Formation of supramolecular structures of alkylamides from Echinacea – implications for cannabinoid type-2 receptor (CB2) interactions in vitro

Various N-alkyl amides (alkylamides) from the medicinal plant Echinacea are cannabinoid type-2 receptor (CB₂)-specific cannabinomimetics. Based on biphasic effects observed in radioligand-based receptor binding assays it was postulated that Echinacea alkylamides may form aggregates. In this study we show that dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) assemble into micelles, whereas no micelle formation occurs for undeca-2E-ene,8,10-diynoic acid isobutylamide (A3) or the structurally related endogenous cannabinoids arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol (2-AG). Light scattering spectroscopy was used to determine the size of premicelle aggregates, micelles and supermicelles. The critical micelle forming concentrations (CMC) of A1 (7.4 nmol/L) and A2 (150 pmol/mL) were determined by fluorescence spectroscopy. The experimental data were complemented by molecular dynamics (MD) simulations of the aggregation phenomenon based on Montecarlo calculations. The results of these studies suggest that both A1 and A2 readily aggregate into premicelles, whereas A2 forms more compact aggregates due to a better alignment of hydrophobic chains and higher curvature. This is in line with microscopic analyses, which show that A2 spontaneously forms both globular and rod-like surfactant micellar superstructures. The data on the self-assembly of A1 and A2 may provide a rationale for the concentration-dependent effects of alkylamides in the radioligand binding assay and suggest a partition between the receptor-bound, monomeric, premicellar, and micellar states.