Exploring the Effects of Toll-Like Receptor 4 Antagonism on Gastrointestinal Mucositis and Tumour Activity

Abstract

Gastrointestinal mucositis (GIM) is a hallmark of chemotherapy-induced gastrointestinal toxicity. It affects up to 80% of patients with cancer depending on their treatment regimen. Symptoms of GIM include weight loss, diarrhoea and bleeding. These symptoms can be so severe and debilitating that it often necessitates a reduction of treatment doses or discontinuation of the treatment which compromises patient survival. Unfortunately, there are no effective treatment strategies for these patients and more studies are required to develop potential intervention strategies. TLR4 is an intra- and extra-cellular receptor expressed on endosomes and cytoplasmic membranes. TLR4 recognises pathogen-associated molecular patterns (PAMPs) (flagellin and LPS) and damage-associated molecular patterns (DAMPs) (calprotectin, S100A8/9 HMGB1 and HSP70) through its co-receptors MD-2 and CD-14. The activation of TLR4 has been proposed to have a major influence on inflammatory signalling pathways and the pathogenesis of GIM. Inhibition of TLR4 has been postulated as an effective way to treat intestinal inflammation. However, there is a limited number of studies looking into the potential of TLR4 antagonism as a therapeutic approach for gastrointestinal (GI) inflammation. The work described in this thesis focussed primarily on the influence of TLR4 antagonism on GI toxicity stemming from irinotecan/CPT-11, a DNA topoisomerase I inhibitor used in the treatment of advanced colorectal cancer. The TLR4 antagonists studied were TAK-242 and IAXO-102, due to their potential to serve as alternative treatment options for GIM. Firstly, I modelled binding sites and affinity of IAXO-102, TAK-242 and SN-38 (the active metabolite of CPT-11) to the human TLR4/MD-2 complex, identifying specific amino acid residues of interaction and performed 3D structural analysis through in silico docking analysis. Computational techniques provide the possibility to explore drug development opportunities in order to rapidly provide structural, chemical, and biological data to improve understanding of potential drugs and their targets. The results from this study could contribute to rational development of therapeutic anti-inflammatory drugs targeting TLR4 in the GI tract. Secondly, I assessed the potential of the TAK-242 and IAXO-102 to attenuate GI inflammation in 2 different models; 1) an in vitro model using intestinal epithelial cell lines (T84, HT-29) and monocyte-like cells (U937), and 2) an ex vivo model using segments of mouse colon. Both models were induced with inflammation using TLR4 agonists and inflammatory mediators. Results from this study did not show significant protection with TAK-242 or IAXO-102 which, highlighted the limitation of in vitro and ex vivo models to accurately simulate GIM. Finally, from the in vitro and ex vivo studies, the TLR4 antagonist with the greatest potential for clinical development, IAXO-102 was evaluated for effectiveness to attenuate GI inflammation as well as supress tumour activity in a colorectal-tumour bearing mouse model of CPT-11-induced GIM. Results showed that IAXO-102 was able to prevent diarrhoea in mice treated with CPT-11 as well as reduce tumour volume. However, it had no effect in protecting the colon from tissue damage or changing proliferation and apoptosis rates in both the colon and tumour. As such, it was concluded TLR4 activation plays a partial role in GIM development but further research is required to understand the specific inflammatory signals underpinning tissue-level changes.