The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

New drugs and methods to efficiently fight carbapenem-resistant gram-negative pathogens are sorely needed. In this study, we characterized the preclinical pharmacokinetics (PK) and pharmacodynamics of the clinical stage drug candidate apramycin in time kill and mouse lung infection models. Based on in vitro and in vivo data, we developed a mathematical model to predict human efficacy. Three pneumonia-inducing gram-negative species Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were studied. Bactericidal kinetics were evaluated with time-kill curves; in vivo PK were studied in healthy and infected mice, with sampling in plasma and epithelial lining fluid after subcutaneous administration; in vivo efficacy was measured in a neutropenic mouse pneumonia model. A pharmacokinetic-pharmacodynamic model, integrating all the data, was developed and simulations were performed. Good lung penetration of apramycin in epithelial lining fluid (ELF) was shown (area under the curve (AUC)_ELF/AUC_plasma = 88%). Plasma clearance was 48% lower in lung infected mice compared to healthy mice. For two out of five strains studied, a delay in growth (∼5 h) was observed in vivo but not in vitro. The mathematical model enabled integration of lung PK to drive mouse PK and pharmacodynamics. Simulations predicted that 30 mg/kg of apramycin once daily would result in bacteriostasis in patients. Apramycin is a candidate for treatment of carbapenem-resistant gram-negative pneumonia as demonstrated in an integrated modeling framework for three bacterial species. We show that mathematical modelling is a useful tool for simultaneous inclusion of multiple data sources, notably plasma and lung in vivo PK and simulation of expected scenarios in a clinical setting, notably lung infections.

The rise of multidrug-resistant (MDR) bacteria, particularly carbapenem-resistant Enterobacterales (CRE), poses a significant threat to public health. Infections caused by CRE, such as Escherichia coli and Klebsiella pneumoniae , are associated with high rates of antibiotic treatment failure. β-lactam antibiotics, like meropenem, remain crucial in treating these infections, but their efficacy is undermined by β-lactamase production. This study investigates the potential of APC24-7, a novel broad-spectrum β-lactamase inhibitor (BLi) with dual activity, to restore antimicrobial activity of meropenem against CRE clinical isolates. The in-vitro analysis of a diverse panel of clinically relevant E. coli and K. pneumoniae isolates expressing both serine- and metallo-β-lactamases demonstrated that APC24-7 effectively restored meropenem activity by reducing the minimum inhibitory concentrations (MICs) to below breakpoint. Time-kill assays confirmed that the combination therapy showed dose-dependent bacterial killing, with significant potentiation of meropenem activity against isolates expressing both serine- and metallo-β-lactamases. In-vivo efficacy evaluation in a murine thigh infection model further confirmed APC24-7’s potential to restore meropenem efficacy against meropenem resistant strains. These findings suggest that APC24-7offers a promising strategy to combat infections caused by β-lactamase-producing Enterobacterales .

The growing threat of antimicrobial resistance highlights the urgent need for new treatment strategies. Reliable animal data are essential to accelerate antibiotic development, and standardized murine infection models, like the neutropenic mouse pneumonia model, can improve the reproducibility and comparability of efficacy data across laboratories—key for clinical translation. This study aims to develop a standardized murine pneumonia model to enhance the clinical relevance of preclinical findings. Using a consensus lung infection protocol, we tested 32 Klebsiella pneumoniae and Pseudomonas aeruginosa isolates. Fifteen met predefined virulence criteria—showing at least a 1 log _₁₀ increase in bacterial load from baseline to endpoint, while maintaining mouse survival for at least 12 h post-inoculation. These isolates are available through the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Follow-up studies at independent sites confirmed the virulence of eight isolates with minimal variability in bacterial growth. These were added to the Collaboration for Prevention and Treatment of MDR Bacterial Infection (COMBINE Preclinical Bacterial Strain Repository at DSMZ. Based on this work, we propose a standardized experimental framework using this isolate panel to support robust preclinical testing of new antibacterial therapies. This model offers a reproducible, well-characterized platform for evaluating anti-infective candidates. We believe the COMBINE protocol can enhance the reliability and consistency of preclinical efficacy assessments and help reduce the number of animals required, aligning with the 3R principles—reduce, refine, replace—in animal research. IMPORTANCE: The rise of antibiotic-resistant bacteria has made it increasingly difficult to treat common infections, such as pneumonia. To develop new antibiotics, scientists rely on animal infection models to test how well potential drugs work before human trials. However, inconsistent methods between laboratories make it hard to compare results and slow the progress of new treatments. This study established and validated a standardized mouse pneumonia model for Klebsiella pneumoniae and Pseudomonas aeruginosa —two major pneumonia pathogens—across three international research centers. By identifying and sharing a set of well-characterized bacterial strains and a common experimental protocol, we provide a reliable foundation for comparing drug efficacy data. This model will help improve the quality and reproducibility of preclinical antibiotic research, reduce unnecessary animal use, and accelerate the discovery of new treatments against life-threatening bacterial infections.