Cyclosporine (CsA) is an eleven amino acid cyclic peptide immunosuppressive agent that has revolutionized organ transplantation. Due to its unique specificity for interaction with T-cells, CsA is projected for use in other autoimmune diseases. The benefits of this powerful immunosuppressant are counterbalanced by a number of untoward side-effects including nephrotoxicity, hypertension, and hepatotoxicity. The pharmacology and toxicity of cyclosporine (CsA) and its metabolites are not well understood. The clinical significance of the complex metabolism of CsA is undetermined but is potentially of great importance. Metabolites could be the principle immunosuppressive agents in vivo. On the other hand, toxic manifestations do not always correlate with concentrations of CsA in the blood and are possibly due to undetected metabolites. A better understanding of the consequences of the distribution and metabolism of CsA would provide more precise immunosuppression and should lessen morbidity and mortality in these patients. An understanding of the nephrotoxicity of the drug could allow its use in other groups of patients. This grant request proposes systematic study of the metabolites of CsA for immunosuppressive activity, for nephrotoxicity in both in vivo animal studies and in cell culture microassay systems, and for hepatotoxicity in mixed hepatocyte-Kupffer cell culture. Several tasks are involved in this study. First, sufficient amounts of metabolite must be isolated to allow testing of the immunosuppressive activity and toxicity. The purification will be accomplished with preparative-scale HPLC separation of bile constituents, and the structural identity of each metabolite will be established by fast atom bombardment mass spectrometry or HPLC/MS and nuclear magnetic resonance where appropriate. Immunosuppressive activity will be measured using both mixed lymphocyte culture and IL-2-dependent cell culture assays. Nephrotoxicity will be determined in an rat model system as well as a renal cell culture model system. A hepatotoxicity model will also be developed using mixed hepatocyte-Kupffer cell cultures. The use of cell culture techniques will provide the ability to assay activity in microgram quantities of metabolite.
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