecan. To analyze the resulting intermediates, suggest control points. Materials and methods. Lipids manufactured by Lipoid, Germany were used to make liposomes. Cholesterol, citric acid monohydrate, solvents were used by Sigma-Aldrich, USA. The lipid film was produced on a rotary evaporator Buchi 210 with a vacuum controller at a residual pressure of 0.02 atm. The pH was monitored on a pH meter of Seven Compact (Mettler Toledo, USA). For homogenization, a high-pressure extrusion method was used, which was carried out on a Microfluidiser M-110P (Microfluidics, USA). The size of the liposomes was determined at 20 °C on a Zetasizer Nano ZS (Malvern Instruments, UK). The technology of the “chemical gradient” was carried out using the ultrafiltration method at the pilot plant ASF-018 (Vladiart, Russia). The level of encapsulation of irinotecan in liposomes, irinotecan concentration, and impurity content were monitored by high performance liquid chromatography (HPLC) using Shimadzu LC-20 chromatographs (Japan). The lyophilization process was performed on Quarco equipment (PRC). Results. Technologies of nano-structured drugs obtaining require an integrated approach to development. The article discusses the order of the liposomal form of irinotecan production, the experience gained in the pharmaceutical development and the introduction of the drug into pilot production is structured. The key stages of production, the necessary points for «in process» control, and quality control of the finished medicinal product are analyzed. The differences between the laboratory scale technology and the technology implemented in pilot scale are considered. The implementation of the presented technology was laid down at the stage of pharmaceutical development and scientifically-practically confirmed by a number of conducted experiments. As a result, product series with stable quality indicators were obtained with reproducible and controlled process parameters. Conclusions. The original experimental-industrial technology of obtaining liposomal form of irinotecan is proposed. The stages ofthe technology are analyzed from the point of view of industrial implementation, control points are proposed.

Objective: to study the nanostructure of red blood cell membranes and erythrocyte index in preterm neonatal infants. Subjects and methods . The trial enrolled 47 neonatal infants, including 33 preterm infants who were included in a study group and 14 fullterm infants who formed a comparative group. The gestational age of the preterm infants was 33.3±1.9 weeks and the birth weight was 2065.4±304.8 g. Red blood cell counts, hemoglobin, and erythrocyte indices were estimat ed and the red blood cells were examined using an atomicforce microscope. Results . At birth, the preterm infants showed macrocytosis, intrauterine poikylocytosis, and the impaired nanostructure of red blood cell membranes. Intrauterine hypoxia affects the red blood cell membrane nanostructures: a phospholipid bilayer and a spectrin matrix, without damaging the membrane protein component. The detected changes are reversible and directed to maintaining the functional ability of red blood cells in a critical situation. At birth, gestational age, a baby's weight, hemoglobin, and blood cholesterol and standard bicarbonate levels influence the parameters of a red blood cell component. The early neonatal period was characterized by an active process on the red blood cell membranes and a change of morphological forms, suggesting the continuing postnatal rearrangement of erythropoiesis and a preterm infant's adaptation to new environmental conditions.

Оксалиплатин и иринотекан являются перспективными веществами для создания липосомальной лекарственной формы. Разработка препаратов проводилась с конечной лиофилизацией для повышения показателей стабильности готовых лекарственных форм. Для успешного применения липосомальных препаратов в терапевтической практике необходимо обеспечить их необходимую стабильность после регидратации, перед введением. Стабильность включает сохранность инкапсуляции и размера в случае каждой липосомы, а также термодинамическую стабильность коллоидной системы в целом. Было изучено влияние холестерина на сохранность размера и степени инкапсуляции на примере липосом с иринотеканом. Липосомы были получены методом «химического градиента», с цитратом натрия в качестве внутреннего буферного раствора. Показано, что холестерин повышает стабильность размеров и инкапсуляции, оптимальным содержанием которого является 20% массовых. Также было изучено влияние дзета-потенциала на термодинамическую стабильность липосом после регидратации на примере липосом с иринотеканом и оксалиплатином. Повышения стабильности для липосом с иринотеканом удалось достигнуть при помощи добавки l-лизина в концентрациях 0,012%, при этом значение дзета-потенциала составило -5,1 mV. Oxaliplatin and irinotecan are prospective active pharmaceutical ingredients (API) for liposomal form creation. Development with the final lyophilization was carried out to improve stability of the eventual dosage forms. It is necessary to provide essential stability of colloidal formulations after rehydration, for the successful application of liposome preparations in therapeutic practice. The stability parameters involves such criteria, as encapsulation degree and liposomes size, as well as the thermodynamic stability of the whole colloidal system. The effect of cholesterol on the liposomes size and the encapsulation degree after rehydratation, with liposomal irinotecan as example, was studied. Liposomes were obtained by “chemical gradient”, with sodium citrate as the internal buffer solution. It was shown that cholesterol improves the stability of size and encapsulation degree, the optimum content was 20% by weight. The influence of the zeta potential on the thermodynamic stability of the liposomes after rehydration was also studied with liposomal irinotecan and liposomal oxaliplatin as examples. Increased stability of irinotecan liposomes was achieved with the addition of l-lysine (concentration 0.012%), the value of the zeta potential was -5,1 mV.

При создании эмульсионных лекарственных средств, в частности, наноструктурированных липосомальных форм, одним из важнейших показателей качества является агрегативная стабильность препарата. Дзета-потенциал является важным критерием термодинамической устойчивости липосомальной системы. Было изучено, что лиофилизированная липосомальная форма иринотекана обладает недостаточной термодинамической устойчивостью после регидратации. Проведены исследования, направленные на увеличение показателя дзета-потенциала и седиментационной устойчивости препарата путём модификации двойного электрического слоя l-аминокислотами. Изучены l-глицин, l-лизин и l-аргинин. Показано, что эффективным является содержание 0,012% l-лизина. Предложен технологический метод введения аминокислоты в липосомальную фармацевтическую композицию. During creation of emulsion medicines, in particular nanostructured liposomal forms, one of the most important indicators of quality is the aggregative stability of the drug. The zeta potential is an important criterion for the thermodynamic stability of the liposomal system. It was studied that the liposomal form of irinotecan possesses insufficient thermodynamic stability after rehydration. An experiment was performed to increase the zeta potential and, in the same time, the stability of the liposomal formulation, by modifying the double electrical layer with the l-amino acids. L-glycine, l-lysine and l-arginine was studied. It was shown that the content of 0.012% l-lysine is optimal. A technological method for introducing an amino acid into a liposome pharmaceutical composition was proposed.

A correct model of lipid molecule (distearoylphosphatidylcholine, DSPC) and lipid membrane in wa- ter was constructed. Model lipid membrane is stable and has a reliable energy distribution among degrees of free- dom. Also after equilibration model system has spatial parameters very similar to those of real DSPC membrane in liquid-crystalline phase. This model was used for studying of lipid membrane permeability to oxygen and water molecules and sodium ion. We obtained the values for transmembrane mobility and diffusion coefficients profiles, which we used for effective permeability coefficients calculation. We found lipid membranes to have significant diffusional resistance to penetration not only by charged particles, such as ions, but also by nonpolar molecules, such as oxygen molecule. We propose theoretical approach for calculation of particle flow across a membrane, as well as methods for estimation of distribution coefficients between bilayer and water phase.

Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089-2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.