California Sea Grant College Program

This dissertation is focused upon the study and pharmacological treatment of an inflammatory hallmark of rheumatoid arthritis. It involved the following phases: 1) The development and study of cellular models of rheumatoid arthritis. 2) The bioassay guided fractionation of 124 marine extracts. 3) The characterization of two purified compounds discovered through the bioassay model.

1 The initial phase of the work studied rheumatoid arthritis synoviocytes. These cells have an altered phenotype in the disease milieu. The production of 14kDa secreted Phospholipase A2 (sPLA2) by these cells was studied, and a secondary cellular model was developed through this study. The bioassay derived from this phase of study employed the human hepatoma cell line HepG2. Interleukin-1β was used to recreate the disease characteristic of the rheumatoid fibroblasts in this second cell type.

2 Through several stages of guided fractionation, a dozen primary candidate extracts were ultimately reduced to two. Subsequently, these two active extracts were purified to homogeneity.

3 The first of these compounds was trans-trans ceratospongamide. This cyclic peptide was first co-purified with a conformer, cis-cis ceratospongamide. The trans-trans form has an ED50 of 32 nM in the HepG2 model. The cis-cis form is inactive. Reporter and electrophoretic mobility shift assays indicated that the AP-1 pathway of cytokine signaling was a potential site of action for the active molecule. The second molecule, kalkitoxin, was purified after six stages of fractionation. In unpublished work, this molecule had previously been found ichthyotoxic and invertibraticidal. In the HepG2 model, this molecule has an ED50 of 7 nM. Subsequent studies showed that this molecule was active in an assay sensitive to microtubule poisons. The IL-1β signaling pathway leading to sPLA2 release is also very susceptible to microtubule poisons, and thus, it appears that kalkitoxin may be acting at this level in the bioassay.