A Novel mTOR Inhibitor Is Efficacious in a Murine Model of Colitis

Abstract

Purpose: Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T-cell activation, increased production of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ), and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess anti-proliferative activities (e.g., rapamycin and its analog everolimus; mammalian target of rapamycin (mTOR) inhibitors) and/or inhibit IFN-γ production (NCX-1015, a nitric oxide derivative of prednisolone) exhibit a therapeutic effect in murine models of colitis. In this study, we report that a novel aminopyridine based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model [the dextran sulfate sodium (DSS) model] of human colitis. Methods: Cell-based ELISAs, Western blot analysis and DSS-model of colitis were employed. Results: In vitro studies using western blot analysis and cell-based ELISA assays each showed that P2281 inhibits mTOR activity (69% inhibition of mTOR phosphorylation at 10 μM in ELISA assays). In vitro and in vivo assays of pro-inflammatory cytokine production revealed that P2281 diminishes induced IFN-γ synthesis/release (IC50: ∼20 μM) but not TNF-α synthesis/release (IC50: > 100 μM). In the disease model of colitis (i) macroscopic colon observations revealed that P2281, administered intraperitoneally, significantly inhibited DSS-induced weight loss (5.70 ± 2.4% for P2281-treated mice vs. 17.75 ± 3.07% for control mice), improved rectal bleeding index (0.42 ± 0.08 for P2281-treated mice vs. 0.67 ± 0.17 for control mice), decreased disease activity index (3.92 ± 0.8 for P2281-treated mice vs. 6.83 ± 0.76 for control mice) and reversed DSS-induced shortening of the colon (colon lengths: 11.15 ± 0.3, 6.85 ± 0.39, and 7.88 ± 0.3 cms for naïve, control and P2281-treated mice, respectively); (ii) histological analyses of colonic tissues revealed that P2281 attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa and resulted in protection against DSS-induced crypt damage. Conclusion: These results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting synthesis/release of pro-inflammatory mediators (e.g, IFN-γ) and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.