Structure-activity relationship of a series of inhibitors of monoacylglycerol hydrolysis--comparison with effects upon fatty acid amide hydrolase.

A series of 32 heterocyclic analogues based on the structure of 2-arachidonoylglycerol (2-AG) were synthesized and tested for their ability to inhibit monoacylglycerol lipase and fatty acid amide hydrolase activities. The designed compounds feature a hydrophobic moiety and different heterocyclic subunits that mimic the glycerol fragment. This series has allowed us to carry out the first systematic structure-activity relationship study on inhibition of 2-AG hydrolysis. The most promising compounds were oxiran-2-ylmethyl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate (1) and tetrahydro-2H-pyran-2-ylmethyl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate (5). They inhibited cytosolic 2-oleoylglycerol (2-OG) hydrolysis completely (IC50 values of 4.5 and 5.6 muM, respectively). They also blocked, albeit less potently, 2-OG hydrolysis in membrane fractions (IC50 values of 19 and 26 muM, respectively) and anandamide hydrolysis (IC50 values of 12 and 51 muM, respectively). These compounds will be useful in delineating the importance of the cytosolic hydrolytic activity in the regulation of 2-AG levels and, hence, its potential as a target for drug development.

Inhibition of fatty acid amide hydrolase, a key endocannabinoid metabolizing enzyme, by analogues of ibuprofen and indomethacin.

There is evidence in the literature that the nonsteroidal anti-inflammatory drugs indomethacin and ibuprofen can interact with the cannabinoid system both in vitro and in vivo. In the present study, a series of analogues of ibuprofen and indomethacin have been investigated with respect to their ability to inhibit fatty acid amide hydrolase, the enzyme responsible for the hydrolysis of the endogenous cannabinoid anandamide. Of the fourteen compounds tested, the 6-methyl-pyridin-2-yl analogue of ibuprofen ("ibu-am5") was selected for further study. This compound inhibited rat brain anandamide hydrolysis in a non-competitive manner, with IC50 values of 4.7 and 2.5 microM being found at pH 6 and 8, respectively. By comparison, the IC50 values for ibuprofen were 130 and 750 microM at pH 6 and 8, respectively. There was no measurable N-acylethanolamine hydrolyzing acid amidase activity in rat brain membrane preparations. In intact C6 glioma cells, ibu-am5 inhibited the hydrolysis of anandamide with an IC50 value of 1.2 microM. There was little difference in the potencies of ibu-am5 and ibuprofen towards cyclooxygenase-1 and -2 enzymes, and neither compound inhibited the activity of monoacylglycerol lipase. Ibu-am5 inhibited the binding of [3H]-CP55,940 to rat brain CB1 and human CB2 cannabinoid receptors more potently than ibuprofen, but the increase in potency was less than the corresponding increase in potency seen for inhibition of FAAH activity. It is concluded that ibu-am5 is an analogue of ibuprofen with a greater potency towards fatty acid amide hydrolase but with a similar cyclooxygenase inhibitory profile, and may be useful for the study of the therapeutic potential of combined fatty acid amide hydrolase-cyclooxygenase inhibitors.

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.

Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for anandamide. It is concluded that VDM11 is an inhibitor of FAAH under the assay conditions used here, and that the inhibition may at least in part be a consequence of the compound acting as an alternative substrate.

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 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.

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.

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.

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

1. 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. 2. 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 microM). 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. 3. 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 microM in the presence of 0, 1, 3 and 10 microM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose - response curves to capsaicin. 4. Palmitoylethylamide was synthesized and found to be a mixed-type inhibitor (K(i(slope)) 4.1 microM, K(i(intercept)) 66 microM) of [3H]-AEA metabolism by rat brain membranes. 5. The -amide, -ethylamide, -isopropylamide, -butylamide, -cyclohexamide and -trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 microM AEA. 6. There was no obvious relation between the abilities of the compounds to enhance the Ca2+ influx response to 1 microM AEA into hVR1-HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. 7. 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.

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.

Overview of the chemical families of fatty acid amide hydrolase and monoacylglycerol lipase inhibitors.

The family of the endogenous agonists of the cannabinoid receptors--i.e., the endocannabinoids--includes several polyunsaturated fatty acid amides and esters. Arachidonoylethanolamide (anandamide, AEA) and 2-arachidon-oylglycerol (2-AG) are, respectively, the leads of these chemical families. So far, two enzymes responsible for the metabolism of AEA and 2-AG have been described: fatty acid amide hydrolase (FAAH) which hydrolyzes AEA and in some cells 2-AG, and Monoacylglycerol Lipase (MAGL) which hydrolyzes 2-AG. In spite of the early characterisation of MAGL and the nearly simultaneous clonings of the two enzymes, most of the efforts were dedicated to the study of FAAH and consequentially, the range of FAAH inhibitors available nowadays exceeds the number of compounds active upon MAGL. FAAH inhibitors can be divided in two major groups, the first one includes the inhibitors inspired by the chemical structures of FAAH substrates, which carry an arachidonoyl-, oleoyl- or palmitoyl-carbon chain that mimic the fatty acid chains of anandamide, oleamide and palmitoylethanolamide. The second group involves compounds that do not share similarities with the endocannabinoids, such as the carbamates, oxazolopyridins, 2-thioxoimidazolidin-4-ones, imidazolidine-2,4-diones and the non-steroidal anti-inflammatory drugs. However, the family of MAGL inhibitors contains few members and most of them exhibit a lack of selectivity. The purpose of this review is to give an overview of the families of synthetic inhibitors of FAAH and MAGL. The synthetic pathways, the chemical features, potencies, selectivities and modes of inhibition are listed and discussed in order to facilitate their comparison.

Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase.

Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.2 microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and 21 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 12 and 32 microM, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.

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.

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 [(5 Z,8 Z,11 Z,14 Z)- 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 IC(50) values of 4.9 and 2.7 microM, 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 microM concentrations. AM404 (1 microM), VDM 11 (1 microM) and palmitoylisopropylamide (3-30 microM), 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.