Immediate and long-term effects of COVID-19 on antibiotic dispensing: increasing use of Watch antibiotics.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic affected antibiotic usage worldwide. However, there is limited data from Serbia. Dispensing of oral antibiotics in Serbian pharmacies was analyzed to calculate monthly and yearly changes between 2018-2021, and to explore immediate and long-term effects of COVID-19 on antibiotic dispensing during this period. METHODOLOGY: The number of antibiotic packages dispensed from pharmacies during the study period was analyzed with a Chi-square test to assess the average change in annual dispensing, and an interrupted time-series analysis was used to evaluate the impact of the pandemic on antibiotic dispensing. The data from 2018-2021 were retrieved from the database of a large community pharmacy chain in Serbia. RESULTS: The average number of antibiotic packages dispensed per day and per pharmacy was higher in 2021 compared to 2018 by one package. However, the dispensing of macrolides increased significantly; 17.7% (2018) vs. 22.5% (2021) (p < 0.05). In general, an increase in antibiotic dispensing was detected during COVID-19 for total antibiotics (16.4%), Watch antibiotics (44.8%), third-generation cephalosporins (80.4%), macrolides (45.5%) and azithromycin (83.7%). However, the immediate effect of COVID-19 was a decrease in the dispensing of Watch antibiotics, penicillin, and third-generation cephalosporins (p < 0.05); and a notable long-term COVID-19 effect was an increase in the dispensing of azithromycin (p < 0.05). CONCLUSIONS: In spite of a relatively stable trend of total antibiotic dispensing before and during COVID-19 pandemic, the use of Watch antibiotics, third-generation cephalosporins, and macrolides (particularly azithromycin) showed an increasing trend in dispensing that should be optimized.

Antimicrobial utilization and resistance in Pseudomonas aeruginosa using segmented regression analysis: a comparative study between Serbia and eight European Countries.

BACKGROUND: In Europe, Serbia occupies a high position in antibiotic utilization and antimicrobial resistance (AMR). AIM: The aim was to analyse utilization trends of meropenem, ceftazidime, aminoglycosides, piperacillin/tazobactam and fluoroquinolones (2006-2020), and the reported AMR in Pseudomonas aeruginosa (2013-2020) in Serbia and to compare with data from eight European countries (2015-2020). METHOD: Joinpoint regression was used to analyse antibiotic utilization data (2006-2020) and the reported AMR in Pseudomonas aeruginosa (2013-2020). Data sources were relevant national and international institutions. Antibiotic utilization and AMR in Pseudomonas aeruginosa data in Serbia were compared with eight European countries. RESULTS: There was a significantly increased trend for ceftazidime utilization and reported resistance in Pseudomonas aeruginosa, Serbia (p < 0.05) (2018-2020). For ceftazidime, piperacillin/tazobactam, and fluoroquinolones resistances in Pseudomonas aeruginosa an increased trend was observed, Serbia (2013-2020). A decrease in both the utilization of aminoglycosides, Serbia (p < 0.05) (2006-2018) and contemporaneous Pseudomonas aeruginosa resistance (p > 0.05) was detected. Fluoroquinolone utilization (2015-2020) was highest in Serbia compared to Netherlands and Finland, 310 and 305% higher, similar compared to Romania, and 2% less compared to Montenegro. Aminoglycosides (2015-2020) were 2550 and 783% more used in Serbia compared to Finland and Netherlands, and 38% less regarding Montenegro. The highest percentage of Pseudomonas aeruginosa resistance was in Romania and Serbia (2015-2020). CONCLUSION: The use of piperacillin/tazobactam, ceftazidime and fluoroquinolones should be carefully monitored in clinical practice due to increased Pseudomonas aeruginosa resistance. The level of utilization and AMR in Pseudomonas aeruginosa is still high in Serbia compared to other European countries.

Comparative analysis of Ag NPs functionalized with olive leaf extract and oleuropein and toxicity in human trophoblast cells and peripheral blood lymphocytes.

Dry olive leaf extract (DOLE) and its active component oleuropein (OLE) were applied as reducing and stabilizing agents to prepare colloidal 20-25 nm silver nanoparticles (Ag NPs). The Ag NPs were characterized using transmission electron microscopy, X-ray diffraction analysis, and absorption spectroscopy. The cytotoxic actions of coated Ag NPs, and their inorganic and organic components, were examined against trophoblast cells and human peripheral blood lymphocytes (PBLs), Gram-positive, Gram-negative bacteria, and yeast. The genotoxic potential was evaluated in PBLs in vitro with the comet assay. Ag/DOLE and Ag/OLE induced cytotoxic effects in both types of cells after 24 h exposure when silver concentrations were 0.025-0.2 mM. However, the most pronounced cytotoxicity exhibits Ag/OLE. Both colloids also caused reduced ROS production in both cell types at 0.1 mM and 0.2 mM, while bare Ag NPs did not alter ROS levels at any of the conditions. Functionalized Ag/DOLE and Ag/OLE did not show genotoxic effects in PBLs, while bare AgNPs increased DNA damage significantly only at 0.2 mM. Regarding the antimicrobial effects, the Ag/OLE had MIC values for all evaluated microorganisms from 0.0625 to less than 0.0312 mM. Also, the antimicrobial effect of Ag/DOLE was significantly higher on Gram-negative bacteria and yeast than on Gram-positive bacteria. Obtained results indicate that Ag/OLE induced the most pronounced biological effects, beneficial for its application as an antimicrobial agent, but with potential risks from exposure to high concentrations that could induce cytotoxicity in healthy human cells.

Chemical vs. Physical Methods to Improve Dermal Drug Delivery: A Case Study with Nanoemulsions and Iontophoresis.

So far, various approaches have been proposed to improve dermal drug delivery. The use of chemical penetration enhancers has a long history of application, while methods based on the electrical current (such as iontophoresis) stand out as promising "active" techniques. Aiming to evaluate the contribution of different approaches to dermal delivery, in this work curcumin-loaded nanoemulsions with and without monoterpenes (eucalyptol or pinene) as chemical penetration enhancers, and a custom-made adhesive dermal delivery system based on iontophoresis were designed and assessed. In an in vivo study applying skin bioengineering techniques, their safety profile was proven. Three examined iontophoresis protocols, with total skin exposure time of 15 min (continuous flow for 15 min (15-0); 3 min of continuous flow and 2 min pause (3-2; 5 cycles) and 5 min of continuous flow and 1 min pause (5-1; 3 cycles) were equally efficient in terms of the total amount of curcumin that penetrated through the superficial skin layers (in vivo tape stripping) (Q3-2 = 7.04 ± 3.21 µg/cm2; Q5-1 = 6.66 ± 2.11 µg/cm2; Q15-0 = 6.96 ± 3.21 µg/cm2), significantly more efficient compared to the referent nanoemulsion and monoterpene-containing nanoemulsions. Further improvement of an efficient mobile adhesive system for iontophoresis would be a practical contribution in the field of dermal drug application.

Curcumin Loaded PEGylated Nanoemulsions Designed for Maintained Antioxidant Effects and Improved Bioavailability: A Pilot Study on Rats.

The current study describes the experimental design guided development of PEGylated nanoemulsions as parenteral delivery systems for curcumin, a powerful antioxidant, as well as the evaluation of their physicochemical characteristics and antioxidant activity during the two years of storage. Experimental design setup helped development of nanoemulsion templates with critical quality attributes in line with parenteral application route. Curcumin-loaded nanoemulsions showed mean droplet size about 105 nm, polydispersity index <0.15, zeta potential of -40 mV, and acceptable osmolality of about 550 mOsm/kg. After two years of storage at room temperature, all formulations remained stable. Moreover, antioxidant activity remained intact, as demonstrated by DPPH (IC50 values 0.078-0.075 mg/mL after two years) and FRAPS assays. In vitro release testing proved that PEGylated phospholipids slowed down the curcumin release from nanoemulsions. The nanoemulsion carrier has been proven safe by the MTT test conducted with MRC-5 cell line, and effective on LS cell line. Results from the pharmacokinetic pilot study implied the PEGylated nanoemulsions improved plasma residence of curcumin 20 min after intravenous administration, compared to the non-PEGylated nanoemulsion (two-fold higher) or curcumin solution (three-fold higher). Overall, conclusion suggests that developed PEGylated nanoemulsions present an acceptable delivery system for parenteral administration of curcumin, being effective in preserving its stability and antioxidant capacity at the level highly comparable to the initial findings.

Polyglycerol Ester-Based Low Energy Nanoemulsions with Red Raspberry Seed Oil and Fruit Extracts: Formulation Development toward Effective In Vitro/In Vivo Bioperformance.

This study focuses on the development of biocompatible oil-in-water (O/W) nanoemulsions based on polyglycerol esters, as promising carriers for natural actives: red raspberry seed oil-RO and hydro-glycolic fruit extracts from red raspberry-RE and French oak-FE. Nanoemulsions were obtained via phase inversion composition (PIC) method at room temperature by dilution of microemulsion phase, confirmed by visual appearance, percentage of transmittance, microscopic, rheological and Differential Scanning Calorimetry (DSC) investigations. The results have shown that the basic RO-loaded formulation could be further enriched with hydro-glycolic fruit extracts from red raspberry or French oak, while keeping a semi-transparent appearance due to the fine droplet size (Z-ave: 50 to 70 nm, PDI value ≤ 0.1). The highest antioxidant activity (~92% inhibition of the DPPH radical) was achieved in the formulation containing both lipophilic (RO) and hydrophilic antioxidants (FE), due to their synergistic effect. The nanoemulsion carrier significantly increased the selective cytotoxic effect of RO towards malignant melanoma (Fem-X) cells, compared to normal human keratinocytes (HaCaT). In vivo study on human volunteers showed satisfactory safety profiles and significant improvement in skin hydration during 2 h after application for all nanoemulsions. Therefore, polyglycerol ester-based nanoemulsions can be promoted as effective carriers for red raspberry seed oil and/or hydro-glycolic fruit extracts in topical formulations intended for skin protection and hydration.

Low-energy nanoemulsions as carriers for red raspberry seed oil: Formulation approach based on Raman spectroscopy and textural analysis, physicochemical properties, stability and in vitro antioxidant/ biological activity.

Considering a growing demand for medicinal/cosmetic products with natural actives, this study focuses on the low-energy nanoemulsions (LE-NEs) prepared via the Phase inversion composition (PIC) method at room temperature as potential carriers for natural oil. Four different red raspberry seed oils (ROs) were tested, as follows: cold-pressed vs. CO2-extracted, organic vs. non-organic, refined vs. unrefined. The oil phase was optimized with Tocopheryl acetate and Isostearyl isostearate, while water phase was adjusted with either glycerol or an antioxidant hydro-glycolic extract. This study has used a combined approach to formulation development, employing both conventional methods (pseudo-ternary phase diagram - PTPD, electrical conductivity, particle size measurements, microscopical analysis, and rheological measurements) and the methods novel to this area, such as textural analysis and Raman spectroscopy. Raman spectroscopy has detected fine differences in chemical composition among ROs, and it detected the interactions within nanoemulsions. It was shown that the cold-pressed, unrefined, organic grade oil (RO2) with 6.62% saturated fatty acids and 92.25% unsaturated fatty acids, was optimal for the LE-NEs. Textural analysis confirmed the existence of cubic gel-like phase as a crucial step in the formation of stable RO2-loaded LE-NEs, with droplets in the narrow nano-range (125 to 135 nm; PDI ≤ 0.1). The DPPH test in methanol and ABTS in aqueous medium have revealed a synergistic free radical scavenging effect between lipophilic and hydrophilic antioxidants in LE-NEs. The nanoemulsion carrier has improved the biological effect of raw materials on HeLa cervical adenocarcinoma cells, while exhibiting good safety profile, as confirmed on MRC-5 normal human lung fibroblasts. Overall, this study has shown that low-energy nanoemulsions present very promising carriers for topical delivery of natural bioactives. Raman spectroscopy and textural analysis have proven to be a useful addition to the arsenal of methods used in the formulation and characterization of nanoemulsion systems.

Metformin effects on malignant cells and healthy PBMC; the influence of metformin on the phenotype of breast cancer cells.

The aim of research was to determine the effects of maximally therapeutically achievable concentrations of metformin on malignant cells and healthy peripheral blood mononuclear cells (PBMC). Eight patients with T2D or hyperglycemia and nine healthy volunteers were included in the study. For determination of the influence of metformin on the phenotype of breast carcinoma, 1,410 patients with surgically removed tumors were included. From this group 37 breast cancer patients had DM type 2 or hyperglycemia and were pretreated with metformin alone or sometimes in combination with other antidiabetic drugs. Our results proved that metformin at low concentrations induced mild decrease in survival of malignant cells and PBMC stimulated for proliferation, but it didn't affect survival of resting PBMC. The effects of plasma of hyperglycemic patients who were under metformin therapy on autologous PBMC-induced decrease in survival of MDA-MB-361 cells, was noticeable in some patients. Metformin pretreatment for 24 h of HER2+ MDA-MB-361 cells, which were subsequently treated for 48 h with Herceptin, induced additional decline in cell survival. The analysis of influence of metformin on phenotype of breast cancer cells revealed significantly lower number of diabetic cancer patients treated with metformin with overexpressed HER2+ tumors (p < 0.013), while the number of patients with ER+PR+ tumors was not significantly changed (p < 0.832). In conclusion, therapeutically used concentrations of metformin exhibit mild cytotoxic action on malignant and dividing normal cells pointing to its preferred role in malignant and autoimmune diseases. The use of metformin was associated with pronounced decrease in HER2 overexpressing tumors.

The cmbT gene encodes a novel major facilitator multidrug resistance transporter in Lactococcus lactis.

Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in this study, it can be concluded that CmbT is a novel major facilitator multidrug resistance transporter candidate in L. lactis, with a possible signaling role in sulfur metabolism.

Target fishing and docking studies of the novel derivatives of aryl-aminopyridines with potential anticancer activity.

A set of 16 previously synthesized aryl-aminopyridine and aryl-aminoquinoline derivatives have been evaluated for cytotoxic activity against three cancer cell lines (human cervical cancer-HeLa; human chronic myeloid leukemia-K562; human melanoma-Fem-x) and two types of normal peripheral blood mononuclear cells, with and without phytohemaglutinin (PBMC-PHA; PBMC+PHA). Twelve of the studied compounds showed moderate cytotoxicity, with selectivity against K562 but not the remaining two cancer cell lines. Four compounds were not active in cytotoxicity assays, presumably due to high predicted lipophilicity and low solubility. To rationalize the observed cytotoxic effects, structure-based virtual screening was carried out against a pool of potential targets constructed using the inverse docking program Tarfisdock and bibliographical references. The putative targets were identified on the basis of the best correlation between docking scores and in vitro cytotoxicity. It is proposed that the mechanism of action of the studied aminopyridines involves the disruption of signaling pathways and cancer cell cycle through the inhibition of cyclin-dependent kinases and several tyrosine kinases, namely Bcr-Abl kinase and KIT receptor kinase. The obtained results can guide further structural modifications of the studied compounds aimed at developing selective agents targeting proteins involved in cancer cell survival and proliferation.

Protective effects of tungstophosphoric acid and sodium tungstate on chemically induced liver necrosis in wistar rats.

PURPOSE: Many chemical compounds and infectious agents such as viruses induce liver damage like necrosis or fulminant hepatic failure which is sometimes difficult to manage by medical therapies. The induced liver necrosis by carbon tetrachloride (CCl4) and thioacetamide (TAA) are exemplary models for experimental liver necrosis caused by oxygen free radicals. The aim of this study was to investigate the effects of tungstophosphoric acid (TPA) and sodium tungstate (ST) on liver injury induced by CCl4 or TAA. METHODS: Hepatoprotective effects of TPA and ST on acute liver necrosis, chemically induced, were evaluated by the activity of serum enzymes (alkaline phosphatase, alanine transaminase and aspartate transaminase), oxidative stress parameters (activity of xanthine oxidase, concentrations of malondialdehyde and production of superoxide anion), antioxidative defence markers (concentration of reduced glutathione), and histopathology in Wistar rats. Liver necrosis was induced by administering a single intraperitoneal (i.p.) injection of CCl4 (1.0 ml/kg b.wt. of 80% CCl4 in corn oil) or a single i.p. injection of TAA (400 mg/kg b.w. dissolved in normal saline). TPA and ST were administrated to rats orally for 7 weeks (50 mg/kg b.wt.) prior to induction of liver necrosis. RESULTS: Induced liver necrosis caused significant elevation of activity of liver enzymes, parameters of oxidative stress and marked changes in histopathology, like necrosis of hepatocytes, hepatocyte degeneration and infiltration of inflammatory cells. In TPA and ST pretreated rats histopathological changes were almost absent, serum enzymes and oxidative stress parameters were decreased, while at the same time the concentration of reduced gluthathione was increased. CONCLUSION: The present findings suggest that treatment with TPA and ST for 7 weeks could be useful for the prevention of hepatic injury in rats.

The role of rat Crry, a complement regulatory protein, in proliferation of thymocytes.

In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 microg/ml and 20 microg/ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6+ antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far.

Differentiation of human dendritic cells from monocytes in vitro using granulocyte-macrophage colony stimulating factor and low concentration of interleukin-4.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC, such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast, adherent cells cultivated with GM-CSF alone were predominantly macrophages, as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.