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dc.contributor.advisorHouston, Todd A
dc.contributor.authorFoulkes, Broderick M
dc.date.accessioned2020-05-19T01:06:50Z
dc.date.available2020-05-19T01:06:50Z
dc.date.issued2020-05-01
dc.identifier.doi10.25904/1912/481
dc.identifier.urihttp://hdl.handle.net/10072/393974
dc.description.abstractBackground: Leishmaniasis is a DNDi listed disease caused by protozoan parasites of the kinetoplastida class. The disease currently spans over 80 countries, across the New World and Old World, potentially affecting 500 million people with 1 million new cases reported annually. Leishmaniasis is a vector-driven disease, utilizing two genus of sand fly, Phlebotomus sand fly is responsible for Old World transmission, whereas the Lutzomyia sand fly is responsible for New World transmission. The two main stages of leishmaniasis, are the diagnostic stage (promastigote in sand fly) and the infective stage (amastigote in mammalian cells). Dependent on strain, geography and location of infection, there are three main forms of leishmaniasis: cutaneous (subdivided into diffuse-cutaneous leishmaniasis (DCL) and disseminated-cutaneous leishmaniasis (DL)); mucocutaneous; and visceral (subdivided into post-kala azar dermal leishmaniasis (PKDL)). The DNDi status of leishmaniasis indicates that the pharmaceutical interest into research and development is shockingly low, resulting in very little progress into new treatments, limited to current therapeutics that suffer from severe toxicities (cardio, nephro, hepato, oto). These issues can be circumvented by utilising liposomal drug-carriers, as part of an increased interest in nanoparticle research across all glycosciences, modifying these drugs to interact better with the target cell or liposomal carrier can be of great benefit. Aims and Objectives: This project investigated the modification of current therapeutics in leishmanial treatment, paromomycin and compare the changes in antimicrobial efficacy. These modifications would revolve around enhanced binding affinity for macrophages and for liposomal carriers. This was achieved by modifying paromomycin at its reactive primary alcohols, using previously explored chemistry to attach long-chain fatty acids (LCFAs) to these reactive groups to create potential prodrugs. These were then subject to comparative kill efficiency studies against S. aureus, P. aeruginosa, and L. donovani DD8 cells in MIC assays and a resazurin based assay. A further objective was to investigate novel drug targets in leishmania, using LCFA-ligase as a potential target, as it has been reported this protein is differentially expressed, showing prominence and a potential for inhibition. This compound was also tested against L. donovani DD8 in the resazurin based assay. Methods: Paromomycin laurate and palmitate-based derivatives were synthesised by simple esterification, and the LCFA directive synthesised tert-butyl (4-(2-(decanesulfonyl)acetamido)butyl)carbamate) (N-Boc DSA) was synthesised by known methods. These compounds were characterised and subjected to MIC assays on S. aureus and P. aeruginosa, and a resazurin-based high content imaging (HCI) assay on axenic amastigotes of L. donovani DD8. Parallel synthesis and testing of neomycin LCFA derivatives were made by Dylan Farr, and tested against the same pathogens, comparatively with paromomycin derivatives. All experiments were conducted in triplicate and quadruplicate, with statistical differences being analysed by two-way analysis of variance (Two-way ANOVA). Values with P<0.05 were considered significant. Results and Discussion: Paromomycin palmitate and dipalmitate were synthesised with preference on dipalmitate testing due to increased binding affinity for liposomal carriers and macrophages. Laurate synthesis was much less effective under a multitude of conditions. Secondary compound Boc-DSA was synthesised for use in conjunction with the paromomycin derivatives. The paromomycin dipalmitate compound was tested against S. aureus and P. aeruginosa, with comparative aminoglycosides: neomycin palmitate, amikacin palmitate, and kanamycin palmitate. Against S. aureus, all compounds showed reduced activity at all concentrations, with paromomycin and amikacin being the least affected. Lower concentrations of antibiotic saw antagonistic effects with the lipid chain synergistically enhancing bacterial growth. P. aeruginosa testing was inconclusive due to increased pyocyanin expression, potentially increasing biofilm aggregation of the bacterial cells, reducing interactable surface-area for the aminoglycosides. Further testing with biofilm disruptors in conjunction may show improved results. Candidates paromomycin dipalmitate, N-Boc DSA, and neomycin palmitate were tested by V.Avery group at GRIDD (Griffith Institute for Drug Discovery) against L. donovani DD8 axenic amastigotes. Results showed <50% activity among derivative candidates, with lower activity even for paromomycin, a known anti-leishmanial agent. Morphological and pathophysiological changes due to geographical variations in leishmanial strains have been reported to have different effects on therapeutic efficacy. Although the reduced activity of the candidates can be noted for the DD8 strain, further testing on a variety of geographically relevant strains may show different activities. Human monocyte cytotoxicity THP-1 assays were performed in conjunction, <50% activity was similarly found for the described compounds. Conclusions and Future Remarks: Overall, the modification of ring-1 C6’ and ring-3 C5’ into a LCFA-derivative via esterification chemistry showed reduced activity at all concentrations against S. aureus, P. aeruginosa, and L. donovani DD8 amastigotes. Similar for comparative aminoglycosides of neomycin, amikacin, and kanamycin, although results against P. aeruginosa indicate potential biofilm aggregation. The reference compounds, including DSA, tested against L. donovani DD8 showed <50% inhibition, this may be indicative of morphological and pathophysiological changes due to geographical differences in the test strain. Future avenues worth pursuing is a range of LCFA-derivatives such as C10,12,14,18 for synergistic studies. The use of biofilm disruptors in conjunction with the reference compounds may improve P. aeruginosa activity, in addition to biofilm disruptors, the addition of surfactants to improve solubility of the LCFAs, these additions may have antagonistic effects and are worth investigating. Further testing among various geographically relevant strains of L. donovani would prove the theory put forth by Stuart et al. and show the efficacy of the reference compound across multiple geographically-dependent strains. Incorporation of the test compounds into liposomes were not achieved within this project, however investigations against the aforementioned L. donovani DD8 amastigotes, and against RAW 264.7 cells using the encapsulated compounds as comparative data is warranted.
dc.languageEnglish
dc.language.isoen
dc.publisherGriffith University
dc.publisher.placeBrisbane
dc.rights.copyrightThe author owns the copyright in this thesis, unless stated otherwise.
dc.subject.keywordsLeishmaniasis
dc.subject.keywordsleishmanial treatment
dc.subject.keywordsparomomycin
dc.subject.keywordslong-chain fatty acids
dc.subject.keywordsnovel drug targets
dc.titleDeveloping novel drug delivery methods for anti-leishmanial drugs.
dc.typeGriffith thesis
gro.facultyGriffith Health
gro.rights.copyrightThe author owns the copyright in this thesis, unless stated otherwise.
gro.hasfulltextFull Text
dc.contributor.otheradvisorGrice, Irwin D
gro.identifier.gurtID000000026554
gro.thesis.degreelevelThesis (Masters)
gro.thesis.degreeprogramMaster of Medical Research (MMedRes)
gro.departmentSchool of Medical Science
gro.griffith.authorFoulkes, Brody M.


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