Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse tissue using LC–MS/MS; and its application to a pharmacodynamic evaluation of MvfR inhibition

摘要

Highlights ? A sensitive method optimised to quantify alkyl-quinolone biomarkers in tissues. ? A chromatographic separation improvement using 2-Picolinic acid with tandem MS/MS. ? A quantitative accuracy flag with samples containing a ‘live’ bacterial sub-population. ? The high metabolic clearance of HHQ and PQS underlies the low in-vivo concentrations. ? A dose dependent reduction in biomarker tissue concentrations was observed. Abstract Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1 H )-quinolone (PQS) are important quorum sensing signals, which play a mediatory role in the pathogenesis of acute and chronic Pseudomonas aeruginosa infection. A targeted approach inhibiting the bacterial ‘multiple virulence factor regulon’ (MvfR) protein complex, offers the possibility to block the synthesis of MvfR-dependant signal molecules. Here, a high throughput bioanalytical method was developed using LC–MS/MS detection for the selective determination of HHQ and PQS in mouse tissue homogenate, over a sensitive range of 1–5000 and 10–5000 pg/mL, respectively. Chromatographic peak distortion of the iron chelator PQS was overcome with the applied use of a bidentate chelator mobile phase additive 2-Picolinic acid at 0.2 mM concentration, giving an improved separation and response for the analyte, whilst maintaining overall MS system robustness. Following thigh infection with P. aeruginosa strain 2-PA14 in mice, the concentration and time course of HHQ and PQS (4-hydroxy-2-alkyl-quinolone (HAQ) biomarkers) residing in the biophase were evaluated, and exhibited a low level combined with a substantial inter-individual variability. Quantifiable levels could be obtained from approximately 15 h post infection, to the study termination at 21–22 h. A dose dependant reduction in HAQ tissue concentrations at selected time points were obtained following MvfR inhibitor administration versus drug vehicle ( p 0.01, Kruskal–Wallis—one way ANOVA) and meta -analyses of several studies enabled an inhibitory concentration (IC 50 ) of 80 nM free drug to be determined. However, due to the experimental limitations a defined time profile for in-vivo HAQ production could not be characterised. Microsomal stability measurements demonstrated a rapid metabolic clearance of both alkyl quinolone biomarkers in the bacterial host, with a hepatic extraction ratio greater than 0.96 (the measurable assay limit). High clearance underpinned the low concentrations present in the well-perfused thigh tissue. Along with method development and validation details, this paper considers the kinetics of in-vivo HAQ bio-synthesis during Pseudomonas infection; and risks of biomarker over-estimation from samples which contain an exogenous population of bacteria.