Self-Assembling Cannabinomimetics: Supramolecular Structures of N-Alkyl Amides Stefan Raduner, ² William Bisson, ‡ Ruben Abagyan, ‡ Karl-Heinz Altmann, ² and Ju ¨rg Gertsch* ,² Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zu ¨rich, Switzerland, and Molecular Biology, The Scripps Research Institute, TSRI, 10550 North Torrey Pines Road, TPC-28, La Jolla, California 92037 ReceiVed NoVember 30, 2006 Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB 2 ) receptors. In this study we show that the CB 2 -binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca- 2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB 2 , or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB 2 receptor, and aggregation of N-alkyl amides thus determines their in Vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds. Purple coneflower (Echinacea purpurea and E. angustifolia) preparations are widely used herbal medicines for the treatment of the common cold and upper respiratory infections, 1,2 with an estimated sales volume in 2002 of $500 million in the U.S. alone. 3 Fatty acid N-alkyl amides (alkylamides) are a class of bioavailable lipophilic compounds, which are generally found in Echinacea species. There is increasing evidence that N-isobutyl amides inhibit acute inflammation and might contribute to the beneficial effects reported for Echinacea. 4-6 We have recently shown that two major alkylamides from Echinacea, dodeca-2E,4E-dienoic acid isobutyl- amide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) (Figure 1), constitute a new class of cannabinomimetics. 4 Compounds 1 and 2 bind to and activate the peripheral cannabinoid receptor (CB 2 ) more strongly than the endogenous cannabinoids arachidonoyl ethanolamine (anandamide; AA) and 2-arachidonoyl- glycerol (2-AG) (Figure 1); 1 also has moderate affinity for the CB 1 receptor. Activation of CB 2 receptors has been associated with distinct immunomodulatory effects, such as inhibition of tumor necrosis factor alpha (TNF-R) expression. 7 During radioligand experiments with compounds 1 and 2 a biphasic displacement of the radioligand [ 3 H]-CP55,940 was observed, which raised the question whether N-alkyl amide CB 2 binding is competing with N-alkyl amide self-aggregation as a function of concentration. 4 In order to determine whether aggrega- tion could be responsible for discontinuous CB 2 receptor binding of 1 and 2, the aim of the current study was to examine the aggregation behavior of N-isobutyl amides 1, 2, and 3 (the latter has no affinity to cannabinoid receptors) and the structurally related endogenous cannabinoids anandamide and 2-AG in aqueous medium. Aggregation of amphiphilic molecules can result in the formation of three types of supramolecular structures: premicelles, which are generally clusters of less than 10 monomers; micelles, which are aggregates of amphiphilic molecules as colloidal disper- sion capable of entrapping lipophilic molecules (e.g., fluorescent dyes); and higher order complexes of micelles (supermicelles). Since N-alkyl amides are likely to contribute to the pharmacological action of Echinacea preparations, it is important to understand aggregation and micellization of these compounds, in particular because such processes are known to influence the pharmacokinetic and phar- macodynamic behavior of drugs. 8 Results and Discussion Aqueous solutions of 1 and 2 were found to display a pronounced Tyndall effect (Rayleigh scattering) at concentrations of >2 μM. At concentrations of <3 μM the colloidal particles formed by 1 and 2 could not be filtered out with 0.2 μm cellulose acetate filters nor did they settle upon standing at room temperature. In contrast, neither compound 3, which is structurally related to 1 and 2 but does not bind to CB 2 , nor arachidonoyl ethanolamine (anandamide) or 2-arachidonoylglycerol (2-AG) (Figure 1) showed a Tyndall effect. Higher concentrations of 1 (>3 μM) resulted in the formation of floating precipitates when stored at room temperature over 24- 72 h. In the dispersed state, the particles were separate and moved erratically with Brownian motion, whereas the flocked state was characterized by particles, which clumped together in aggregates under the influence of van der Waals forces. 9 In order to study * Corresponding author. E-mail: juerg.gertsch@pharma.ethz.ch. ² ETH Zurich. ‡ Scripps Research Institute. Figure 1. Structures of N-alkyl amides from Echinacea purpurea (1-3) and endogenous cannabinoids arachidonoyl ethanolamine (AA) and 2-arachidonoylglycerol (2-AG). 1010 J. Nat. Prod. 2007, 70, 1010-1015 10.1021/np060598+ CCC: $37.00 © 2007 American Chemical Society and American Society of Pharmacognosy Published on Web 05/11/2007