Abstract
The emergence of antimicrobial resistance and the impact of broad-spectrum antibiotics on the host microbiota and the environment has prompted the need for targeted antibacterial therapies. This thesis explores the development of novel, triggerable prodrugs of cloxacillin, an antibiotic widely used in the treatment of bovine mastitis in the agricultural sector in Aotearoa New Zealand. The overarching goal of this research was to design cloxacillin prodrugs that are selectively activated in the presence of bacterial microenvironment stimuli present at elevated levels at sites of infection, thereby limiting off-target effects and reducing antimicrobial pressure on commensal microbial communities. Three stimuli-responsive cloxacillin prodrugs were successfully designed and synthesised, incorporating cleavable linkers sensitive to infection-associated triggers: reactive oxygen species (ROS), hydrogen sulphide (H2S), and nitroreductase (NTR) activity.
Prodrug stability was evaluated under a range of physiologically and agriculturally relevant conditions (Chapter Two). The NTR- and H2S-triggerable prodrugs exhibited robust stability over a seven-day period in both PBS and raw milk, supporting their suitability for on-farm storage and application. In contrast, the ROS-triggerable prodrug displayed instability under several test conditions, with evidence of partial spontaneous conversion. An untriggerable negative control likewise proved insufficiently stable, highlighting the need for further development of appropriate experimental controls.
Prodrugs were characterised using chromatographic and spectrometric analytical techniques, and their activation profiles were evaluated in vitro using chemical trigger systems (Chapter Three). Antimicrobial activity was assessed against the clinically relevant mastitis-associated pathogens Staphylococcus aureus Newman and Streptococcus uberis to confirm reduced activity in the masked state and restoration of antibacterial efficacy following triggering. The NTR- and H2S-triggerable prodrugs demonstrated the most effective masking, with untriggered MIC values of 250 µg/mL against both organisms (3200-fold above cloxacillin; MIC 0.078 µg/mL), and near-complete restoration of potency upon activation. The NTR-triggerable prodrug achieved MICs of 0.078 µg/mL against S. aureus (equivalent to cloxacillin) and 0.156 µg/mL against S. uberis (two-fold above cloxacillin), and the H2S-triggerable prodrug achieving MICs of 0.156 µg/mL and 0.313 µg/mL against S. aureus and S. uberis respectively. The untriggerable control showed no measurable antimicrobial activity (MIC >500 µg/mL; >6400-fold relative to cloxacillin), confirming the integrity of the prodrug design. The ROS-triggerable prodrug exhibited partial spontaneous activation in its untriggered state (MIC 1.25 µg/mL; 16-fold above cloxacillin), likely reflecting its intrinsic instability, though full potency was restored upon triggering (MIC 0.078 µg/mL). MIC values remained consistent in a 50:50 raw milk:MHB medium, indicating that milk components did not substantially alter antimicrobial performance.
The most effective prodrugs were assessed in an in vivo murine skin abscess model, allowing evaluation of therapeutic efficacy and activation under physiologically relevant conditions (Chapter Four). At an initial dose of 8 mg/kg cloxacillin equivalent, the NTR-triggerable prodrug demonstrated encouraging antimicrobial activity, prompting further evaluation at 25 mg/kg. At this elevated dose, both the NTR- and H2S-triggerable prodrugs significantly reduced abscess size and bacterial burden in S. aureus-infected mice, with outcomes comparable to or exceeding those of the parent antibiotic administered at equivalent doses. No increase in local tissue toxicity or adverse effects was observed, supporting the suitability of this dose for further preclinical development. The ROS-triggerable prodrug demonstrated some reduction in abscess size and bacterial load at 8 mg/kg but was excluded from further investigation owing to its poor stability under physiological conditions.
Selected prodrugs were incorporated into injectable Pluronic™ F-127 hydrogel formulations to enable sustained local delivery (Chapter Five). Rheological analysis confirmed that prodrug loading did not significantly alter the mechanical properties of the hydrogel, preserving its thermoresponsive, in situ-gelling behaviour. The hydrogel fully degraded within ten days under aqueous conditions, and HPLC analysis confirmed sustained cloxacillin release over this period. In vivo, the formulation was well tolerated, with no observed toxicity and complete dissipation from the injection site within 24 hours. Mice treated with NTR-triggerable prodrug-loaded hydrogels exhibited significant reductions in both abscess size and bacterial burden relative to controls, supporting in vivo prodrug activation within the hydrogel system. The H2S-triggerable prodrug formulation did not achieve equivalent bacterial clearance, indicating the need for further optimisation of activation kinetics and release profiles. Notably, across all treatments, hydrogel formulation appeared to preserve reductions in abscess size whilst consistently attenuating bacterial killing relative to PBS-based dosing, a key limitation identified for future investigation.
Collectively, this work presents a platform for site-specific antibiotic delivery with the potential to improve treatment outcomes and reduce ecological impact. The NTR-triggerable prodrug emerged as the most promising candidate, demonstrating effective masking of antimicrobial activity in its untriggered state (3200-fold MIC elevation), near-complete restoration of cloxacillin potency upon activation, and significant in vivo efficacy at 25 mg/kg in both solution and hydrogel formulations. The integration of triggerable chemistry with tailored delivery systems marks a step forward in the development of next-generation antimicrobial strategies, with particular relevance to agricultural biosecurity and sustainable antibiotic use in Aotearoa New Zealand.