(T1130-05-26) Developing a Dual Prodrug for Triggered Antibiotic Activation

Purpose: The overuse of antibiotics promotes bacterial resistance (1). Limiting antibiotic use in medicine and agriculture is helpful in reducing the development of resistance, but there is also a need for new therapeutic approaches for the treatment of infections. Our research focuses on the development of triggerable antibiotic prodrugs that can be used to reduce unnecessary antibiotic exposure in people and animals. Prodrugs allow chemists to install a temporary moiety on a drug, removable by enzymes or other environmental stimuli (2). They can be used to target the release of active drug in specific organs or disease conditions (3). We have developed an antibiotic prodrug using two masking moieties. The stimuli we have chosen to focus on will trigger prodrug activation in infected tissues, thereby providing targeted activation. This dual prodrug requires two different triggers found in the area of infection for activation which we hypothesize will improve the specificity of release.

Methods: The dual prodrug, corresponding single prodrugs and non-triggerable prodrugs were designed and synthesised, followed by characterisation using Nuclear Magnetic Resonance (NMR), Mass Spectrometry, Infrared spectra and Melting Point analysis. Triggering of the prodrugs with their respective stimuli was confirmed using analytical High-Performance Liquid Chromatography (HPLC). The ability of the prodrugs and the active parent drug to kill Escherichia coli, before and after triggering, was determine in vitro using a Minimum Inhibitory Concentration (MIC) assay. The effect of prolonged exposure of E. coli to increasing concentrations of drug or prodrug over 8 days was investigated in a resistance assay (4). To investigate activation in infection conditions we used an in vivo murine skin infection where mice were infected with E. coli then treated with the parent antibiotic or prodrugs one hour post infection. After three days abscesses were measured, excised and homogenised to determine the number of viable bacteria.

Results: We have successfully synthesised a dual prodrug, based on a model antibiotic, as well as a small compound library of corresponding single masked prodrugs and uncleavable controls. HPLC studies show activation of single and dual prodrugs, and conversion to the parent antibiotic. In MIC assays the single prodrugs showed 100-fold reduced activity compared to the parent antibiotic whereas the dual prodrug showed an 8000-fold reduction. When activated in vitro by their corresponding triggers the prodrugs ability to kill bacteria was restored, suggesting the release of the active antibiotic. Bacteria exposed to increasing prodrug concentrations over eight days did not show an increase in the MIC while prolonged exposure to active antibiotic required eight times the MIC of the parent drug to eliminate growth. In vivo the prodrug reduced the number of viable bacteria suggesting that the activity of the parent antibiotic was restored.

Conclusion: New methods of antibiotic administration are needed to slow the development of antimicrobial resistance. Antibiotic prodrugs can reduce unnecessary exposure of non-pathogenic bacteria to antibiotics, reducing the selective pressure on the bacteria to develop resistance. We have successfully synthesised a dual prodrug using a model antibiotic. The combination of the two capping groups reduced the activity of the antibiotic more than single capping groups as the two masking moieties better hinder binding to the target enzymes. Both triggers were required to regain activity similar to that demonstrated by the parent antibiotic. This dual prodrug holds promise as a site-specific antibiotic treatment reducing unnecessary antibiotic exposure.

References: 1. Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nature Medicine. 2004;10(12):S122-S9.
2. Cheng AV, Wuest WM. Signed, Sealed, Delivered: Conjugate and Prodrug Strategies as Targeted Delivery Vectors for Antibiotics. ACS Infectious Diseases. 2019;5(6):816-28.
3. Clas S-D, Sanchez RI, Nofsinger R. Chemistry-enabled drug delivery (prodrugs): recent progress and challenges. Drug Discovery Today. 2014;19(1):79-87.
4. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015;517(7535):455-9.

Acknowledgements:

Funding: Otago Innovation Limited (OIL), New Zealand Pharmacy Education and Research Foundation, University of Otago School of Pharmacy and Department of Microbiology and Immunology.
All animal experiments have been approved by the University of Otago Animal Ethics Committee under Animal Use Protocol 20-74


Figure 1: Schematic of the proposed mechanism of the Dual Prodrug


Figure 2: Colony forming units per millilitre (CFU/mL) of solutions incubated with prodrugs and prodrugs pre-incubated with their triggers. (A) Dual prodrug. (B) Single Prodrug A. (C) Single Prodrug B. (D) Uncleavable Single Prodrug Control. Solid lines indicate the mean (n=3). Dashed line indicates limit of detection. Data shown are mean +/- SD (n=3)