Pharmacology of Palmitoylethanolamide and Related Compounds

Anandamide (AEA) is an endogenous fatty acid which activates the same cannabinoid receptors as ∆9-tetrahydrocannabinol, the psychoactive substance in marijuana. In vivo, anandamide exerts a number of actions including effects upon pain and inflammation. However, AEA has a short duration of action since it is rapidly metabolised, primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH). The general aim of this thesis has been to identify and characterize compounds capable of preventing the metabolism of anandamide. The chemical approach was based on the endogenous anti-inflammatory compound palmitoylethanolamide (PEA), a compound related to anandamide with the ability to inhibit anandamide degradation by substrate competition, but without the ability to directly activate cannabinoid receptors. A number of compounds were identified as inhibitors of rat brain FAAH in the initial in vitro studies, without having major affinity for the cannabinoid receptors. In particular, palmitoylisopropylamide (PIA) was found to reduce the metabolism of AEA in intact C6 glioma cells with potency similar to the prototypical AEA reuptake inhibitor AM404. This compound was in addition found to exert less effect upon C6 glioma cell proliferation than either AM404 or the closely related uptake inhibitor VDM11. To evaluate if PIA was effective in vivo, a model of mast cell dependent inflammation, oedema of the ear following local injection of compound 48/80, was set up using anaesthetised mice. Initially, a CB2 cannabinoid receptor selective agonist was used to probe the model and demonstrated to produce an anti-oedema effect. In contrast, the compound was inactive in vitro in skin slice preparations. PIA showed a similar pattern, although there was a large variation in responses which affected the significance of the result obtained, as did the vehicle used to dissolve the compound. Taken together, the present data would suggest that PIA can inhibit the degradation of AEA without having deleterious effects upon cell proliferation or affinity for the cannabinoid receptors. Further experimentation is necessary to elucidate the usefulness of this compound in vivo

Modifications of the ethanolamine head in N-palmitoylethanolamine: synthesis and evaluation of new agents interfering with the metabolism of anandamide

The endogenous fatty acid amide anandamide (AEA) has, as a result of its actions on cannabinoid and vanilloid receptors, a number of important pharmacological properties including effects on nociception, memory processes, spasticity, and cell proliferation. Inhibition of the metabolism of AEA, catalyzed by fatty acid amide hydrolase (FAAH), potentiates the actions of AEA in vivo and therefore may be a useful target for drug development. In the present study, we have investigated whether substitution of the headgroup of the endogenous alternative FAAH substrate palmitoylethanolamide (PEA) can result in the identification of novel compounds preventing AEA metabolism. Thirty-seven derivatives of PEA were synthesized, with the C16 long chain of palmitic acid kept intact, and comprising 20 alkylated, 12 aromatic, and 4 halogenated amides. The ability of the PEA derivatives to inhibit FAAH-catalyzed hydrolysis of [(3)H]AEA was investigated using rat brain homogenates as a source of FAAH. Inhibition curves were analyzed to determine the potency of the inhibitable fraction (pI(50) values) and the maximal attained inhibition for the compound, given that solubility in an aqueous environment is a major issue for these compounds. In the alkylamide family, palmitoylethylamide and palmitoylallylamide were inhibitors of AEA metabolism with pI(50) values of 5.45 and 5.47, respectively. Halogenated derivatives (Cl and Br) exhibit pI(50) values of approximately 5.5 but rather low percentages of maximal inhibition. The -OH group of the ethyl head chain of N-palmitoylethanolamine was not necessary for interaction with FAAH. Amides containing aromatic moieties were less potent inhibitors of AEA metabolism. Compounds containing amide and ester bonds, 13 and 37, showed pI(50) values of 4.99 and 5.08, respectively. None of the compounds showed obvious affinity for CB(1) or CB(2) receptors expressed on Chinese hamster ovary (CHO) cells. It is concluded that although none of the compounds were dramatically more potent than PEA itself at reducing the metabolism of AEA, the lack of effect of the compounds at CB(1) and CB(2) receptors makes them useful templates for development of possible therapeutic FAAH inhibitors

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

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

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

1. The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [(3)H]-anandamide ([(3)H]-AEA) has been investigated. 2. Palmitoylethanolamide and homologues with chain lengths from 12–18 carbon atoms inhibited rat brain [(3)H]-AEA metabolism with pI(50) values of ∼5. Homologues with chain lengths ⩽eight carbon atoms gave <20% inhibition at 100 μM. 3. R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [(3)H]-AEA metabolism with pI(50) values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. 4. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [(3)H]-WIN 55,212-2 binding to human CB(2) receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [(3)H]-CP 55,940 binding to human CB(1) receptors expressed on CHO cells. 5. Most of the compounds had little effect upon the uptake of [(3)H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 μM) and palmitoylisopropylamide (30 and 100 μM) produced more inhibition of [(3)H]-AEA uptake than expected to result from inhibition of [(3)H]-AEA metabolism alone. 6. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [(3)H]-ethanolamine from [(3)H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective. 7. 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 CB(1) or CB(2) receptors

Esters, retroesters, and a retroamide of palmitic acid: pool for the first selective inhibitors of N-palmitoylethanolamine-selective acid amidase.

Cyclohexyl hexadecanoate, hexadecyl propionate, and N-(3-hydroxypropionyl)pentadecanamide, respectively ester, retroester, and retroamide derivatives of N-palmitoylethanolamine, represent the first selective inhibitors of "N-palmitoylethanolamine hydrolase" described so far. These compounds are devoid of affinity for CB(1) and CB(2) receptors and characterized by high percentages of inhibition of N-palmitoylethanolamine-selective acid amidase (84.0, 70.5, and 76.7% inhibition at 100 microM, respectively) with much lower inhibitory effect on either fatty acid amide hydrolase or the uptake of anandamide

AM404 and VDM 11 non-specifically inhibit C6 glioma cell proliferation at concentrations used to block the cellular accumulation of the endocannabinoid anandamide

AM404 [N-(4-hydroxyphenyl)arachidonylamide] and VDM 11 [(5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] are commonly used to prevent the cellular accumulation of the endocannabinoid anandamide, and thereby to potentiate its actions. However, it has been reported that AM404 can produce an influx of calcium into cells, which might be expected to have deleterious effects on cell proliferation. In the present study, AM404 and VDM 11 were found to reduce C6 glioma cell proliferation with IC50 values of 4.9 and 2.7 muM, respectively. The inhibition of cell proliferation following a 96-h exposure was not accompanied by dramatic caspase activation, and was not prevented by either a combination of cannabinoid and vanilloid receptor antagonists, or by the antioxidant alpha-tocopherol, suggestive of a non-specific mode of action. Similar results were seen with palmitoylisopropylamide, although this compound only produced significant inhibition of cell proliferation at 30 muM concentrations. AM404 (1 muM), VDM 11 (1 muM) and palmitoylisopropylamide (3-30 muM), i.e. concentrations producing relatively modest effects on cell proliferation per se, reduced the vanilloid receptor-mediated antiproliferative effects of anandamide, as would be expected for compounds preventing the cellular accumulation of anandamide (and thereby access to its binding site on the vanilloid receptor). It is concluded that concentrations of AM404 and VDM 11 that are generally used to reduce the cellular accumulation of anandamide have deleterious effects upon cell proliferation, and that lower concentrations of these compounds may be more appropriate to use in vitro

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 Ca2+ 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 Ca2+ 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. (C) 2003 Elsevier Inc. All rights reserved