Séverine Vandevoorde

The palmitoylethanolamide family: a new class of anti-inflammatory agents?

The discovery of anandamide as an endogenous ligand for the cannabinoid receptors has led to a resurgence of interest in the fatty acid amides. However, N-palmitoylethanolamine (PEA), a shorter and fully saturated analogue of anandamide, has been known since the fifties. This endogenous compound is a member of the N-acylethanolamines, found in most mammalian tissues. PEA is accumulated during inflammation and has been demonstrated to have a number of anti-inflammatory effects, including beneficial effects in clinically relevant animal models of inflammatory pain. It is now engaged in phase II clinical development, and two studies regarding the treatment of chronic lumbosciatalgia and multiple sclerosis are in progress. However, its precise mechanism of action remains debated. In the present review, the biochemical and pharmacological properties of PEA are discussed, in particular with respect to its analgesic and anti-inflammatory properties.

Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide

The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)‐catalysed hydrolysis of [3H]‐anandamide ([3H]‐AEA) has been investigated. Palmitoylethanolamide and homologues with chain lengths from 12–18 carbon atoms inhibited rat brain [3H]‐AEA metabolism with pI50 values of ∼5. Homologues with chain lengths eight carbon atoms gave <20% inhibition at 100 μM. R‐palmitoyl‐(2‐methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [3H]‐AEA metabolism with pI50 values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [3H]‐WIN 55,212‐2 binding to human CB2 receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R‐palmitoyl‐(2‐methyl)ethanolamide had modest effects upon [3H]‐CP 55,940 binding to human CB1 receptors expressed on CHO cells. Most of the compounds had little effect upon the uptake of [3H]‐AEA into C6 and/or RBL‐2H3 cells. However, palmitoylcyclohexamide (100 μM) and palmitoylisopropylamide (30 and 100 μM) produced more inhibition of [3H]‐AEA uptake than expected to result from inhibition of [3H]‐AEA metabolism alone. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [3H]‐ethanolamine from [3H]‐AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R‐palmitoyl‐(2‐methyl)ethanolamide were less effective. These data provide useful information upon the ability of palmitoylethanolamide analogues to act as ‘entourage’ compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB1 or CB2 receptors.

'Entourage' effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism

The abilities of a series of saturated N‐acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca2+ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1‐HEK293 cells) has been investigated. The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca2+‐influx, nor the influx in response to a submaximal concentration of AEA (1 μM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA. Lauroylethanolamide (C12:0) produced a leftward shift in the dose‐response curve for AEA. EC50 values for AEA to produce Ca2+ influx into hVR1‐HEK293 cells were 1.8, 1.5, 1.1 and 0.22 μM in the presence of 0, 1, 3 and 10 μM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose – response curves to capsaicin. Palmitoylethylamide was synthesized and found to be a mixed‐type inhibitor (Ki(slope) 4.1 μM, Ki(intercept) 66 μM) of [3H]‐AEA metabolism by rat brain membranes. The ‐amide, ‐ethylamide, ‐isopropylamide, ‐butylamide, ‐cyclohexamide and ‐trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 μM AEA. There was no obvious relation between the abilities of the compounds to enhance the Ca2+ influx response to 1 μM AEA into hVR1‐HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. It is concluded that although palmitoylethanolamide has entourage‐like effects at VR1 receptors expressed on hVR1‐HEK293 cells, other N‐acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.

Anticonvulsant Activity of N‐Palmitoylethanolamide, a Putative Endocannabinoid, in Mice

Summary:  Purpose: The purpose of this study was to evaluate in mice the anticonvulsant potential of N‐palmitoylethanolamide, a putative endocannabinoid that accumulates in the body during inflammatory processes.

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro.

Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2‐arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated.

N-Cyclohexanecarbonylpentadecylamine: a selective inhibitor of the acid amidase hydrolysing N-acylethanolamines, as a tool to distinguish acid amidase from fatty acid amide hydrolase

Anandamide ( N-arachidonoylethanolamine) and other bioactive N-acylethanolamines are degraded to their corresponding fatty acids and ethanolamine. This hydrolysis is mostly attributed to catalysis by FAAH (fatty acid amide hydrolase), which exhibits an alkaline pH optimum. In addition, we have identified another amidase which catalyses the same reaction exclusively at acidic pH values [Ueda, Yamanaka and Yamamoto (2001) J. Biol. Chem. 276, 35552-35557]. In attempts to find selective inhibitors of this acid amidase, we screened various derivatives of palmitic acid, 1-hexadecanol, and 1-pentadecylamine with N-palmitoylethanolamine as substrate. Here we show that N-cyclohexanecarbonylpentadecylamine inhibits the acid amidase from rat lung with an IC50 of 4.5 microM, without inhibiting FAAH at concentrations up to 100 microM. The inhibition was reversible and non-competitive. This compound also inhibited the acid amidase in intact alveolar macrophages. With the aid of this inhibitor, it was revealed that rat basophilic leukaemia cells possess the acid amidase as well as FAAH. Thus the inhibitor may be a useful tool to distinguish the acid amidase from FAAH in various tissues and cells and to elucidate the physiological role of the enzyme.

The effect of the palmitoylethanolamide analogue, palmitoylallylamide (L-29) on pain behaviour in rodent models of neuropathy

Cannabinoids are associated with analgesia in acute and chronic pain states. A spectrum of central cannabinoid (CB1) receptor‐mediated motor and psychotropic side effects limit their therapeutic potential. Here, we investigate the analgesic effect of the palmitoylethanolamide (PEA) analogue, palmitoylallylamide (L‐29), which via inhibition of fatty acid amide hydrolase (FAAH) may potentiate endocannabinoids thereby avoiding psychotropic side effects.

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol, and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.

Focus on the three key enzymes hydrolysing endocannabinoids as new drug targets.

The family of endocannabinoids (i.e., the endogenous agonists of cannabinoid receptors) contains several polyunsaturated fatty acid amides such as anandamide (AEA) and oleamide but also esters such as 2-arachidonoylglycerol (2-AG). These compounds are the subject of growing interest in pharmacology for their multiple therapeutic potentials. Unfortunately, they are rapidly inactivated by enzymatic hydrolysis, which prevents their effective medical use. Inhibitors of endocannabinoid degradation seem to be necessary tools for the development of endocannabinoid therapeutics. But hitting this target is inconceivable without good knowledge of the enzymes. Fatty acid amide hydrolase (FAAH) is the oldest and the best characterised enzyme involved in the degradation of endocannabinoids. Cloning, distribution in the body and crystal structure of FAAH have been described. A large number of FAAH inhibitors have also been synthesised and tested. For a long time, FAAH was considered as the only key enzyme hydrolysing endocannabinoids. But recent findings indicate that at least two other enzymes have critical role in the endocannabinoids degradation. Monoglyceride lipase participates in 2-AG degradation and some data indicate that it is the primary mechanism for 2-AG inactivation in intact neurons. N-palmitoylethanolamine-selective acid amidase (NPAA) is a second fatty acid amide hydrolase more active with N-palmitoylethanolamine, an anti-inflammatory substance. The purpose of this review is to collect and compare the catalytic properties of these 3 key enzymes hydrolysing endocannabinoids.

N-Morpholino- and N-diethyl-analogues of palmitoylethanolamide increase the sensitivity of transfected human vanilloid receptors to activation by anandamide without affecting fatty acid amidohydrolase activity

The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca(2+) influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anandamide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoylethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA was retained when the N-CH(2)CH(2)OH group was replaced by N-CH(2)CH(2)Cl,whereas replacement with N-alkyl substituents [from -H up to -(CH(2))(12)CH(3)] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH(2)CH(3))(2) (19) and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 microM AEA to a greater degree than the parent compound, whereas the N-(CH(3))(2) analogue was inactive. 19 and 20 produced leftward shifts in the dose-response curve for AEA activation of Ca(2+) influx into hVR1-HEK293 cells. EC(50) values for AEA to produce Ca(2+) influx into hVR1-HEK293 cells were 1.1, 1.1, 0.54 and 0.36 microM in the presence of 0, 1, 3 and 10 microM 19, respectively. The corresponding values for 20 were 1.5, 1.3, 0.77 and 0.17 microM, respectively. The compounds did not affect the dose-response curves to capsaicin. The ability of oleoylethanolamide to potentiate AEA is retained by the N-CH(2)CH(3) and N-CH(CH(3))(2) analogues (22 and 23, respectively). 22 and 23 produced a small ( approximately 25%) inhibition of the binding of [(3)H]-CP55,940 and [(3)H]-WIN 55,212-2 to CB(1) and CB(2) receptors, respectively, expressed in CHO cells. The compounds inhibited the metabolism of 2 microM [(3)H]-AEA by rat brain fatty acid amidohydrolase with IC(50) values of 5.6 and 11 microM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid amidohydrolase activity. Minor reductions in the accumulation of 10 microM [(3)H]-AEA into C6 glioma cells were seen at 10 microM concentrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially confounding effects upon CB receptors or fatty acid amidohydrolase activity.