Abstract
5-Aminosalicylic acid (5-ASA) is widely prescribed for treating inflammatory bowel disease (IBD), yet its mechanism of action remains unclear. IBD, a chronic inflammatory disorder affecting the gastrointestinal tract, includes both Crohn’s disease and ulcerative colitis. While the exact cause of IBD is uncertain, an overactive immune response, particularly involving neutrophils at sites of intestinal inflammation, may play a significant role. Myeloperoxidase, an enzyme found in neutrophil granules, could contribute to IBD pathology by producing reactive oxidants, such as hypochlorous acid (HOCl), that lead to further tissue damage of the intestinal epithelium. Previous findings have shown an association between 5-ASA and myeloperoxidase. Thus, I hypothesised that 5-ASA may reduce inflammation by inhibiting myeloperoxidase production of HOCl, thereby limiting the resulting tissue damage.
To determine the interaction between 5-ASA and myeloperoxidase, various in vitro techniques were used, including UV-Vis spectrophotometry, mass spectrometry, and nuclear magnetic resonance (NMR). Spectrophotometric analysis revealed that 5-ASA reversibly inhibited myeloperoxidase production of HOCl at physiological concentrations. Evidence for this was observed with both purified myeloperoxidase and isolated neutrophils. It is likely this inhibitory effect was due to 5-ASA acting as a competitive peroxidase substrate that outcompetes the oxidation of chloride by myeloperoxidase. 5-ASA may also scavenge HOCl. Analysis of 5-ASA reacted with HOCl revealed oxidation products that formed conjugates with glutathione. These products could be formed in the gut of IBD patients and may be important indicators of HOCl levels, and thus inflammation, in the intestine. Conjugation with glutathione occurred via a thiol linkage, suggesting that protein thiols could be similarly targeted by oxidised 5-ASA. Enzyme activity can be greatly influenced by a thiol moiety. Thus, modulating the activity of inflammatory enzymes could provide an additional mechanism of 5-ASA.
This thesis provides novel insights into the anti-inflammatory mechanisms of 5-ASA, demonstrating its ability to inhibit myeloperoxidase in vitro and scavenge reactive oxygen species. Oxidised 5-ASA may also interact with thiol-dependent enzymes, which could provide additional therapeutic benefits. 5-ASA oxidation products with and without glutathione conjugation should be further investigated as potential biomarkers of IBD inflammation.