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P056. Inhibition of matrix metalloproteinase-9 in intestinal inflammation by barbiturate–nitrate hybrids

S. O'Sullivan, J. Wang, J. Gilmer, C. Medina, Trinity College Dublin, School of Pharmacy and Pharmaceutical Sciences, Dublin, Ireland

Background

Matrix metalloproteinases (MMPs) are a family of endopeptidases involved in the turnover of the extracellular matrix. MMP-9 is known to be up-regulated in inflammatory bowel disease (IBD) and is associated with disruption of the epithelial barrier and progression of inflammation. Inhibition of this enzyme may aid in reducing the severity of the disease; however, the use of MMP inhibitors has been disappointing in clinical trials due to the lack of selectivity. Previous studies from our group have identified a series of barbiturate–nitrate compounds as potential MMP-9 inhibitors (J Med Chem 2012;55(5): 2154–62). Here, we aim to study the effects of barbiturate–nitrate compounds and their component parts (non-nitrate analogues and nitrate side chains) in intestinal inflammation, both in vitro and in vivo.

Methods

TNF-α and IL-1 β stimulated Caco-2 cells were used in all in vitro experiments and co-incubated with the compounds for 24 hours. Mechanistic elucidation was carried out using the soluble guanylate cyclase (sGC) inhibitor, ODQ, and the cGMP analogue, 8-Br-cGMP. In vivo experiments were carried out using male wistar rats given DSS 5% ad libitum in drinking water for 5 days and inhibitor compound or vehicle administered rectally, twice daily. MMP-9 activity was measured by gelatin zymography and analysis of gene expression was carried out using RT-qPCR. Taq-Man low density arrays (TLDA) were used to evaluate the expression of 90 inflammatory genes in the rat colon.

Results

The barbiturate–nitrate hybrid compounds dramatically attenuated MMP-9 expression in cytokine stimulated Caco-2 cells which is not mirrored in the non-nitrate analogues or the nitrate side chains alone. Furthermore, MMP-9 down-regulation by the barbiturate–nitrate hybrid compounds was cGMP dependent. ODQ abolished the inhibition of MMP-9 expression and this effect was reversed when the cells were co-incubated with 8-Br-cGMP. A representative compound shows promise in the DSS model of IBD by reducing various clinical and histological scores of inflammation and inhibiting the expression of pro-inflammatory mediators by reducing MMP-9 expression and activity.

Conclusion

This study proves the efficacy of the barbiturate–nitrate hybrids in MMP-9 inhibition. The compounds inhibit MMP-9 expression in both in vitro and in vivo models of IBD with potential to improve the currently available therapies.