Developing Drugs for Multi-Resistant Gram-Negative Infections in the Era of Antimicrobial Resistance.

Background: Several clinical trials have failed for drugs being considered for pneumonia for a variety of reasons. Antibiotic penetration into lung parenchyma is variable and differential exposure at the effect site is likely to be a risk factor for the emergence of antimicrobial resistance, treatment failure and poor clinical outcomes. Earlier identification and an improved understanding of the pulmonary spatial distribution of antibiotics in the preclinical phase will aid development of drugs for pneumonia. This thesis aims to investigate drugs and metabolites in tissue sections and provide semi-quantification to assess the lung penetration of meropenem using Matrix-Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI). Carbapenemase-producing Enterobacterales (CPE) are recognised as priority pathogens for research and drug development by the World Health Organisation. In particular, Enterobacterales that produce OXA-48-like β-lactamases have emerged as a global threat to public health due to the paucity of data on effective therapeutic options. Controversy surrounds the use of carbapenems for the treatment of infections caused by these bacteria, with both CLSI and EUCAST recommending susceptibility is inferred as reported by the MIC. This thesis aims to improve understanding of the pharmacokinetics-pharmacodynamics (PK-PD) of meropenem against OXA-48 producing Enterobacterales. Methods: To explore the use of MALDI MSI, new spatially resolved data of meropenem and polymyxin B in murine lung and kidney were generated. For meropenem, a semi-quantitative method to estimate concentration-time profiles within murine lung was developed. Differences between the semi-quantitative estimates obtained using MALDI MSI were compared to values determined using Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS). To further understand MALDI MSI data, the pharmacokinetics of meropenem and polymyxin B were determined in murine plasma, epithelial lining fluid (ELF) and lung using a single dose (i.v. meropenem; s/c polymyxin B) with sequential sacrifice design. To improve understanding of the PK-PD of meropenem against Enterobacterales producing OXA-48 the dynamic in vitro hollow fibre model of infection (HFIM) and in vivo murine thigh infection model have been used. Dose-ranging studies extending beyond exposures relevant to the licensed clinical regimens were performed. Results: Polymyxin B1 (PB1) is reported to be the most abundant component and this correlated with the MALDI MSI findings. The spatial distribution of PB1 was variable across lung parenchyma in different anatomical sections with the intensity of parent adducts decreasing over time while its metabolites (e.g. m/z 784.4581) were most clearly observed after 12 h. MALDI MSI data for meropenem were consistent with reported the pharmacokinetics of meropenem in mice i.e. short half-life and rapid clearance. The drug was distributed throughout lung within the first 15 min and appeared most intense centrally. The main metabolite was not visualized. As was the case for polymyxin B, spatial distribution was variable across different slices. Estimated concentrations of meropenem were higher than homogenate or ELF values when semi-quantitation was attempted. Pharmacodynamic data from the HFIM revealed meropenem monotherapy was not efficacious against Enterobacterales with blaOXA-48 (E. cloacae, meropenem MIC =1; K. oxytoca, meropenem MIC = 0.25). In all drug-exposed arms meropenem failed to cause sustained bacterial kill. For E. cloacae, the total bacterial population expanded and a resistant sub-population emerged. The mechanism(s) of resistance were explored using RT-qPCR and Whole Genome Sequencing (WGS). RT-qPCR data failed to show an increase in expression of blaOXA-48 in response to meropenem exposure for either challenge strain. WGS data suggested a high impact frameshift mutation in the gene encoding the OmpC porin. Other mutations potentially relevant to the pharmacodynamics, were also observed. The failure to consistently obtain the relevant pharmacodynamic target required for bacterial kill in the murine thigh infection model mean definite conclusions could not be drawn. Discussion: To our knowledge, this is the first time MALDI-MSI has been used to image polymyxin B or meropenem in murine lung after in vivo dosing. Numerous parent products, metabolites, and their associated adducts, made polymyxin B a particularly challenging drug to image, whereas rapid clearance and limited sensitivity of the instrument made meropenem challenging. This thesis presents MALDI MSI as an innovative tool with the aim of enhancing our understanding of spatial distribution and pharmacokinetics at select effect sites. These data provide important information on advantages, disadvantages and considerations for MALDI MSI in antimicrobial drug development. Data presented provide strong evidence to caution the use of meropenem monotherapy in clinical infections caused by Enterobacterales producing OXA-48. However, further preclinical and clinical PK-PD research is urgently needed across a range of Enterobacterales and non-fermenters to determine the appropriateness of current clinical breakpoints for OXA-48 producers, and to assess the role of combination therapy or new agents against these pathogens