A cellular model of inflammation for identifying TNF-α synthesis inhibitors

Neuroinflammation is a common facet of both acute and chronic neurodegenerative conditions, exemplified by stroke and by Alzheimer’s and Parkinson’s disease, and the presence of elevated levels of the proinflammatory cytokine, tumor necrosis factor-α (TNF-α) has been documented in each. Although initial TNF-α generation is associated with a protective compensatory response, its unregulated chronic elevation is generally detrimental and can drive the disease process. In such circumstances, therapeutic strategies that can both gain access to the brain and target the production of TNF-α are predicted to be of clinical benefit. An in vitro mouse macrophage-like cellular screen, utilizing RAW 264.7 cells, was hence developed to identify novel TNF-α lowering agents incorporating lipophilic physicochemical characteristics predicted to allow penetration of the blood-brain barrier. Cultured RAW 264.7 cells exposed to lipopolysaccharide (LPS) induced a rapid, marked and concentration-dependent cellular release of TNF-α into the cell culture media, which was readily detected by Enzyme Linked ImmunoSorbent Assay (ELISA). The effects of four characterized thalidomide-based TNF-α lowering agents were assessed alongside 10 novel uncharacterized compounds synthesized on the same backbone. One of these new analogs possessed activity of sufficient magnitude to warrant further investigation. Activity determined in the cellular model translated to an in vivo rodent model of acute LPS-induced TNF-α elevation. The utility of the TNF-α cellular assay lies in its simplicity and robust nature, providing a tool for initial pharmacological screening to allow for the rapid identification novel TNF-α lowering agents