Alkylamides (alkamides) from Echinacea modulate
tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the
cannabinoid type 2 (
CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic
acid isobutylamide (A1) and dodeca-2E,4E-dienoic
acid isobutylamide (A2) bind to the
CB2 receptor more strongly than the endogenous
cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity
cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the
solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist
SR144528. At 50 nM, A1, A2, and the endogenous
cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive
interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2,
anandamide, the CB2 antagonist
SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic
acid isobutylamide all significantly inhibited
lipopolysaccharide-induced
tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and
anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated
cytokine expression in human whole blood. Overall, alkylamides,
anandamide, and
SR144528 potently inhibited
lipopolysaccharide-induced
inflammation in human whole blood and exerted modulatory effects on
cytokine expression, but these effects are not exclusively related to CB2 binding.