Tu1616 Rpc1063 Is a Potent S1p1r Agonist With Efficacy in the Samp1yitfc Ileitis Model: New Evidence for Dysregulation of S1p Metabolism in Preclinical Models and Patients With Inflammatory Bowel Disease

Abstract

Body: Synthetic triterpenoids are effective anti-cancer agents. They also inhibit IL17 and improve autoimmune disease in mice, but have not been tested in murine IBD models. One prototype triterpenoid, CDDO-Im, inhibits STAT3 pathway activation. This transcription factor plays a role in the pathogenesis of IBD. Aims: The goals of the study were two-fold: To determine if ex vivo treatment with CDDO-Im would be effective for attenuating colonic IL-17 isoform secretion; and to determine if oral treatment with CDDOIm would modulate colonic IL-17 and improve DSS-induced colitis in mice. Methods: Study 1: Colitis was induced in male C57BL/6 mice (n = 9) by adding DSS 2% to the drinking water for 6 days. A control group of mice (n = 4) received filtered water. On day 6, colons were collected and organ culture was performed. Colonic strips (3 mm) were exposed to CDDO-Im at 0.5, 1, and 2 μM concentrations in the presence of IL-23 + IL-1 β (10 ng/ml of each). After 24 hours, supernatant was collected. IL-17 andIL-17F were measured by ELISA. Study 2: Using the same colitis paradigm described above, 20 mice were randomized into 3 groups (table 1). Mice were dosed by orogastric gavage with vehicle (10% Cremophor, 10% DMSO, 80% PBS) or CDDO-Im (20mg/kg) on study days 0, 1, 3 and 5. Body weights, water consumption and disease activity index (DAI) scores (0-12 scale) were recorded daily. Colons were collected on day 6. Distal colon was retained for colonic histology. Using coded slides, histology scores were determined with a 40-point severity scale. The proximal colon samples were analyzed for IL-17, IL-17F, IL-6 (by ELISA), and colonic MPO. Colonic nuclear protein extracts were used to measure the binding of STAT3 to a consensus site (specific Trans-Am® kit). Results: In colonic strips from DSS-treated mice, ex vivo treatment with CDDO-Im dose-dependently decreased both IL-17 and IL-17F secretion. The calculated IC50 values were 0.51 μM and 0.62 μM respectively. A similar inhibitory profile was found for CDDO-Im when using colonic strips from non-DSS-treated mice. In vivo, vehicle-treated mice lost weight between study days 2 and 6, while CDDO-treated animals gained weight (table 1). On study day 6, mean DAI, MPO, histology score, IL-17 and IL-6 values were all lower in the CDDO-Im group. These results are consistent with decreased colonic inflammation, compared to Vehicle/DSS treatment. Nuclear binding of STAT3 was also significantly attenuated (p , 0.05) in the CDDO-Im group of mice, compared to the corresponding vehicle cohort of animals. Summary: CDDO-Im attenuated IL-17 isoform secretion in an ex vivo model of colonic inflammation. In vivo, CDDO-Im improved multiple parameters of murine DSS-induced colitis, including STAT3 and IL-17. Conclusion: CDDO-Im has a unique pharmacological profile, which supports further testing in animal models of IBD. Table 1. Treatment Groups S-807 AGA Abstracts Values are Mean ± SEM. + p , 0.05 vs. Vehicle/Water or * p , 0.05 vs. Vehicle/DSS