Date of Award

Winter 11-6-2015

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Marc Devocelle

Second Supervisor

Deirdre Fitzgerald-Hughes

Funder/Sponsor

HEA

Keywords

Peptide antimicrobial cystic fibrosis prodrug

Abstract

Host defence peptides (HDPs) are short antimicrobial peptides that form a crucial component of the innate immune system. Deficiencies in HDP activity contribute to enhanced susceptibility to infections; this is the case in cystic fibrosis (CF). Exogenously-applied HDPs can compensate for these deficiencies and are attractive as new antiinfectives, but their development as antimicrobials is limited by cytotoxicity. This thesis describes the development of a series of HDP prodrugs whose cytotoxicity is reduced by net-charge decrease. As high levels of the host protease neutrophil elastase (NE) are colocalised in the lung with Pseudomonas aeruginosa infection, the enzyme is the target for prodrug activation. This system limits antimicrobial activity and cytotoxicity to areas infected with the bacterium. It is demonstrated here using in vitro assays that the prodrug modification reduces the antimicrobial activity against CF P. aeruginosa clinical isolates and reference strains. This activity is specifically restored with NE, both purified and in CF bronchoalveolar lavage (BAL) fluid. In vitro assays against epithelial cells, erythrocytes, and neutrophils demonstrate how the modification reduces cytotoxicity. Refinement of the model has produced pro-HDPs, with very low cytotoxicity, which are active in BAL fluid, can kill bacterial biofilms, and display low immunogenicity. The requirements for CF lung activity, such as the need for D-amino acids are also demonstrated. We show that the pro-HDPs are deliverable by nebulisation in a model of the human lung using a vibrating mesh nebuliser. In vivo results in mice show the toxicity and inflammatory effects of the pro-HDPs are reduced compared to the active peptides, in agreement with in vitro results. This thesis describes the design, synthesis, in vitro/in vivo evaluation, and drug delivery of pro-HDPs. It describes how the large and growing library of active HDPs may be harnessed for use in CF. The final candidate pro-WMR has the potential to be used in CF to treat infection and warrants further investigation.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

4,465 KB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2015.

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