The effects of phosphodiesterase type 4 inhibitors on tumour necrosis factor‐α and leukotriene B4 in a novel human whole blood assay

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Abstract
1. The aim of this study was to assess the inhibitory activities of phosphodiesterase type 4 (PDE4) inhibitors on tumour necrosis factor-alpha (TNF-alpha) and leukotriene B4 (LTB4) production in a novel human whole blood assay. 2. Lipopolysaccharide (LPS) stimulation of human whole blood caused a time dependent increase in TNF-alpha and prostaglandin E2 (PGE2) plasma levels. Inhibition of LPS-induced TNF-alpha by the selective PDE4 inhibitor RP73401 was proportionally enhanced with endogenous PGE2 (maximal after 24 h). In contrast, blocking endogenous PGE2 production with indomethacin in blood stimulated with LPS for 24 h decreased the potency of RP73401 to that observed with a 4 h LPS incubation. 3. Non-selective and selective PDE4 inhibitors showed greater inhibition of LPS-induced TNF-alpha after 24 h compared to 4 h. Stereoselectivity was only achieved in the 24 h method. 4. LPS-stimulation of whole blood for either 30 min or 24 h followed by N-formyl-Met-Leu-Phe (fMLP) activation resulted in low plasma LTB4 levels. Combination of both treatments resulted in a greater than 7 fold increase in plasma LTB4 levels. Inhibition of the double LPS and fMLP-activated LTB4 production was observed with non-selective and PDE4-selective inhibitors. Their LTB4 inhibitory potencies were similar to that observed in the 24 h LPS-induced TNF-alpha assay. Thus, stimulation of human whole blood with two LPS stimulations followed by fMLP gives rise to both TNF-alpha and LTB4 and their inhibition by various compounds can be assessed in the same blood sample. 5. Calcium ionophore (A23187) stimulation of whole blood resulted in plasma LTB4 levels similar to the double LPS and fMLP method. Inhibition of A23187-induced LTB4 biosynthesis was also achieved by PDE4-selective inhibitors as well as the direct 5-lipoxygenase (5-LO) inhibitor L-739,010. 6. These results confirm the anti-inflammatory properties of PDE4 inhibitors. Thus, this novel human whole blood can be used to assess the biochemical efficacy of PDE4 inhibitors in human subjects.

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