Acidic nonsteroidal anti-inflammatory drugs inhibit rat brain fatty acid amide hydrolase in a pH-dependent manner

Previous studies have demonstrated that fatty acid amide hydrolase, the enzyme responsible for the metabolism of anandamide, is inhibited by the acidic non-steroidal anti-inflammatory drug (NSAID) ibuprofen with a potency that increases as the assay pH is reduced. Here we show that (R)-, (S)- and (R,S)-flurbiprofen, indomethacin and niflumic acid show similar pH-dependent shifts in potency to that seen with ibuprofen. Thus, (S)-flurbiprofen inhibited 2 microM [3H]anandamide metabolism with IC50 values of 13 and 50 microM at assay pH values of 6 and 8, respectively. In contrast, the neutral compound celecoxib was a weak fatty acid amide hydrolase inhibitor and showed no pH dependency (IC50 values approximately 300 microM at both assay pH). The cyclooxygenase-2-selective inhibitors nimesulide and SC-58125 did not inhibit fatty acid amide hydrolase activity at either pH. The data are consistent with the conclusion that the non-ionised forms of the acidic NSAIDs are responsible for the inhibition of fatty acid amide hydrolase.     

Arachidonylsulfonyl derivatives as cannabinoid CB1 receptor and fatty acid amide hydrolase inhibitors

Arachidonylsulfonyl fluoride (3), reported here for the first time, is similar in potency to its known methyl arachidonylfluorophosphonate (2) analogue as an inhibitor of mouse brain fatty acid amide hydrolase activity (IC(50) 0.1 nM) and cannabinoid CB1 agonist [3H]CP 55,940 binding (IC(50) 304-530 nM). Interestingly, 3 is much more selective than 2 as an inhibitor for fatty acid amide hydrolase relative to acetylcholinesterase, butyrylcholinesterase and neuropathy target esterase. N-(2-Hydroxyethyl)arachidonylsulfonamide (4) is at least 2500-fold less potent than N-(2-hydroxyethyl)arachidonamide (anandamide) (1) at the CB1 agonist site.     

Fatty acid amide hydrolase substrate specificity

Fatty acid amide hydrolase (FAAH), also referred to as oleamide hydrolase and anandamide amidohydrolase, is a serine hydrolase responsible for the degradation of endogenous oleamide and anandamide, fatty acid amides that function as chemical messengers. FAAH hydrolyzes a range of fatty acid amides, and the present study examines the relative rates of hydrolysis of a variety of natural and unnatural fatty acid primary amide substrates using pure recombinant rat FAAH.     

Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor alpha into the bronchoalveolar lavage (BAL) fluid, which was not accompanied by epithelial cell injury. The treatment, however, did not change significantly the levels of anandamide and the related compound palmitoylethanolamide in the cell-free fraction of the BAL fluid. The activities of the anandamide synthetic enzymes N-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity.     

Lipophilic amines as potent inhibitors of N-acylethanolamine-hydrolyzing acid amidase

N-Acylethanolamines (NAEs) including N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine are endogenous lipid mediators. These molecules are degraded to the corresponding fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH) or NAE-hydrolyzing acid amidase (NAAA). Lipophilic amines, especially pentadecylamine (2c) and tridecyl 2-aminoacetate (11b), were found to exhibit potent NAAA inhibitory activities (IC(50)=5.7 and 11.8μM), with much weaker effects on FAAH. These simple structures would provide a scaffold for further improvement in NAAA inhibitory activity.     

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.     

alpha-Keto heterocycle inhibitors of fatty acid amide hydrolase: carbonyl group modification and alpha-substitution.

Two sets of novel analogues of the recently disclosed alpha-keto heterocycle inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for regulation of endogenous oleamide and anandamide, were synthesized and evaluated in order to clarify a role of the electrophilic carbonyl group and structural features important for their activity. Both the electrophilic carbonyl and the degree of alpha-substitution markedly affect inhibitor potency.     

Heterocyclic sulfoxide and sulfone inhibitors of fatty acid amide hydrolase

A novel series of heterocyclic sulfoxides and sulfones was prepared and examined as potential inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for inactivation of neuromodulating fatty acid amides including anandamide and oleamide.     

Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K., and Yamamoto, S. (2001) J. Biol. Chem. 276, 35552-35557). Here we report complementary DNA cloning and functional expression of the enzyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" from human, rat, and mouse. The deduced primary structures revealed that NAAA had no homology to fatty acid amide hydrolase but belonged to the choloylglycine hydrolase family. Human NAAA was essentially identical to a gene product that had been noted to resemble acid ceramidase but lacked ceramide hydrolyzing activity. The recombinant human NAAA overexpressed in HEK293 cells hydrolyzed various N-acylethanolamines with N-palmitoylethanolamine as the most reactive substrate. Most interestingly, a very low ceramide hydrolyzing activity was also detected with NAAA, and N-lauroylethanolamine hydrolyzing activity was observed with acid ceramidase. By the use of tunicamycin and endoglycosidase, NAAA was found to be a glycoprotein. Furthermore, the enzyme was proteolytically processed to a shorter form at pH 4.5 but not at pH 7.4. Expression analysis of a green fluorescent protein-NAAA fusion protein showed a lysosome-like distribution in HEK293 cells. The organ distribution of the messenger RNA in rats revealed its wide distribution with the highest expression in lung. These results demonstrated that NAAA is a novel N-acylethanolamine-hydrolyzing enzyme that shows structural and functional similarity to acid ceramidase.     

Inhibition of Fatty Acid Amidohydrolase,the Enzyme Responsible for the Metabolism of the Endocannabinoid Anandamide,by Analogues of Arachidonoyl-serotonin

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (K_i(slope) 1.3 µM; K_i(intercept) 44 µM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.     

A simple stopped assay for fatty acid amide hydrolase avoiding the use of a chloroform extraction phase

A stopped assay for fatty acid amide hydrolase (FAAH) has been developed, whereby the enzyme reaction product ([(3)H]ethanolamine) was separated from substrate (anandamide [ethanolamine-1-(3)H]), by differential adsorption to charcoal. The assay gave a better extraction efficiency when acidic rather than alkaline charcoal solutions were used to stop the reaction, and a very good ratio of sample/blank was also seen. The acidic charcoal assay gave the expected sensitivities to compounds known to inhibit FAAH (palmitoyltrifluoromethyl ketone, arvanil, AM404 and indomethacin). It is concluded that the acidic charcoal extraction method provides a robust and simple stopped assay for FAAH without the need to use potentially hazardous solvents like chloroform.     

Synthesis of all-trans anandamide: a substrate for fatty acid amide hydrolase with dual effects on rabbit platelet activation

Anandamide (AEA) presents the four double bonds in the cis configuration, deriving from the arachidonic acid moiety. In the context of an antisense strategy based on the double bond configuration, all-trans AEA (t-AEA) was synthesized in high yield starting from all-trans methyl arachidonate and ethanolamine in the presence of KCN. t-AEA was assayed on rabbit platelet aggregation, obtaining effect only at high concentrations (>10(-4) M) after an also concentration-dependent lag phase. At lower concentrations it inhibited PAF-induced rabbit platelet aggregation with an IC(50)=4.6 x 10(-6) M. In contrast to anandamide, the activation of platelets was not due to the conversion of t-AEA to trans arachidonic acid, as ascertained by negative results with FAAH inhibitors. However, t-AEA was found to be a substrate for fatty acid amide hydrolase (FAAH), the enzyme that cleaves anandamide and regulates in vivo the magnitude and duration of the signaling induced by this lipid messenger.     

Inhibition of Endocannabinoid Metabolism by the Metabolites of Ibuprofen and Flurbiprofen

Background

In addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen.

Methodology/Principal Findings

COX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4′-hydroxyflurbiprofen and possibly also 3′-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds.

Conclusions/Significance

It is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo.     

Inhibition of fatty acid amidohydrolase, the enzyme responsible for the metabolism of the endocannabinoid anandamide, by analogues of arachidonoyl-serotonin

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (Ki(slope) 1.3 microM; Ki(intercept) 44 microM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.     

Modulation of anxiety through blockade of anandamide hydrolysis

The psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol, produces in humans subjective responses mediated by CB1 cannabinoid receptors, indicating that endogenous cannabinoids may contribute to the control of emotion. But the variable effects of Delta(9)-tetrahydrocannabinol obscure the interpretation of these results and limit the therapeutic potential of direct cannabinoid agonists. An alternative approach may be to develop drugs that amplify the effects of endogenous cannabinoids by preventing their inactivation. Here we describe a class of potent, selective and systemically active inhibitors of fatty acid amide hydrolase, the enzyme responsible for the degradation of the endogenous cannabinoid anandamide. Like clinically used anti-anxiety drugs, in rats the inhibitors exhibit benzodiazepine-like properties in the elevated zero-maze test and suppress isolation-induced vocalizations. These effects are accompanied by augmented brain levels of anandamide and are prevented by CB1 receptor blockade. Our results indicate that anandamide participates in the modulation of emotional states and point to fatty acid amide hydrolase inhibition as an innovative approach to anti-anxiety therapy.     

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.     

A structure-activity relationship study on N-arachidonoyl-amino acids as possible endogenous inhibitors of fatty acid amide hydrolase

N-arachidonoyl-glycine (NAGly) has been recently identified in rodent tissues and found to exhibit analgesic activity in vivo. NAGly is a potent inhibitor of the fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for the degradation of the endocannabinoid N-arachidonoyl-ethanolamine (anandamide), and was shown recently to elevate the blood levels of the this analgesic compound. We have synthesized several N-arachidonoyl-amino acids of potential natural occurrence, as well as the D- and L-isomers of N-arachidonoyl-alanine, and have tested their activity on FAAH preparations from mouse, rat, and human cell lines, and from mouse or rat brain. The results indicate that the relative potency and enantioselectivity of N-arachidonoyl-amino acids as FAAH inhibitors depend on the animal species. Thus, whilst NAGly is the most potent compound on the rat and mouse enzymes, N-arachidonoyl-isoleucine is active only on human FAAH and N-arachidonoyl-alanine enantiomers show a varying degree of potency. Taken together, these data support the view that an enhancement of endogenous anandamide levels underlies in part the analgesic effects of NAGly in rodents.     

Depolarizing and calcium-mobilizing stimuli fail to enhance synthesis and release of endocannabinoids from rat brain cerebral cortex slices

The concentrations of the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (anandamide) were examined in rat brain cerebral cortex slices and surrounding medium. Basal concentrations of endocannabinoids were similar to those identified previously in rat brain, with anandamide content being much lower (19 pmol/g) than that of 2-AG (7300 pmol/g). In contrast, basal concentrations in the surrounding medium were proportionally much lower for 2-arachidonoylglycerol (16 pmol/mL) compared to anandamide (0.6 pmol/mL). Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue concentrations of endocannabinoids, while endocannabinoids in the medium were unaltered by elevated KCl. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue concentrations of anandamide (and related N-acylethanolamines), without altering 2-AG, while evoking proportional elevations of anandamide in the medium. Removal of extracellular calcium ions failed to alter tissue concentrations of anandamide, but significantly reduced 2-AG in the tissue by 90% and levels in the medium to below the detection limit. Supplementation of the medium with 50 μM N-oleoylethanolamine only raised tissue concentrations of N-oleoylethanolamine in the presence of cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester and failed to alter either tissue or medium anandamide or 2-AG concentrations. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-AG concentrations in this tissue.     

A role for caveolae/lipid rafts in the uptake and recycling of the endogenous cannabinoid anandamide

The mechanisms responsible for the uptake and cellular processing of the endogenous cannabinoid anandamide are not well understood. We propose that anandamide uptake may occur via a caveola/lipid raft-related endocytic process in RBL-2H3 cells. Inhibitors of caveola-related (clathrin-independent) endocytosis reduced anandamide transport by approximately 50% compared with the control. Fluorescein derived from fluorescently labeled anandamide colocalized with protein markers of caveolae at early time points following transport. In this study, we have also identified a yet unrecognized process involved in trafficking events affecting anandamide following its uptake. Following uptake of [(3)H]anandamide by RBL-2H3 cells, we found an accumulation of tritium in the caveolin-rich membranes. Inhibitors of both anandamide uptake and metabolism blocked the observed enrichment of tritium in the caveolin-rich membranes. Mass spectrometry of subcellular membrane fractions revealed that the tritium accumulation observed in the caveolin-rich membrane fraction was not representative of intact anandamide, suggesting that following metabolism by the enzyme fatty acid amide hydrolase (FAAH), anandamide metabolites are rapidly enriched in caveolae. Furthermore, HeLa cells, which do not express high levels of FAAH, showed an accumulation of tritium in the caveolin-rich membrane fraction only when transfected with FAAH cDNA. Western blot and immunocytochemistry analyses of RBL-2H3 cells revealed that FAAH was localized in intracellular compartments distinct from caveolin-1 localization. Together, these data suggest that following uptake via caveola/lipid raft-related endocytosis, anandamide is rapidly metabolized by FAAH, with the metabolites efficiently recycled to caveolin-rich membrane domains.     

Anandamide hydrolysis: a new target for anti-anxiety drugs?

The major psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol, affects emotional states in humans and laboratory animals by activating brain cannabinoid receptors. A primary endogenous ligand of these receptors is anandamide, the amide of arachidonic acid with ethanolamine. Anandamide is released in selected regions of the brain and is deactivated through a two-step process consisting of transport into cells followed by intracellular hydrolysis. Pharmacological blockade of the enzyme fatty acid amide hydrolase (FAAH), which is responsible for intracellular anandamide degradation, produces anxiolytic-like effects in rats without causing the wide spectrum of behavioral responses typical of direct-acting cannabinoid agonists. These findings suggest that anandamide contributes to the regulation of emotion and anxiety, and that FAAH might be the target for a novel class of anxiolytic drugs.