Abstract
Antibiotic use in medicine and agriculture requires caution to reduce the development of antimicrobial resistance (AMR). Antibiotics limit the impact and spread of bacterial infection, however use promotes AMR reducing their effectiveness. Regulating antibiotic use in humans and agriculture are useful tools to prevent the development of AMR but there is also a need for new therapeutic approaches.
The hypothesis being tested in this thesis is that responsive prodrugs, that allow for selective activation of an antibiotic, could target activity to the site of infection, reducing exposure of non-pathogenic bacteria to antibiotics, and slowing the development of AMR. This involves the installation of temporary moieties on the antibiotic that are removable by enzymes or environmental conditions found in increased concentrations in infections.
Single responsive antibiotic prodrugs and a dual responsive prodrug were synthesised and characterised using NMR, HRMS, IR and melting point. The dual responsive prodrug masked two functional groups of the antibiotic with masking groups responsive to two different triggers, this was hypothesised to increase the specificity of activation. HPLC was used to investigate the cleavage of the prodrugs, including uncleavable control compounds, to antibiotic upon exposure to trigger(s).
The effect of the masking groups on the antibacterial activity of the antibiotic was investigated by determining the minimum inhibitory concentration (MIC) of the prodrugs as compared to unmodified antibiotic. Single responsive prodrugs demonstrated a 100-fold reduction in the activity of the antibiotic, whereas the dual responsive prodrug showed a 4000-fold reduction in ability to inhibit the growth of E. coli. Repeated exposure to untriggered prodrug did not impact on susceptibility to the antibiotic, suggesting responsive prodrugs may reduce the development of resistance. The antimicrobial activity of the prodrugs was then examined in bacteria exposed to stress or following direct exposure to triggers experienced by bacteria in vivo. While the results from the stressed bacteria were inconclusive, exposure to triggers resulted in a return of antimicrobial activity.
Proof-of-concept studies were carried out using an in vivo murine skin abscess model. The responsive prodrugs reduced bacterial count to a similar degree to the reduction observed with the antibiotic, suggesting cleavage of the prodrugs in the infection microenvironment. Treatment with the uncleavable single prodrug gave a bacterial count similar to treatment with saline alone, suggesting if the masking group was not cleaved the antibacterial activity of the prodrugs was reduced.
A second generation of prodrugs responsive to a third trigger were investigated as a means of further increasing specificity of activation and the potential development of an oral therapy. Results from MIC assays confirmed dual masking reduces the activity of the antibiotic to a greater degree than single masking, however there were suggestions the third masking group investigated was not stable or specific.
Responsive antibiotic prodrugs have shown promise, selectively reducing antibacterial activity which is regained in the presence of infection. Further investigation is needed to investigate the effect of the masking groups on pharmacokinetics and confirm the prodrugs remain uncleaved in healthy tissues in animals.