The effects of phosphodiesterase type 4 inhibitors on tumour necrosis factor-alpha and leukotriene B-4 in a novel human whole blood assay

1 The aim of this study was to assess the inhibitory activities of phosphod iesterase type 4 (PDE4) inhibitors on tumour necrosis factor-alpha (TNF-alp ha) and leukotriene B-4 (LTB4) production in a novel human whole blood assa y. 2 Lipopolysaccharide (LPS) stimulation of human whole blood caused a time d ependent increase in TNF-alpha and prostaglandin E-2 (PGE(2)) plasma levels . Inhibition of LPS-induced TNF-alpha by the selective PDE4 inhibitor RP734 01 was proportionally enhanced with endogenous PGE(2) (maximal after 24 h). In contrast, blocking endogenous PGE(2) production with indomethacin in bl ood stimulated with LPS for 24 h decreased the potency of RP73401 to that o bserved 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 onl y achieved in the 24 h method. 4 LPS-stimulation of whole blood for either 30 min or 24 h followed by N-fo rmyl-Met-Leu-Phe (fMLP) activation resulted in low plasma LTB4 levels. Comb ination of both treatments resulted in a greater than 7 fold increase in pl asma LTB4 levels. Inhibition of the double LPS and fMLP-activated LTB4 prod uction was observed with non-selective and PDE4-selective inhibitors. Their LTB4 inhibitory potencies were similar to that observed in the 24 h LPS-in duced 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 the ir 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 a s 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.