Objective. To evaluate the role of the MEK/ERK MAP kinase pathway in murine collagen-induced arthritis (CIA) using the selective MEK inhibitor PD184352. We examined the effects of the inhibitor in cytokine-stimulated synovial fibroblasts and in cytokine-induced arthritis in rabbits to investigate its antiinflammatory mechanisms.Methods. Murine CIA was used to assess the effects of the selective MEK inhibitor on paw edema, clinical scores, weight loss, histopathologic features, and joint levels of p-ERK. Western blotting and immunohistochemistry techniques were used to assess p-ERK in human and rabbit synovial fibroblasts and synovial tissue from rheumatoid arthritis (RA) patients. Interleukin-1␣ (IL-1␣)-stimulated stromelysin production in rabbit synovial fibroblasts was assessed by enzyme-linked immunosorbent assay. A rabbit IL-1␣-induced arthritis model was used to assess the effects of the inhibitor on IL-1␣-induced MEK activity, stromelysin production, and cartilage degradation.Results. In the CIA model, PD184352 inhibited paw edema and clinical arthritis scores in a dosedependent manner. Disease-induced weight loss and histopathologic changes were also significantly improved by treatment. Inhibition of disease-induced p-ERK levels in the joints was seen with the inhibitor. Levels of p-ERK in the synovium were higher in RA patients than in normal individuals. PD184352 reduced IL-1␣-induced p-ERK levels in human RA synovial fibroblasts. The production of p-ERK and stromelysin was also inhibited in IL-1␣-stimulated rabbit synovial fibroblasts. We observed IL-1␣-induced p-ERK in the synovial lining, subsynovial vasculature, and articular chondrocytes. IL-1␣-induced stromelysin production and proteoglycan loss from the articular cartilage were reduced by PD184352.Conclusion. These data demonstrate the inhibition of murine CIA by PD184352, support the hypothesis that antiinflammatory activity contributes to the mechanism of action of the inhibitor, and suggest that a selective inhibitor may effectively treat RA and other inflammatory disorders.

Objective To evaluate the decrease of cartilage destruction by a novel orally active and specifi c matrix metalloproteinase 13 (MMP-13) inhibitor in three different animal models of rheumatoid arthritis (RA). Materials and methods The SCID mouse co-implantation model of RA, the collagen-induced arthritis (CIA) model in mice and the antigen-induced arthritis model (AIA) in rabbits were used. Results In the SCID mouse co-implantation model, the MMP-13 inhibitor reduced cartilage destruction by 75%. In the CIA model of RA, the MMP-13 inhibitor resulted in a signifi cant and dose-dependent decrease in clinical symptoms as well as of cartilage erosion by 38% (30 mg/kg), 28% (10 mg/kg) and 21% (3 mg/kg). No signifi cant effects were seen in the AIA model. No toxic effects were seen in all three animal models. Conclusion Although several MMPs in concert with other proteinases have a role in the process of cartilage destruction, there is a need for highly selective MMP inhibitors to reduce severe side effects that occur with non-specifi c inhibitors. Signifi cant inhibition of MMP-13 reduced cartilage erosions in two of three tested animal models of RA. These results strongly support the development of this class of drugs to reduce or halt joint destruction in patients with RA.

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.

No abstract

Subcutaneous (sc) transposition of the spleen followed by fine needle biopsies (FNB) from the organ allows the evaluation of relative numbers of splenic cells in Lewis lung tumor-bearing mice. Absolute cell numbers (N) can be calculated when the aspirated cells are weighed on a precision balance (m) and counted (c) after resuspension in a defined volume (v): N = C X V/m X M. The weight from the whole spleen (M) is derived from a calibration curve. Splenic nucleated cells and their subsets--as determined by the use of monoclonal antibodies--can thus probably be evaluated with a degree of accuracy which was not possible before. Shifts of splenic cells from and to the organ can be monitored.