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Title: Novel small molecule modulator of the antioxidant response pathway : potential for therapy in inflammatory diseases/cancer
Author: Obliers, Miriam
ISNI:       0000 0004 5993 2267
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2016
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Development of safe and effective therapies for atherosclerosis has lagged behind the increasing epidemic health and economic impact of chronic inflammatory diseases. Inflammation and oxidative stress are consistent features and major mediators of the progression of chronic inflammatory diseases such as atherosclerosis. The activity of nuclear factor erythroid-2-related factor-2 (Nrf2), a redox sensitive transcription factor, increases in response to electrophilic and oxidative stress. Nrf2 plays a pivotal role in redox homeostasis maintenance and protection against oxidative stress through the transcriptional activation of antioxidants and detoxification enzymes. A novel Nrf2 activating chemical entity, AQP-3935, was identified from an industrial drug screen of marine microorganisms. AQP-3935 belongs to the chemical family of butenolides, a class of lactones with a heterocyclic ring structure consisting of four carbons. The aim of this study was to investigate the in vitro safety profile, its efficacy and mode of action as immune response modulator, and its potential as therapeutic drug for inflammatory diseases in an in vitro macrophage atherosclerosis model. In chapter 3, drug metabolism and pharmacokinetic studies showed favourable biochemical characteristics including solubility, membrane permeability, and absorption. Liver microsomes metabolised AQP-3935 and revealed a high intrinsic clearance. Potential drug-drug interactions such as cytochrome P450 inhibition or activation were not evident with CYP isoforms that are most commonly involved in drug-drug interactions. AQP-3935 was tested negative for genotoxicity. Despite glutathione adduct formation and yet unidentified metabolites, the biochemical properties and the pharmacokinetics profile of AQP-3935 indicated a potential use as therapeutic drug. In chapter 4, AQP-3935 upregulated Nrf2 and Nrf2 downstream antioxidants and detoxifying enzymes HO-1 and NQO1 in murine and primary human macrophages, which are key inflammatory cells. The Nrf2 activation by AQP-3935 was attenuated compared to clinically approved Nrf2 modulating agents such as dimethyl fumarate (DMF). Pro-inflammatory cytokine secretion, TNF-a and IL-6, was suppressed with AQP-3935 treatment in LPS stimulated human and murine macrophages with distinct effects in human and mouse, indicating the importance of translational studies from mouse into man. Distinct modes of action were identified for AQP-3935 and DMF, with AQP-3935 inhibiting phosphorylation of IRF3 and MAPK p38. Thus AQP-3935 demonstrated weaker Nrf2 modulatory activity than DMF, but strong and distinct antiinflammatory actions in macrophages, suggesting AQP-3935 as an alternative candidate to treat chronic inflammatory diseases. In chapter 5, a human in vitro macrophage atherosclerosis model was developed by establishing and optimising human macrophage foam cell formation by oxLDL uptake. The hypothesised modulatory effects of AQP-3935 to inhibit initiation or progression of atherosclerosis were not confirmed in our model as AQP-3935 did not significantly influence oxLDL uptake or expression levels of the scavenger receptor CD36, responsible for oxLDL uptake. Unchanged oxidation levels of human LDL suggested that the molecule AQP-3935 itself did not have antioxidant properties. However, it does not exclude efficacy and therapeutic application for other chronic inflammatory diseases as it attenuated the pro-inflammatory response in LPS stimulated macrophages. In chapter 6, global analysis of the transcriptome in LPS stimulated macrophages showed that AQP-3935 had weaker transcriptional effects compared to DMF. Minimal changes in gene expression were detected in response to either Nrf2 modulator, indicating that these agents mainly act post-transcriptionally since attenuation of proinflammatory signalling was demonstrated. In summary, AQP-3935 was not effective in our in vitro atherosclerosis model and therefore a repositioning of AQP-3935 to a different disease model should be explored. However, the favourable DMPK properties and the distinct modulation of inflammatory effects in LPS stimulated murine and primary human macrophages by AQP-3935 could open a potential new avenue for putative therapeutic targets to control chronic inflammatory diseases. Current treatments for chronic inflammatory diseases, such as anti-TNF-a biologics or Nrf2 activating molecules, have shown promising therapeutic results, but are limited to certain subgroups of patients, suggesting that molecular stratification of disease may be required.
Supervisor: Not available Sponsor: Medical Research Scotland
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Inflammation ; Antioxidants