The long-term goal of this program is to develop novel inhibitors of human sphingosine kinase (SK) that are effective for the treatment of arthritis. Sphingolipids are being increasingly recognized as key mediators of apoptosis, stress responses, cell differentiation and proliferation, and are known to regulate the inflammatory cascade of central importance in arthritis. Specifically, sphingosine-1-phosphate produced by SK mediates the effects of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), pro-inflammatory cytokines that are central to pathologic processes in this disease. In spite of accumulating evidence for a pivotal role of SK in regulating immune function, pharmacological inhibition of SK is an untested means of treating inflammatory diseases, including arthritis. This is largely due to the heretofore lack of pharmacologically useful SK inhibitors. To overcome this problem, we have recently identified novel inhibitors of human SK. We hypothesize that these SK inhibitors will block the effects of inflammatory cytokines and thereby ameliorate injury in arthritis. To provide proof-of-principle evaluations of the utility of these compounds, the following Specific Aims will be addressed in Phase I of this project: 1) To synthesize sufficient amounts of three SK inhibitors for in vitro and in vivo studies. Approximately 3 g each of three newly-identified small molecule inhibitors of SK will be synthesized following methods we have developed. The identity and purity of each compound will be confirmed by HPLC and spectroscopy. 2) To determine the in vitro effects of these SK inhibitors on the actions of the pro-inflammatory cytokines TNFalpha and IL-1beta. Each SK inhibitor will be tested in several in vitro assays for responses to TNFalpha and IL-1beta, including: SK activation, S1P generation, NFkappaB activation, and induction of adhesion molecules, VEGF and COX-2 expression. These studies will utilize human umbilical vein endothelial cells (HUVEC), human chondrocytes, human fibroblast-like synoviocytes, HL-60 human promyelocytic leukemia cells, and U937 human histiocytic lymphoma cells that model immune and target cells involved in the inflammatory response. 3) To evaluate the in vivo toxicities, pharmacokinetics and efficacies of SK inhibitors in models of arthritis. Each SK inhibitor will be tested for overt toxicity and pharmacokinetics in mice and rats following intraperitoneal and oral administration. The compounds will then be tested for efficacy in an acute inflammation model (topical phorbol ester), and in the collagen-induced arthritis model in mice and the adjuvant (Mycobacterium butyricum) arthritis model in rats.
