oxalylglycine and Colitis

Inflammatory bowel disease (IBD) is characterized by epithelial barrier dysfunction with resultant inflammation as the mucosal immune system becomes exposed to luminal antigens. The hydroxylase inhibitor dimethyloxalylglycine (DMOG) reduces symptoms in experimental colitis through the upregulation of genes promoting barrier function and inhibition of epithelial cell apoptosis. The immunosuppressive drug cyclosporine reduces inflammation associated with IBD via suppression of immune cell activation. Given the distinct barrier protective effect of DMOG and the anti-inflammatory properties of cyclosporine, we hypothesized that combining these drugs may provide an enhanced protective effect by targeting both barrier dysfunction and inflammation simultaneously. We used the dextran sulfate sodium model of colitis in C57BL/6 mice to determine the combinatorial efficacy of cyclosporine and DMOG. While cyclosporine and DMOG ameliorated disease progression, in combination they had an additive protective effect that surpassed the level of protection afforded by either drug alone. The ability of DMOG to augment the anti-inflammatory effects of cyclosporine was largely due to preservation of barrier function and at least in part due to zonula occludens-1 regulation. We propose that combining the barrier protective effects of a hydroxylase inhibitor with the anti-inflammatory effects of cyclosporine provides added therapeutic benefit in colitis.

Topics: Amino Acids, Dicarboxylic; Animals; Apoptosis; Colitis; Cyclosporine; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Hypoxia; Hypoxia-Inducible Factor 1; Immunosuppressive Agents; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Protective Agents; Up-Regulation

Targeting hypoxia-sensitive pathways has recently been proposed as a new therapeutic approach to the treatment of intestinal inflammation. HIF-hydroxylases are enzymes which confer hypoxic-sensitivity upon the hypoxia-inducible factor (HIF), a major regulator of the adaptive response to hypoxia. Previous studies have shown that systemic (intraperitoneal) administration of hydroxylase inhibitors such as dimethyloxalylglycine (DMOG) is profoundly protective in multiple models of colitis, however the therapeutic potential of this approach is limited due to potential side-effects associated with systemic drug exposure and the fact that orally delivered DMOG is ineffective (likely due to drug inactivation by gastric acid). In order to overcome these issues, we formulated DMOG in a liquid emulsion drug delivery system which, when coated with specific polymer coatings, permits oral delivery of a reduced dose which is released locally throughout the colon. This colon-targeted DMOG formulation demonstrated increased relative colonic bioactivity with reduced systemic exposure and provided a similar degree of protection to systemic (intraperitoneal) administration at a 40-fold lower dose in DSS-induced colitis. In summary, targeted delivery of DMOG to the colon provides local protection resulting in enhanced efficacy with reduced systemic exposure in the treatment of colitis. This novel approach to targeting hydroxylase inhibitors to specific diseased regions of the GI tract may improve it's potential as a new therapeutic in inflammatory bowel diseases such as ulcerative colitis.

Topics: Administration, Oral; Amino Acids, Dicarboxylic; Animals; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Drug Delivery Systems; Female; HeLa Cells; Humans; Luciferases, Firefly; Mice; Mice, Transgenic; Mixed Function Oxygenases; NF-kappa B; Treatment Outcome

Intestinal ischemia/reperfusion injury (IR) is characterized by intermittent loss of perfusion to the gut, resulting in dramatic increases in morbidity and mortality. Based on previous studies indicating an anti-inflammatory role for hypoxia-inducible factor (HIF)-1-elicited enhancement of extracellular adenosine production via ecto-5'-nucleotidase (CD73) and signaling through the A2B adenosine receptor (A2BAR), we targeted HIF-1 during IR using pharmacological or genetic approaches. Initial studies with pharmacological HIF activation indicated attenuation of intestinal injury with dimethyloxallyl glycine (DMOG) treatment during murine IR. Although DMOG treatment was associated with induction of CD73 transcript and protein, DMOG protection was abolished in cd73(-/-) mice. Similarly, DMOG treatment enhanced A2BAR transcript and protein levels, whereas DMOG protection was abolished in A2BAR(-/-) mice. Finally, studies of mice with conditional HIF-1α deletion in intestinal epithelia or pharmacological inhibition of HIF-1 with 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin revealed enhanced tissue injury during IR. These studies indicated a tissue-protective role of HIF-dependent enhancement of intestinal adenosine generation and signaling during intestinal IR.

Topics: 5'-Nucleotidase; Amino Acids, Dicarboxylic; Animals; Colitis; Glycine; Hypoxia-Inducible Factor 1, alpha Subunit; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Receptor, Adenosine A2B; Reperfusion Injury; RNA, Messenger; Signal Transduction

This study investigates whether deoxy-2-[18F]fluoro-d-glucose (FDG) micro-positron emission tomography (μPET)/computed tomography (CT) can serve as a tool for monitoring of the commonly used dextran sodium sulfate (DSS)-induced murine model of inflammatory bowel disease (IBD).. DSS-colitis was induced in Sv129 mice. In a first experiment, four animals were serially scanned with CT and FDG-μPET on days 0, 3, 7, 11, and 14. The ratio of the mean voxel count of the PET images in the colon and the brain was compared with the histological inflammation score and the colonic myeloperoxidase levels. A second experiment was performed to investigate whether FDG-μPET was able to detect differences in inflammation between two DSS-treated groups, one receiving placebo (n = 4) and one receiving dimethyloxalylglycine (DMOG) (n = 4), a compound that protects against DSS-induced colitis.. The progression of the colonic/brain FDG-signal ratio (over days 0-14) agreed with the predicted histological inflammation score, obtained from a parallel DSS-experiment. Moreover, the quantification of normalized colonic FDG-activity at the final timepoint (day 14) showed an excellent correlation with both the MPO levels (Spearman's rho = 1) and the histological inflammation score (Spearman's rho = 0.949) of the scanned mice. The protective action of DMOG in DSS colitis was clearly demonstrated with FDG-μPET/CT (normalized colonic FDG-activity DMOG versus placebo: P < 0.05).. FDG-μPET-CT is a feasible and reliable noninvasive method to monitor murine DSS-induced colitis. The implementation of this technique in this widely used IBD model opens a new window for pathophysiological research and high-throughput screening of potential therapeutic compounds in preclinical IBD research.

Topics: Amino Acids, Dicarboxylic; Animals; Colitis; Dextran Sulfate; Disease Models, Animal; Female; Image Processing, Computer-Assisted; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Positron-Emission Tomography; Tomography, X-Ray Computed

A positive-feedback mechanism between hypoxia-inducible factor 1 alpha (HIF-1α) and metallothioneins (MTs) has been identified in different diseases. Both proteins have been independently proposed in the pathogenesis of inflammatory bowel disease (IBD); however, their relation has never been studied in the gut. In this study, we investigated the interaction between HIF-1α and MTs in colonic epithelial cells and during experimental colitis. Dimethyloxalylglycine (DMOG) was used to subject colonocytes to hydroxylase inhibition and HIF-1α stabilization in three experimental models (in vitro, in vivo and ex vivo). Small interfering RNA targeting HIF-1α (siRNA-HIF) and MT (siRNA-MT) together with zinc-mediated MT induction were used to study the interaction between HIF-1α and MT in HT29 cells. Acute colitis was induced in C57BL/6 mice using dextran sulphate sodium. MT expression and HIF-1α protein levels were measured using quantitative real-time PCR and ELISA, respectively. Vascular endothelial growth factor (VEGF) expression was quantified as an indirect measure of HIF-1 transcriptional activity. DMOG down-regulated MT expression in HT29 cells, in freshly isolated human colonocytes and in colonocytes isolated from mice treated with DMOG (p<0.05). SiRNA-HIF-treated cells displayed significant higher basal MT levels (p<0.05) and an attenuated MT down-regulation after DMOG treatment. In turn, HIF-1α stabilization was significantly lower in zinc-treated control cells, displaying high MT levels, compared to siRNA-MT cells treated with DMOG (p<0.05). In the course of experimental colitis, MT and VEGF mRNA expression were inversely related. MTs were induced in the acute phase and down-regulated during recovery. Opposing results were observed for VEGF expression levels (p<0.05). The present study underscores the inverse relation between HIF-1α and MT expression in colonocytes and during experimental colitis. The manipulation of MTs may represent a novel therapeutic strategy for patients suffering from IBD.

Topics: Amino Acids, Dicarboxylic; Animals; Colitis; Colon; Down-Regulation; HT29 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammatory Bowel Diseases; Metallothionein; Mice; Mice, Inbred C57BL; Models, Animal; RNA, Small Interfering; Vascular Endothelial Growth Factor A; Zinc

Prolyl and asparaginyl hydroxylases are key oxygen-sensing enzymes that confer hypoxic sensitivity to transcriptional regulatory pathways including the hypoxia inducible factor 1 (HIF-1) and nuclear factor-kappaB (NF-kappaB). Knockout of either HIF-1 or (IKKbeta-dependent) NF-kappaB pathways in intestinal epithelial cells promotes inflammatory disease in murine models of colitis. Both HIF-1 and NF-kappaB pathways are repressed by the action of hydroxylases through the hydroxylation of key regulatory molecules.. In this study we have investigated the effects of the hydroxylase inhibitor dimethyloxalylglycine (DMOG) on Caco-2 intestinal epithelial cells in vitro and in a dextran sodium sulfate-induced model of murine colitis.. DMOG induces both HIF-1 and NF-kappaB activity in cultured intestinal epithelial cells, and is profoundly protective in dextran-sodium sulfate colitis in a manner that is at least in part reflected by the development of an anti-apoptotic phenotype in intestinal epithelial cells, which we propose reduces epithelial barrier dysfunction.. These data show that hydroxylase inhibitors such as DMOG represent a new strategy for the treatment of inflammatory bowel disease.

Topics: Amino Acids, Dicarboxylic; Animals; Caco-2 Cells; Cell Culture Techniques; Colitis; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred C57BL; Mixed Function Oxygenases; NF-kappa B