Infection of bronchial epithelial cells with pre-grown P. aeruginosa biofilms at Air-Liquid Interface for testing nebulized anti-infectives in vitro

Patients suffering from lung diseases as cystic fibrosis are especially at risk contracting chronic bacterial infections1,2. In anti-infective research, one of the alternatives to experiments on animals are novel in vitro models3,4. In contrast to traditional tests, that comprise bacteria and the agent in one well, novel co-cultures with mammalian cells help to get a deeper understanding of host-pathogen interactions3,5. This work is aimed at creating and testing a realistic in vitro bronchial model of a P. aeruginosa6 biofilm infected patient and its treatment with anti-infectives as inhaled tobramycin. In the first of two experimental parts, the technical procedure for depositing antibiotics on top of Transwell® inserts by using a device developed at HIPS is presented. The deposition of a metered dose is thereby controlled by the invested volume. The system is tested for its applicability for infection research by assessing toxicity on cells, amongst others. The second part presents the development and testing of a biofilm infected in vitro model of the lung with cells growing at the air-liquid interface. Either tobramycin and a novel pathoblocker7 was used for testing. On the basis of the previous thesis of Dr. Juntke8,9, a chronic-like system could be established via multiple consecutive aerosolized treatments with tobramycin4.Patienten, die an Lungenkrankheiten wie der Mukoviszidose leiden, sind besonders anfällig, bakterielle Erkrankungen zu entwickeln1,2. In vitro Modelle sind dabei eine der Alternativen zu in vivo Tierversuchen3,4. Anders als traditionelle Tests, die nur Bakterien und Antibiotika in vitro untersuchen, können neue Co-Kulturen mit humanen Zelllinien zum Verständnis der „Host-Pathogen Interaction“ beitragen3,5. Diese Arbeit befasst sich mit der Erstellung und Testung eines möglichst realistischen in vitro Bronchialmodells eines mit einem P. aeruginosa6 Biofilm infizierten Patienten und dessen Behandlung durch Inhalation von Anti-Infektiva wie z.B. Tobramycin. Im ersten von zwei experimentell erarbeiteten Teilen wird ein neu entwickeltes System vorgestellt, mit dem man Antibiotika auf Transwell® Einsätzen als Aerosol applizieren kann. Die deponierte Dosis wird dabei durch das Volumen der zu vernebelnden Flüssigkeit kontrolliert. Die Eignung für ein entsprechendes Infektionsmodell wird unter anderem durch die Bestimmung der Toxizität auf Zellen geprüft. Der zweite Teil befasst sich mit der Entwicklung und Testung eines mit Biofilm infizierten Modells der Lunge mit an der Luft ausgesetzten Bronchialepithelzellen. Dabei wurde neben Tobramycin auch ein neuartiger Pathoblocker7 eingesetzt. Aufbauend auf die vorausgegangene Dissertation von Dr. Juntke8,9 konnte durch konsekutive Applikationen von aerosolisiertem Tobramycin ein über mehrere Tage infiziertes Modell erreicht werden4

A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development

A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development.H2020 Marie Skłodowska-Curie Action

Evaluation of the anti-peristaltic effect of glucagon and hyoscine on the small bowel: comparison of intravenous and intramuscular drug administration

PURPOSE: To evaluate prospectively duration and effectiveness of aperistalsis achieved by glucagon(GLU) or hyoscine N-butylbromide(HBB) following various administration routes. MATERIALS AND METHODS: Six volunteers underwent Magnetic Resonance Imaging (MRI) after standardized oral preparation in random order five separate MR examinations with both spasmolytic agents (HBB intravenous(i.v.) or intramuscular(i.m.), GLU i.v. or i.m., and a combined scheme). The MR protocol included a sagittal 2D cross-section of the small bowel with a temporal resolution of 0.55 s acquired over 60 to 90 min. To quantify bowel motility, small bowel cross-sectional areas were summated over time. RESULTS: The anti-peristaltic i.v. effects of HBB and glucagon started on average after 85 s/65 s and ended after 21 min/23.3 min, respectively. By comparison, the anti-peristaltic effects of i.m. HBB and glucagon started significantly later 5.1/11.6 min (P = 0.001; Wilcoxon signed ranks test) and lasted for 17.7/28.2 min with greater inter-individual differences (P = 0.012; Brown-Forsythe test). The combined scheme resulted in a rapid onset after 65 s with effect duration of 31 min. CONCLUSION: Anti-peristaltic effects on the small bowel are drug dependant, i.e., their onset is faster and more reliable when administering i.v. than i.m.. Combining i.v. GLU with i.m. HBB provides an early onset of effect, sustained spasmolysis and the highest degree of motility impairment. KEY POINTS: • Anti-persitaltic agents are widely used before various diagnostic procedures of the abdomen. • The combination of iv-glucagon with im-hyoscine provides reliable spasmolysis with early onset. • Intravenous spasmolysis is more reliable compared to intramuscular administration. • Intravenous glucagon has a prolonged spasmolytic effect compared to intravenous hyoscine