Neurobehavioral activity in mice of N-vanillyl-arachidonyl-amide

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Abstract

We studied the cannabimimetic properties of N-vanillyl-arachidonoyl-amide (arvanil), a potential agonist of cannabinoid CB1 and capsaicin VR1 receptors, and an inhibitor of the facilitated transport of the endocannabinoid anandamide. Arvanil and anandamide exhibited similar affinities for the cannabinoid CB1 receptor, but arvanil was less efficacious in inducing cannabinoid CB1 receptor-mediated GTPγS binding. The Ki of arvanil for the vanilloid VR1 receptor was 0.28 μM. Administered i.v. to mice, arvanil was 100 times more potent than anandamide in producing hypothermia, analgesia, catalepsy and inhibiting spontaneous activity. These effects were not attenuated by the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide·HCl (SR141716A). Arvanil (i.t. administration) induced analgesia in the tail-flick test that was not blocked by either SR141716A or the vanilloid VR1 antagonist capsazepine. Conversely, capsaicin was less potent as an analgesic (ED50 180 ng/mouse, i.t.) and its effects attenuated by capsazepine. The analgesic effect of anandamide (i.t.) was also unaffected by SR141716A but was 750-fold less potent (ED50 20.5 μg/mouse) than capsaicin. These data indicate that the neurobehavioral effects exerted by arvanil are not due to activation of cannabinoid CB1 or vanilloid VR1 receptors.

Introduction

Arachidonoylethanolamide (anandamide) was isolated from porcine brain and shown to function as a ligand of cannabinoid receptors (Devane et al., 1992). Since the discovery of anandamide, other polyunsaturated fatty acid derivatives including two anandamide congeners (Hanus et al., 1993) and 2-arachidonoyl-glycerol Mechoulam et al., 1995, Sugiura et al., 1995 were shown to bind to cannabinoid receptors and to elicit cannabimimetic responses in vivo and in vitro (for reviews see Di Marzo, 1998, Felder and Glass, 1998, Mechoulam et al., 1998, Martin et al., 1999). Although anandamide's capability to functionally activate cannabinoid CB1 and, to a lesser extent, cannabinoid CB2 subtypes of cannabinoid receptors is supported by several findings (for a recent review on cannabinoid receptor pharmacology, see Pertwee, 1999), numerous reports have suggested that this lipid behaves as a partial agonist at these receptors Breivogel et al., 1998, Griffin et al., 1998, Sugiura et al., 1999. Furthermore, recent data (reviewed by Di Marzo et al., 1999) suggest that anandamide may have also other molecular targets different from the cannabinoid receptor subtypes reported so far. For example, anandamide was shown to interact with 5-hydroxy-tryptamine receptors Fan, 1995, Kimura et al., 1998, N-methyl-d-aspartate receptors (Hampson et al., 1998), vanilloid receptors (Zygmunt et al., 1999) as well as with l-type Ca2+ channels Johnson et al., 1993, Jarrahian and Hillard, 1997 and Shaker-related K+ channels (Poling et al., 1996). Most of these effects are quite selective for anandamide over other fatty acid derivatives, although, in some cases, they are observed at concentrations higher than those required to activate cannabinoid CB1 receptors.

Many pharmacological actions of anandamide are attenuated by the cannabinoid CB1 receptor antagonist (N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide·HCl (SR141716A). However, the selectivity of this compound, especially when used at concentrations higher than those required to bind to cannabinoid CB1 receptors, has been recently questioned White and Hiley, 1998, Zygmunt et al., 1999. Indeed, SR141716A was recently shown to counteract also those pharmacological actions of anandamide or non-psychotropic cannabinoids that are not mediated by cannabinoid CB1 receptors Chaytor et al., 1999, Járai et al., 1999, Wagner et al., 1999. On the other hand, in mice, anandamide typical tetrahydrocannabinol-like effects on spontaneous activity, body temperature and nociception are not affected by SR141716A (Adams et al., 1998). On the basis of our current knowledge, it is reasonable to assume that, although the rapid degradation of this compound both in vitro (Deutsch and Chin, 1993) and in vivo (Willoughby et al., 1997) may explain some of the controversy regarding its mechanism of action, the pharmacological actions of anandamide may be due also to the interaction with macromolecules other than the cannabinoid CB1 and CB2 receptors. Indeed, evidence for a non-CB1, G-protein-coupled anandamide receptor in rat astrocytes (Sagan et al., 1999) as well as for a novel endothelial site of action for anandamide (Járai et al., 1999) was recently reported.

We have recently synthesized a series of unsaturated fatty acid derivatives of the hot chili pepper ingredient, capsaicin, and assessed their biochemical properties in intact cells and cell-free homogenates Di Marzo et al., 1998, Melck et al., 1999. At low μM concentrations, these unsaturated N-acyl-vanillyl-amides, and in particular the arachidonic acid homologue, that we named arvanil (Fig. 1), bind to cannabinoid CB1, but not CB2, receptors, inhibit the uptake (and therefore the inactivation) of anandamide by intact cells, and activate the vanilloid VR1 receptors for capsaicin. We proposed that arvanil could behave as a ‘hybrid’ ligand for cannabinoid CB1 and vanilloid VR1 receptors and gained in vitro evidence that this compound could be up to five-fold more potent than ‘pure’ agonists of either receptor type (Melck et al., 1999). Indeed, other recent observations showed that anandamide also activates vanilloid receptors resulting in an endothelium-independent vasodilator action in rat mesenteric arteries (Zygmunt et al., 1999). Given the known pharmacological properties in vivo of both anandamide Di Marzo, 1998, Pertwee, 1999 and capsaicin (Szallasi and Blumberg, 1999), it is likely that arvanil may have a high potential for therapeutic use as an analgesic agent. Therefore, we undertook the present study with the aim of investigating for the first time the capasicin-like and cannabinoid pharmacological activity of arvanil in vivo. Furthermore, we carried out binding assays with membrane preparations from rat and mouse brain and cells transfected with vanilloid VR1 receptors to re-assess the ability of arvanil to bind and/or activate cannabinoid CB1 and vanilloid receptors. We report that arvanil exhibits a pharmacological profile that is more similar to that of anandamide than capsaicin. However, arvanil is much more potent in vivo than could be expected from its affinity and efficacy at cannabinoid CB1 receptors, thus suggesting that this compound may act, inter alia, through novel sites of action for anandamide.

Section snippets

Animals and reagents

ICR male mice (Harlan Laboratories, Indianapolis, IN) weighing 24 to 26 g were used in all in vivo experiments. Mice were maintained on a 14:10-h light/dark cycle with free access to food and water. Arvanil was synthesized as described previously (Melck et al., 1999). O-1839 and d8-2-arachidonoyl-glycerol were synthesized in our laboratory (RKR) as will be described elsewhere. The chemical structure of O-1839 (Fig. 1) was confirmed by means of nuclear magnetic resonance. SR141716A was obtained

Arvanil as a ligand/agonist of cannabinoid CB1 and vanilloid VR1 receptors

The Ki values for the displacement of the high affinity cannabinoid CB1 receptor ligand [3H]SR141716A from rat and mouse brain membranes by anandamide, arvanil and the dimethylheptyl analogue of arvanil (O-1839, Fig. 1) are shown in Table 1. Arvanil's affinity was slightly less than that of anandamide, whereas O-1839's affinity was less than that of arvanil. If the hydrolase inhibitor phenylmethylsulfonylfluoride was omitted during the assay incubation, the ability of anandamide, but not

Arvanil is a partial agonist at stimulating GTP-γ-S binding to rat brain membranes

Previously, it was shown that μM concentrations of olvanil, a potent ligand of vanilloid receptors (Szallasi and Blumberg, 1999), could inhibit anandamide facilitated transport into cells and activate cannabinoid CB1, but not CB2, receptors as a partial agonist (Di Marzo et al., 1998). More recently, in a study carried out by using isolated cells in culture, the C20:4 n-6 analogue of olvanil, which we named arvanil, was also shown to inhibit the anandamide transporter and to exhibit an affinity

Acknowledgements

This work was supported by a grant from the MURST (3393 to VDM) and by NIH grants DA05274 and DA09789. VDM is the recipient of a short-term fellowship from the Human Frontier Science Program Organization. The authors thank Drs. Daniel N. Cortright and James E. Krause, Neurogen, Branford, CT, for supplying the CHO/rVR1 cells for the experiments.

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