title: 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.
abstract: Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-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 10-5000pg/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 0.2mM 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 15h post infection, to the study termination at 21-22h. 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 (IC50) of 80nM 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.
authors.affiliation: Evotec, Drug Metabolism and Pharmacokinetics Department, Abingdon, Oxon, United Kingdom. Electronic address: paul.turnpenny@evotec.com.
authors.forename: Paul
authors.initials: P
authors.lastname: Turnpenny
authors.affiliation: Evotec, Drug Metabolism and Pharmacokinetics Department, Abingdon, Oxon, United Kingdom.
authors.forename: Anthony
authors.initials: A
authors.lastname: Padfield
authors.affiliation: Evotec, Drug Metabolism and Pharmacokinetics Department, Abingdon, Oxon, United Kingdom.
authors.forename: Patrick
authors.initials: P
authors.lastname: Barton
authors.affiliation: Evotec, Anti-infective Research Unit, Manchester, United Kingdom.
authors.forename: Joanne
authors.initials: J
authors.lastname: Teague
authors.affiliation: Department of Surgery, Massachusetts General Hospital, Boston, MA, United States; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, United States; Shriners Hospitals for Children, Boston, MA, United States.
authors.forename: Laurence G
authors.initials: LG
authors.lastname: Rahme
authors.affiliation: Spero Therapeutics, Cambridge, MA, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pucci
authors.affiliation: Spero Therapeutics, Cambridge, MA, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Zahler
authors.affiliation: Spero Therapeutics, Cambridge, MA, United States.
authors.forename: Aileen
authors.initials: A
authors.lastname: Rubio
mesh_terms: D042462:4-Quinolones / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism / Q000382:microbiology; D011552:Pseudomonas Infections / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism; D053038:Quorum Sensing / Q000502:physiology; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C528166:2-heptyl-4-quinolone; D042462:4-Quinolones
keywords: Alkyl-quinolone; Bioanalysis; LC–MS/MS; Pseudomonas aeruginosa