Background and purpose: The adenosine 2B (A 2B ) receptor is the predominant adenosine receptor expressed in the colon. Acting through the A 2B receptor, adenosine mediates chloride secretion, as well as fibronectin and interleukin (IL)-6 synthesis and secretion in intestinal epithelial cells. A 2B receptor mRNA and protein expression are increased during human and murine colitis. However, the effect of the A 2B receptor in the activation of the intestinal inflammatory response is not known. In this study, we examined the effect of A 2B receptor antagonism on murine colitis. Experimental approach: Dextran sodium sulphate (DSS)-treated mice and piroxicam-treated IL-10 À/À mice were used as animal models of colitis. The A 2B receptor-selective antagonist, ATL-801, was given in the diet. Key results: Mice fed ATL-801 along with DSS showed a significantly lower extent and severity of colitis than mice treated with DSS alone, as shown by reduced clinical symptoms, histological scores, IL-6 levels and proliferation indices. The administration of ATL-801 prevented weight loss, suppressed the inflammatory infiltrate into colonic mucosa and decreased epithelial hyperplasia in piroxicam-treated IL-10 À/À mice. IL-6 and keratinocyte-derived chemokine (KC) concentrations in the supernatants of colonic organ cultures from colitic mice were significantly reduced by ATL-801 administration. Conclusions and implications: Taken together, these data demonstrate that the intestinal epithelial A 2B receptor is an important mediator of pro-inflammatory responses in the intestine and that A 2B receptor blockade may be an effective therapeutic strategy to treat inflammatory bowel disease.

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A(1) adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A(1)AR (hA(1)AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A(1)AR agonist [(125)I]ABA and membranes from CHO-K1 cells stably expressing the hA(1)AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A(1)AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH(2))(n)()- bridge, n = 3 < n = 4 < n = 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA(1)AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.