Evgeny N. Antonov

Micronization of the pharmaceutical substance levofloxacin hemihydrate (LFC) was studied by the RESS method with trifluoromethane (CHF₃) as a polar supercritical solvent. Scanning electron microscopy showed that, at a CHF₃ temperature of 50°C, particles of micronized LFC have the shape of microgranules of submicron size. At a temperature of 100°C, the particles take the shape of thin (less than 0.1-μm-thick) elongated plates with rounded edges. At this temperature, an increase in the CHF₃ pressure from 20 to 35 MPa leads to an increase in the average particle size from 1 to 2 μm, respectively. X-ray powder diffraction analysis of samples obtained at a temperature of 50°C determined the identity of the original and micronized LFC substances, which correspond to the structure of levofloxacin hemihydrate C₃₆H₄₀F₂N₆O₈⋅H₂O. An increase in the process temperature to 100°C leads to the formation of one of pseudopolymorphs of levofloxacin—levofloxacin monohydrate C₁₈H₂₀F₂N₃O₄⋅H₂O—in the micronized LFC.

Исследована микронизация фармацевтической субстанции левофлоксацина полугидрата (ЛФЦ) методом RESS c трифторметаном (СНЕ3) в качестве полярноuо сверхкритического растворителя. Сканирующей электронной микроскопией показано, что при температуре СНЕ3, равной 50°С, частицы микронизированного ЛФЦ имеют вид микрогранул субмикронного размера. При температуре 100 °С частицы приобретают форму тонких (менее 0,1 мкм) продолговатых пластинок с закругленными краями. При этой температуре увеличение давления СНЕ3 от 20 МПа до 35 МПа приводит к росту среднего размера частиц от 1 до 2 мкм соответственно. Рентгенофазовый анализ образцов, полученных при температуре 50 °С, установил идентичность исходной и микронизированной субстанции ЛФЦ, соответствующих структуре левофлоксацина полугидрата - С36Н40F2N6O8⋅H2O. Повышение температуры процесса до 100 °С приводит к появлению в составе микронизированного ЛФЦ одной из его псевдополиморфных форм — левофлоксацина моногидрата С18Н20F2N3O4⋅H2O Micronization of levofloxacin hemihydate drug substance (LFC) by RESS using trifluoromethane (CHF3) as a polar supercritical solvent was investigated. By scanning electron microscopy, it was shown that at CHF3 temperature of 50°C, micronized LFC particles have the form of submicron-sized microbeads. At a temperature of 100°C, the particles take the form of thin (less than 0.1 microns) elongated plates with rounded edges. At this temperature, an increase in CHF3 pressure from 20 MPa to 35 MPa increases the average particle size of these particles from 1 to 2 um, respectively. X-ray phase analysis of samples obtained at 50 °C showed the identity of the original and micronized LFC substance corresponding to the structure of levofloxacin hemihydrate — С36Н40F2N6O8⋅H2O. At the same time, it was shown that an increase in the process temperature to 100°C leads to the appearance of one of its polymorphic forms, levofloxacin monohydrate С18Н20F2N3O4⋅H2O in the micronized LFC

Levofloxacin (LFX) is a highly effective anti-tuberculosis drug with a pronounced bactericidal activity against Mycobacterium tuberculosis (Mtb). In this work, an “organic solvent-free” approach has been used for the development of polylactic-co-glycolic acid (PLGA) microparticles and scaffolds containing LFX at a therapeutically significant concentration, providing for its sustained release. To achieve the target, both nonpolar supercritical carbon dioxide and polar supercritical trifluoromethane have been used. By changing the composition, surface morphology, size, and internal structure of the polymer carriers, one can control the kinetics of the LFX release into phosphate buffered saline solutions and physiological media, providing for its acceptable burst and desirable concentration in the prolonged phase. The biocompatibility and bactericidal efficacy of PLGA/LFX carriers assessed both in vitro (against Mtb phagocytosed by macrophages) and in vivo (against inbred BALB/c mice aerogenically infected with Mtb) demonstrated their anti-tuberculosis activity comparable with that of the standard daily intragastric levofloxacin administration. These results make it possible to consider the developed compositions as a promising candidate for anti-tuberculosis control release formulations providing for the further evaluation of their activity against Mtb and their metabolism in vivo over long periods of tuberculosis infection.

Supercritical fluid encapsulation of gentamicin, levofloxacin and tetracycline into bioresorbable poly(lactic-co-glycolic)acid (PLGA) scaffolds at 20 wt % was performed by PLGA plasticization in supercritical carbon dioxide with its subsequent foaming. The effect of incorporated antibiotics on the rate of PLGA hydrolytic degradation, determined by weekly measurements of the polymer molecular weight and decrease in gravitational masses during 6 weeks of incubation of scaffolds in a phosphate-buffered saline (PBS) solution, was studied. Measurements of pH of PBS containing scaffolds were carried out in the same way. The rate constants of PLGA hydrolysis for different scaffolds comprising various drugs were determined. It was shown that tetracycline significantly reduced the rate of PLGA degradation compared to the rate of degradation of control (pure polymer) scaffolds. At the same time, the presence of gentamicin and levofloxacin in the scaffolds had no visible effect on their degradation. These results enhance the predicting potential for the kinetics of release of the considered antibiotics from bioresorbable polymer carriers into bioactive media, which is necessary for the development of highly efficient sustained-release dosage forms.

Методом сверхкритической флюидной пластификации в сверхкритическом диоксиде углерода с последующим вспениванием проведена инкапсуляция гентамицина, левофлоксацина и тетрациклина в биорезорбируемые полилактогликолидные (ПЛГ) матриксы в количестве 20 масс. %. Исследовано влияние, инкорпорированных в матриксы антибиотиков на скорость гидролитической деградации ПЛГ в растворе фосфатно-солевого буфера (PBS), определявшейся по изменению молекулярной массы полимера в зависимости от времени его инкубации в PBS. Измерения молекулярной и гравитационной масс матриксов, а также pH содержавших их растворов проводились еженедельно в течение 6 недель. Определены константы реакции гидролиза макромолекул полилактогликолида в матриксах различного состава. Показано, что тетрациклин, содержащейся в матриксах, существенно уменьшает скорость их деградации по сравнению со скоростью деградации контрольных (чистых) ПЛГ матриксов. В то же время присутствие в матриксах гентамицина и левофлоксацина, практически, не влияло на скорости деградации их полимерной основы. Результаты исследований расширяют возможности прогнозирования кинетики высвобождения рассмотренных антибиотиков из биорезорбируемых полимерных носителей в биоактивные среды, необходимого для разработки высокоэффективных лекарственных форм пролонгированного...

Gentamicin and levofloxacin are encapsulated into bioresorbable polymer scaffolds and microparticles by the SCF methods: particles from gas saturated solutions (PGSS) and plasticization, with the subsequent foaming of amorphous polymers using supercritical carbon dioxide. The kinetics of the release of the incorporated drug substances into model physiological media are studied. It is shown that the use of the developed methods of SCF-encapsulation of drugs in bioresorbable carriers allows varying the size, shape, and morphology of the formed structures and, accordingly, the rate of release of the drugs into the physiological environments. In our opinion, this approach can be very promising for the development of components of new highly effective antibacterial prolonged action dosage forms.

Sustained-release drugs against tuberculosis are a promising approach to therapy since they positively affect patient compliance with long regimens, especially when it comes to the multidrug-resistant form of the disease. Conventional UV-visible spectroscopy does not work well with multicomponential culture media used for growing M. tuberculosis. The aim of this study was to develop a method for evaluating the kinetics of anti-tuberculosis drug released from bioresorbable polymeric carriers suitable for screening a wide range of encapsulated prolonged-release drugs and identifying the best performing candidate. While studying the growth dynamics of the laboratory susceptible strain M. tuberculosis H37Rv in the presence of different levofloxacin concentrations (from 0.03 to 0.4 μg/ml), we developed a model, which is essentially a set of 2 parallel experiments evaluating the kinetics of drug release into the culture medium. The results of these 2 experiments conducted on 3 encapsulated forms of levofloxacin loaded onto bioresorbable polymeric PLGA carriers (particles sized 50 μm and 100 μm and the matrix) revealed that release kinetics of the drug largely depended on the type of polymeric carrier. The best encapsulation of the antibiotic and its gradual release into the culture medium was observed for the matrix. All experiments were run in 3 replicates. The obtained data were analyzed using descriptive statistics.

Effective treatment of tuberculosis requires long-term use of advanced anti-tuberculosis control released drug formulations. The aim of our work is R&D of such formulation based on supercritical fluid (SCF) encapsulation of levofloxacin (LFX) into bioresorbable polylactic-co-glycolic acid (PLGA) carriers and adjustment of its release kinetics in cultural medium according to MTB strain growth dynamics. Methods: 10 wt.% of LFX (Sigma-Aldrich) was encapsulated into PLGA (PDLG7502, Corbion Purac) by PGSS (Particles from Gas Saturated Solution) method using supercritical carbon dioxide. 1.2 mg of fabricated microparticles (mean diameter ca 5 to 20 µm) was introduced into 8.4 ml Middlebrook 7H9 Broth cultural medium. After 1 hour, 2, 5 and 6 days medium aliquots were added to the MTB culture. Assessment of MTB culture growth under the action of the released LFX was carried out using BACTEC MGIT 960 system. Results: Dynamics of MTB culture growth in presence of aliquots of the medium in which LFX containing PLGA particles were incubated is shown in Fig. Conclusions: Our studies demonstrated the controlled release of LFX encapsulated in PLGA particles and its effect on MTB culture. SCF drug encapsulation eliminates the use of toxic organic solvents and high temperatures, and can be effectively applied for R&D of advanced ATD formulations. Funding: RFBR №18-29-06062

New methods for the synthesis of aliphatic polyesters were proposed and realized with highly effective and low-toxic 1,5,7-triazabicyclo[4.4.0]decene-5 and (2,6-di-tert-butyl-4-methylphenoxy)-butyl-magnesium as catalysts and ethanol as an initiator. The interaction processes between D,L-polylactides of different molecular weights, obtained through these methods, and supercritical carbon dioxide were studied to provide “soft and dry” formation (without high temperatures and toxic organic solvents) of highly porous (up to 90 vol %) bioresorbable matrices for tissue engineering constructions. The chemical structure, molecular weight characteristics, morphology, cytotoxicity, and matrix properties of the samples synthesized from D,L-polylactides were studied with nuclear magnetic resonance spectroscopy, gel permeation chromatography, scanning electron microscopy, colorimetric testing to estimate the metabolic activity of cells (MTT test), and coloration with a vital fluorescent dye of in vitro cultures of multipotent stromal cells of human adipose tissue. A comparative analysis for biocompatibility of these matrices with control samples made from “medical grade” polylactide analog produced industrially indicates that the materials developed can be recommended for tissue engineering.