Alkylamides from Echinacea Are a New Class of Cannabinomimetics

Abstract

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor α 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 K_i values of A1 and A2 (CB₂ ∼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 π -π 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 K_i >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 (K_i <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 α, IL-1β, 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.