Ticagrelor alters the membrane of Staphylococcus aureus and enhances the activity of vancomycin and daptomycin without eliciting cross-resistance.

Infections with multidrug-resistant bacteria pose a major healthcare problem which urges the need for novel treatment options. Besides its potent antiplatelet properties, ticagrelor has antibacterial activity against Gram-positive bacteria, including methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA). Several retrospective studies in cardiovascular patients support an antibacterial effect of this drug which is not related to its antiplatelet activity. We investigated the mechanism of action of ticagrelor in Staphylococcus aureus and model Bacillus subtilis, and assessed cross-resistance with two conventional anti-MRSA antibiotics, vancomycin and daptomycin. Bacillus subtilis bioreporter strains revealed ticagrelor-induced cell envelope-related stress responses. Sub-inhibitory drug concentrations caused membrane depolarization, impaired positioning of both the peripheral membrane protein MinD and the peptidoglycan precursor lipid II, and it affected cell shape. At the MIC, ticagrelor destroyed membrane integrity, indicated by the influx of membrane impermeable dyes, and lipid aggregate formation. Whole-genome sequencing of in vitro-generated ticagrelor-resistant MRSA clones revealed mutations in genes encoding ClpP, ClpX, and YjbH. Lipidomic analysis of resistant clones displayed changes in levels of the most abundant lipids of the Staphylococcus aureus membrane, for example, cardiolipins, phosphatidylglycerols, and diacylglycerols. Exogeneous cardiolipin, phosphatidylglycerol, or diacylglycerol antagonized the antibacterial properties of ticagrelor. Ticagrelor enhanced MRSA growth inhibition and killing by vancomycin and daptomycin in both exponential and stationary phases. Finally, no cross-resistance was observed between ticagrelor and daptomycin, or vancomycin. Our study demonstrates that ticagrelor targets multiple lipids in the cytoplasmic membrane of Gram-positive bacteria, thereby retaining activity against multidrug-resistant staphylococci including daptomycin- and vancomycin-resistant strains.IMPORTANCEInfections with multidrug-resistant bacteria pose a major healthcare problem with an urgent need for novel treatment options. The antiplatelet drug ticagrelor possesses antibacterial activity against Gram-positive bacteria including methicillin-resistant and vancomycin-resistant Staphylococcus aureus strains. We report a unique, dose-dependent, antibacterial mechanism of action of ticagrelor, which alters the properties and integrity of the bacterial cytoplasmic membrane. Ticagrelor retains activity against multidrug-resistant staphylococci, including isolates carrying the most common in vivo selected daptomycin resistance mutations and vancomycin-intermediate Staphylococcus aureus. Our data support the use of ticagrelor as adjunct therapy against multidrug-resistant strains. Because of the presence of multiple non-protein targets of this drug within the bacterial membrane, resistance development is expected to be slow. All these findings corroborate the accumulating observational clinical evidence for a beneficial anti-bacterial effect of ticagrelor in cardiovascular patients in need of such treatment.

Development and optimization of a one step process for the production and sterilization of liposomes using supercritical CO2.

Liposomes are very interesting drug delivery systems for pharmaceutical and therapeutic purposes. However, liposome sterilization as well as their industrial manufacturing remain challenging. Supercritical carbon dioxide is an innovative technology that can potentially overcome these limitations. The aim of this study was to optimize a one-step process for producing and sterilizing liposomes using supercritical CO2. For this purpose, a design of experiment was conducted. The analysis of the experimental design showed that the temperature is the most influential parameter to achieve the sterility assurance level (SAL) required for liposomes (≤10-6). Optimal conditions (80 °C, 240 bar, 30 min) were identified to obtain the fixed critical quality attributes of liposomes. The conditions for preparing and sterilizing empty liposomes of various compositions, as well as liposomes containing the poorly water-soluble drug budesonide, were validated. The results indicate that the liposomes have appropriate physicochemical characteristics for drug delivery, with a size of 200 nm or less and a PdI of 0.35 or less. Additionally, all liposome formulations demonstrated the required SAL and sterility at concentrations of 5 and 45 mM, with high encapsulation efficiency.

Use of supercritical CO2 for the sterilization of liposomes: Study of the influence of sterilization conditions on the chemical and physical stability of phospholipids and liposomes.

The effects of four potential supercritical carbon dioxide (ScCO2) sterilization conditions on the chemical stability of 9 phospholipids and on the physicochemical characteristics of liposomes consisting of stable phospholipids, as well as their sterilization efficiency were evaluated. These conditions were : C1 (ScCO2/70 °C/150 bar/240 min), C2 (ScCO2/0.25 % water/ 0.15% H2O2/ 0.5% acetic anhydride/38° C/85 bar/45 min), C3 (ScCO2/0.08 % peracetic acid/35° C/104 bar/180 min) and C4 (ScCO2/200 ppm H2O2/40 °C/270 bar/90 min). The results showed for phospholipids, a significant increase in hydrolysis products of 3.77 to 14.50 % and an increase in oxidation index of 6.10 to 430.50 % with unsaturated phospholipids for all tested conditions while with saturated phospholipids, no significant degradation was observed. Concerning the liposome formulation, no change in dispersion color and no phospholipid degradation were observed. However, a decrease in liposome size from 126.90 nm to 111.80 nm, 96.27 nm, 99.60 nm and 109.13 nm and an increase in the PdI from 0.208 to 0.271, 0.233, 0.285, and 0.298 were found with conditions C1, C2, C3 and C4 respectively. For the sterilization efficiency, conditions C1, C2 and C3 achieved the required sterility assurance level (SAL) of 10-6 for liposomes.

An innovative one step green supercritical CO2 process for the production of liposomes co-encapsulating both a hydrophobic and a hydrophilic compound for pulmonary administration.

Budesonide and salbutamol-loaded liposomes were prepared using an innovative one step supercritical CO2 method without any use of organic solvents. Liposomes composed of soybean phosphatidylcholine, cholesterol and PEGylated lipid (65/30/5% (m/m)) were produced with a size less than 200 nm, a PdI within the range of 0.3 and 0.35 and encapsulation efficiency for budesonide and salbutamol reaching to 94% and 40% respectively. The physical stability of the formulation was improved by optimizing a dry form by freeze-drying with trehalose in a 20:1 (trehalose:lipid) ratio and an increase in the percentage of PEGylated lipid from 5% to 15%. This dry form stored at 4 °C maintains 90-110% of the initial concentration of active compounds. The concentration of budesonide and salbutamol after 15 weeks was 522.92 ± 73.01 µg/mL and 144.86 ± 31.22 µg/mL respectively. These concentrations are close to the concentrations of these molecules in the pharmaceutical products Pulmicort® (500 µg/mL of budesonide) and Ventolin® (100 µg/dose). The formulation tested on lung cells, allows a cell viability of 71 ± 6%, which is not significantly different from untreated cells.

A supercritical fluid technology for liposome production and comparison with the film hydration method.

Liposomes were produced by an innovative method using supercritical carbon dioxide as a dispersing agent. A quality by design strategy was used to find optimal production conditions with specific parameters (lipid concentration, dispersion volume, agitation rate, temperature and pressure) allowing the production of liposomes with predicted physicochemical characteristics (particles size and PdI). Two conditions were determined with specific production parameters. It was shown that these two conditions allowed the production of liposomes of different compositions and that most of the liposome formulations had size and dispersity in accordance with the prediction values. The condition involving the higher lipid concentration showed a higher variability in terms of size and dispersity. However, this variability remained acceptable. This innovative supercritical method allowed the production of liposomes with physicochemical characteristics similar to those obtained by the conventional thin film hydration method. This new supercritical carbon dioxide method easily scalable in GMP conditions is a one-step production method contrarily to conventional methods which generally need an additional step as extrusion to homogenize the size of liposomes.