Discovery of a Gut-Restricted JAK Inhibitor for the Treatment of Inflammatory Bowel Disease.

To identify Janus kinase (JAK) inhibitors that selectively target gastrointestinal tissues with limited systemic exposures, a class of imidazopyrrolopyridines with a range of physical properties was prepared and evaluated. We identified compounds with low intrinsic permeability and determined a correlation between permeability and physicochemical properties, clogP and tPSA, for a subset of compounds. This low intrinsic permeability translated into compounds displaying high colonic exposure and low systemic exposure after oral dosing at 25 mg/kg in mouse. In a mouse PK/PD model, oral dosing of lead compound 2 demonstrated dose-dependent inhibition of pSTAT phosphorylation in colonic explants post-oral dose but low systemic exposure and no measurable systemic pharmacodynamic activity. We thus demonstrate the utility of JAK inhibitors with low intrinsic permeability as a feasible approach to develop gut-restricted, pharmacologically active molecules with a potential advantage over systemically available compounds that are limited by systemic on-target adverse events.

LC/MS/MS Profiling of Tissue Oxysterols and its Application in Dextran Sodium Sulphate Induced Mouse Colitis Models.

We have developed a workflow to extract, separate, and semi-quantify bioactive oxysterols from mouse colon tissues and fecal matters using solid- and liquid-phase extractions, enzymatic and chemical modifications, and stable-isotope dilution LC/MS/MS. The method was applied to a dextran sodium sulphate (DSS)-induced mouse colitis model, which revealed that one particular dihydroxycholesterol (diOHC), 7α,25-diOHC, was significantly elevated in both colon tissue and fecal matters of mice with colitis compared to that in naïve mice. The extent of 7α,25-diOHC elevation was positively correlated with colitis severity.

Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine.

DNA vaccines show efficacy in many preclinical models, but these results have not yet translated to consistent clinical efficacy. Co-administration of molecularly encoded adjuvants is one approach that may enable DNA vaccines to achieve enhanced immune response induction in humans. Interleukin-18 (IL-18) is a Th1-type cytokine that has been shown to augment the activity of DNA vaccines in some preclinical models. A prostate-specific antigen (PSA) DNA vaccine was tested in a mouse tumor model system to explore the impact of co-administration of a pIL-18 plasmid. Low doses of the pPSA vaccine were not capable of inducing tumor protection, but when pIL-18 was co-administered, complete tumor protection was observed in all mice. Tumor protection was mediated by both CD4(+) and CD8(+) T cells. Detailed analysis of the immune response in mice immunized with either pPSA or pPSA/pIL-18 demonstrated that pIL-18 skewed the PSA-specific immune response toward Th1. More importantly, stronger CD4(+) and CD8(+) T cell responses developed in the pPSA/pIL-18-immunized mice, with faster kinetics. These results suggest that IL-18 is a powerful adjuvant molecule that can enhance the development of antigen-specific immunity and vaccine efficacy.

A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice.

MUC1 (mucin 1) is a tumor-associated antigen that is overexpressed in many adenocarcinomas. Active immunotherapy targeting tumors expressing MUC1 could have great treatment value. MUC1 DNA vaccines were evaluated in MUC1 transgenic (MUC1.Tg) mice challenged with MC38/MUC1+ tumor cells. Vaccination with MUC1 plasmid DNA (pMUC1) alone was insufficient to induce tumor protection. However, co-administration of pMUC1 with a plasmid encoding murine interleukin-18 (pmuIL-18) resulted in significant tumor protection and survival after tumor challenge. Protection was durable in the absence of additional vaccination, as demonstrated by continued protection of vaccinated mice following tumor rechallenge. Mice surviving challenges with MC38/MUC1+ cells showed significant protection after challenge with MUC1(-) MC38 tumor cells, suggesting that these mice had developed immune responses to epitopes shared between the tumor cell lines. Antibody-mediated depletion of lymphocyte subsets demonstrated that protection was due largely to CD4+ T cells. This work demonstrates that a naked DNA vaccine can break tolerance to MUC1 and induce an immune response capable of mediating both significant protection from tumor challenge and increased survival.

Induction of Th1-type immunity and tumor protection with a prostate-specific antigen DNA vaccine.

Prostate specific antigen (PSA) is a serum marker that is widely used in the detection and monitoring of prostate cancer. Though PSA is a self-antigen, T cell responses to PSA epitopes have been detected in healthy men and prostate cancer patients, suggesting it may be used as a target for active immunotherapy of prostate cancer. A PSA DNA vaccine (pPSA) was evaluated in mice and monkeys for its ability to induce antigen-specific immune responses. Mice immunized intradermally with pPSA demonstrated strong PSA-specific humoral and cellular immunity. The anti-PSA immune responses were skewed toward Th1, as shown by high IFNgamma and IL-2 production. The immune response was sufficient to protect mice from challenge with PSA-expressing tumor cells. Tumor protection was durable in the absence of additional vaccination, as demonstrated by protection of vaccinated mice from tumor rechallenge. Furthermore, pPSA vaccination induced PSA-specific antibody titers in male cynomolgus monkeys, which express a closely related PSA gene. These results demonstrate that vaccination with pPSA may be able to break tolerance and can induce an immune response that mediates tumor protection.