Fatty Acid Amide Hydrolase Substrates Research Articles

Fatty acid amide hydrolase inhibition alleviates anxiety-like symptoms in a rat model used to study post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD), a chronic debilitating condition that affects nearly 5-10% of American adults, is treated with a handful of FDA-approved drugs that provide at best symptomatic relief and exert multiple side effects. Preclinical and clinical evidence shows that inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which deactivates the endocannabinoid anandamide, exhibit anxiolytic-like properties in animal models. In the present study, we investigated the effects of two novel brain-permeable FAAH inhibitors - the compounds ARN14633 and ARN14280 - in a rat model of predator stress-induced long-term anxiety used to study PTSD. We exposed male Sprague-Dawley rats to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a volatile constituent of fox feces, and assessed anxiety-like behaviors in the elevated plus maze (EPM) test seven days later. We measured FAAH activity using a radiometric assay and brain levels of FAAH substrates by liquid chromatography/tandem mass spectrometry. Rats challenged with TMT developed persistent (≥ 7days) anxiety-like symptoms in the EPM test. Intraperitoneal administration of ARN14633 or ARN14280 1h before testing suppressed TMT-induced anxiety-like behaviors with median effective doses (ED50) of 0.23 and 0.33mg/kg, respectively. The effects were negatively correlated (ARN14663: R2 = 0.455; ARN14280: R2 = 0.655) with the inhibition of brain FAAH activity and were accompanied by increases in brain FAAH substrate levels. The results support the hypothesis that FAAH-regulated lipid signaling serves important regulatory functions in the response to stress and confirm that FAAH inhibitors may be useful for the management of PTSD.

Synergistic antinociceptive effects of concomitant NAAA and peripheral FAAH inhibition

The intracellular lipid amidases, fatty acid amide hydrolase (FAAH) and N-acylethanolamine acid amidase (NAAA), terminate the actions of anandamide and palmitoylethanolamide (PEA), two antinociceptive and anti-inflammatory lipid-derived mediators. Here we show, confirming prior research, that small-molecule inhibitors of peripheral FAAH (compound URB937) and systemic NAAA (compound ARN19702) individually attenuate, in male CD-1 mice, pain-related behaviors and paw inflammation in the formalin and carrageenan tests. More importantly, isobolographic analyses revealed that the combination of URB937 and ARN19702 produced substantial synergistic (greater than additive) antinociceptive effects in both models as well as additive anti-inflammatory effects in the carrageenan test. Together, the findings uncover a functional interplay between FAAH and NAAA substrates in the control of nociception, which might be exploited clinically to develop safe and effective pain management strategies

Potentiation of endocannabinoids and other lipid amides prevents hyperalgesia and inflammation in a pre-clinical model of migraine

Targeting fatty acid amide hydrolase (FAAH) is a promising therapeutic strategy to combat certain forms of pain, including migraine headache. FAAH inhibitors, such as the O-biphenyl-3-yl carbamate URB597, have been shown to produce anti-hyperalgesic effects in animal models of migraine. The objective of this study was to investigate the behavioral and biochemical effects of compounds ARN14633 and ARN14280, two URB597 analogs with improved solubility and bioavailability, in a migraine-specific rat model in which trigeminal hyperalgesia is induced by nitroglycerin (NTG) administration. ARN14633 (1 mg/kg, i.p.) and ARN14280 (3 mg/kg, i.p.) were administered to adult male Sprague-Dawley rats 3 hours after NTG injection. One hour after the administration of either compound, rats were subjected to the orofacial formalin test. ARN14633 and ARN14280 attenuated NTG-induced nocifensive behavior and reduced transcription of genes encoding neuronal nitric oxide synthase, pain mediators peptides (calcitonin gene-related peptide, substance P) and pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and 6) in the trigeminal ganglion, cervical spinal cord and medulla. Finally, both compounds strongly elevated levels of endocannabinoids and/or other FAAH substrates in cervical spinal cord and medulla, and, to a lesser extent, in the trigeminal ganglia. The results indicate that the novel global FAAH inhibitors ARN14633 and ARN14280 elicit significant anti-hyperalgesic effects in a migraine-specific animal model and inhibit the associated peptidergic-inflammatory response. Although the precise mechanism underlying these effects remains to be elucidated, our results support further investigational studies of FAAH blockade as a potential therapeutic strategy to treat migraine conditions.

Human sperm cells can form paracetamol metabolite AM404 that directly interferes with sperm calcium signalling and function through a CatSper-dependent mechanism

Abstract STUDY QUESTION Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? SUMMARY ANSWER While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. WHAT IS KNOWN ALREADY Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. STUDY DESIGN, SIZE, DURATION This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. PARTICIPANTS/MATERIALS, SETTING, METHODS Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. MAIN RESULTS AND THE ROLE OF CHANCE Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). LIMITATIONS, REASONS FOR CAUTION The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. WIDER IMPLICATIONS OF THE FINDINGS We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. STUDY FUNDING/COMPETING INTEREST(S) D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE—Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

N-acylethanolamine regulation of TLR3-induced hyperthermia and neuroinflammatory gene expression: A role for PPARα

Increasing evidence suggests that SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is associated with increased risk of developing neurological or psychiatric conditions such as depression, anxiety or dementia. While the precise mechanism underlying this association is unknown, aberrant activation of toll-like receptor (TLR)3, a viral recognizing pattern recognition receptor, may play a key role. Synthetic cannabinoids and enhancing cannabinoid tone via inhibition of fatty acid amide hydrolase (FAAH) has been demonstrated to modulate TLR3-induced neuroimmune responses and associated sickness behaviour. However, the role of individual FAAH substrates, and the receptor mechanisms mediating these effects, are unknown. The present study examined the effects of intracerebral or systemic administration of the FAAH substrates N-oleoylethanolamide (OEA), N-palmitoylethanolamide (PEA) or the anandamide (AEA) analogue meth-AEA on hyperthermia and hypothalamic inflammatory gene expression following administration of the TLR3 agonist, and viral mimetic, poly I:C. The data demonstrate that meth-AEA does not alter TLR3-induced hyperthermia or hypothalamic inflammatory gene expression. In comparison, OEA and PEA attenuated the TLR3-induced hyperthermia, although only OEA attenuated the expression of hyperthermia-related genes (IL-1β, iNOS, COX2 and m-PGES) in the hypothalamus. OEA, but not PEA, attenuated TLR3-induced increases in the expression of all IRF- and NFκB-related genes examined in the hypothalamus, but not in the spleen. Antagonism of PPARα prevented the OEA-induced attenuation of IRF- and NFκB-related genes in the hypothalamus following TLR3 activation but did not significantly alter temperature. PPARα agonism did not alter TLR3-induced hyperthermia or hypothalamic inflammatory gene expression. These data indicate that OEA may be the primary FAAH substrate that modulates TLR3-induced neuroinflammation and hyperthermia, effects partially mediated by PPARα.

Paracetamol analogues conjugated by FAAH induce TRPV1-mediated antinociception without causing acute liver toxicity.

Paracetamol, one of the most widely used pain-relieving drugs, is deacetylated to 4-aminophenol (4-AP) that undergoes fatty acid amide hydrolase (FAAH)-dependent biotransformation into N-arachidonoylphenolamine (AM404), which mediates TRPV1-dependent antinociception in the brain of rodents. However, paracetamol is also converted to the liver-toxic metabolite N-acetyl-p-benzoquinone imine already at therapeutic doses, urging for safer paracetamol analogues. Primary amine analogues with chemical structures similar to paracetamol were evaluated for their propensity to undergo FAAH-dependent N-arachidonoyl conjugation into TRPV1 activators both invitro and invivo in rodents. The antinociceptive and antipyretic activity of paracetamol and primary amine analogues was examined with regard to FAAH and TRPV1 as well as if these analogues produced acute liver toxicity. 5-Amino-2-methoxyphenol (2) and 5-aminoindazole (3) displayed efficient target protein interactions with a dose-dependent antinociceptive effect in the mice formalin test, which in the second phase was dependent on FAAH and TRPV1. No hepatotoxicity of the FAAH substrates transformed into TRPV1 activators was observed. While paracetamol attenuates pyrexia via inhibition of brain cyclooxygenase, its antinociceptive FAAH substrate 4-AP was not antipyretic, suggesting separate mechanisms for the antipyretic and antinociceptive effect of paracetamol. Furthermore, compound 3 reduced fever without a brain cyclooxygenase inhibitory action. The data support our view that analgesics and antipyretics without liver toxicity can be derived from paracetamol. Thus, research into the molecular actions of paracetamol could pave the way for the discovery of analgesics and antipyretics with a better benefit-to-risk ratio.

Characterization of the peripheral FAAH inhibitor, URB937, in animal models of acute and chronic migraine

Inhibiting the activity of fatty-acid amide hydrolase (FAAH), the enzyme that deactivates the endocannabinoid anandamide, enhances anandamide-mediated signaling and holds promise as a molecular target for the treatment of human pathologies such as anxiety and pain. We have previously shown that the peripherally restricted FAAH inhibitor, URB937, prevents nitroglycerin-induced hyperalgesia – an animal model of migraine - and attenuates the activation of brain areas that are relevant for migraine pain, e.g. trigeminal nucleus caudalis and locus coeruleus. The current study is aimed at profiling the behavioral and biochemical effects of URB937 in animal models of acute and chronic migraine. We evaluated the effects of URB937 in two rat models that capture aspects of acute and chronic migraine, and are based on single or repeated administration of the vasodilating drug, nitroglycerin (NTG). In addition to nocifensive behavior, in trigeminal ganglia and medulla, we measured mRNA levels of neuropeptides and pro-inflammatory cytokines along with tissue levels of anandamide and palmitoylethanolamide (PEA), an endogenous agonist of peroxisome proliferator-activated receptor type-a (PPAR-a), which is also a FAAH substrate. In the acute migraine model, we also investigated the effect of subtype-selective antagonist for cannabinoid receptors 1 and 2 (AM251 and AM630, respectively) on nocifensive behavior and on levels of neuropeptides and pro-inflammatory cytokines. In the acute migraine paradigm, URB937 significantly reduced hyperalgesia in the orofacial formalin test when administered either before or after NTG. This effect was accompanied by an increase in anandamide and PEA levels in target neural tissue, depended upon CB1 receptor activation, and was associated with a decrease in calcitonin gene-related peptide (CGRP), substance P and cytokines TNF-alpha and IL-6 mRNA. Similar effects were observed in the chronic migraine paradigm, where URB937 counteracted NTG-induced trigeminal hyperalgesia and prevented the increase in neuropeptide and cytokine transcription.The results show that peripheral FAAH inhibition by URB937 effectively reduces both acute and chronic NTG-induced trigeminal hyperalgesia, likely via augmented anandamide-mediated CB1 receptor activation. These effects are associated with inhibition of neuropeptidergic and inflammatory pathways.

A CNS-Targeting Prodrug Strategy for Nuclear Receptor Modulators.

The blood-brain barrier is a major impediment for targeted central nervous system (CNS) therapeutics, especially with carboxylic acid-containing drugs. Nuclear receptor modulators, which often feature carboxylic acid motifs for target engagement, have emerged as a class of potentially powerful therapeutics for neurodegenerative CNS diseases. Herein is described a prodrug strategy that directs the biodistribution of parent drug nuclear receptor modulators into the CNS while masking them as functional receptor ligands in the periphery. This prodrug strategy targets a specific amidase, fatty acid amide hydrolase (FAAH), an enzyme with enriched expression in the CNS. Our results demonstrate that this prodrug strategy can be generalized to a variety of carboxylic acid-containing drug structures that satisfy the structural requirements of blood-brain barrier diffusion and FAAH substrate recognition.

Lower brain fatty acid amide hydrolase in treatment-seeking patients with alcohol use disorder: a positron emission tomography study with [C-11]CURB.

The endocannabinoid enzyme, fatty acid amide hydrolase (FAAH), has been proposed as a therapeutic target for alcohol use disorder (AUD) and co-morbid psychiatric illnesses. Investigating this target in the living human brain and its relationship to clinical outcome is a critical step of informed drug development. Our objective was to establish whether brain FAAH levels are low in individuals with AUD and related to drinking behavior. In this pilot study, treatment-seeking patients with AUD completed two PET scans with the FAAH radiotracer [C-11]CURB after 3-7 days (n = 14) and 2-4 weeks (n = 9) of monitored abstinence. Healthy controls (n = 25) completed one scan. FAAH genetic polymorphism (rs324420) and blood concentrations of anandamide and other N-acylethanolamines metabolized by FAAH were determined and AUD symptoms assessed. In AUD, brain FAAH levels were globally lower than controls during early abstinence (F(1,36) = 5.447; p = 0.025)) and FAAH substrates (anandamide, oleoylethanolamide, and N-docosahexaenoylethanolamide) were significantly elevated (30-67%). No significant differences in FAAH or FAAH substrates were noted after 2-4 weeks abstinence. FAAH levels negatively correlated with drinks per week (r = -0.57, p = 0.032) and plasma concentrations of the three FAAH substrates (r > 0.57; p < 0.04)). Our findings suggest that early abstinence from alcohol in AUD is associated with transiently low brain FAAH levels, which are inversely related to heavier alcohol use and elevated plasma levels of FAAH substrates. Whether low FAAH is an adaptive beneficial response to chronic alcohol is unknown. Therapeutic strategies focusing on FAAH inhibition should consider the possibility that low FAAH during early abstinence may be related to drinking.

Pharmacokinetics, pharmacodynamics and safety studies on URB937, a peripherally restricted fatty acid amide hydrolase inhibitor, in rats.

URB937, a peripheral fatty acid amide hydrolase (FAAH) inhibitor, exerts profound analgesic effects in animal models. We examined, in rats, (1) the pharmacokinetic profile of oral URB937; (2) the compound's ability to elevate levels of the representative FAAH substrate, oleoylethanolamide (OEA); and (3) the compound's tolerability after oral administration. We developed a liquid chromatography/tandem mass spectrometry (LC/MS-MS) method to measure URB937 and used a pre-existing LC/MS-MS assay to quantify OEA. FAAH activity was measured using a radioactive substrate. The tolerability of single or repeated (once daily for 2weeks) oral administration of supramaximal doses of URB937 (100, 300, 1000mg/kg) was assessed by monitoring food intake, water intake and body weight, followed by post-mortem evaluation of organ structure. URB937 was orally available in male rats (F=36%), but remained undetectable in brain when administered at doses that maximally inhibit FAAH activity and elevate OEA in plasma and liver. Acute and subchronic treatment with high doses of URB937 was well-tolerated and resulted in FAAH inhibition in brain. Pain remains a major unmet medical need. The favourable pharmacokinetic and pharmacodynamic properties of URB937, along with its tolerability, encourage further development studies on this compound.

Effects of fatty acid amide hydrolase inhibitor URB597 in a rat model of trauma-induced long-term anxiety

The endocannabinoid neurotransmitter, anandamide, has been implicated in the central modulation of stress responses. Previous animal experiments have shown that inhibitors of the anandamide-degrading enzyme, fatty acid amide hydrolase (FAAH), enhance the ability to cope with acute and chronic stress. Here, we investigated the effects of the globally active FAAH inhibitor URB597 in a rat model of predator stress-induced long-term anxiety. Rats exposed to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a chemical constituent of fox feces, developed a persistent anxiety-like state, which was assessed 7days after exposure using the elevated plus maze (EPM) test. Systemic administration of URB597 [0.03-0.1-0.3mg/kg, intraperitoneal (ip)] 2h before testing suppressed TMT-induced behaviors with a median effective dose (IC50) of 0.075mg/kg. This effect was strongly correlated with inhibition of brain FAAH activity (r2 = 1.0) and was accompanied by increased brain levels of three FAAH substrates: the endocannabinoid anandamide and the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonists, oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The anxiolytic-like effects of URB597 were blocked by co-administration of the CB1 receptor antagonist rimonabant (1mg/kg, ip), but not of the PPAR-α antagonist GW6471 (1mg/kg, ip). Finally, when administered 18h after TMT exposure (i.e., 6days before the EPM test), URB597 (0.3mg/kg, ip) prevented the consolidation of anxiety-like behavior in a CB1-dependent manner. The results support the hypothesis that anandamide-mediated signaling at CB1 receptors serves an important regulatory function in the stress response, and confirm that FAAH inhibition may offer a potential therapeutic strategy for post-traumatic stress disorder.

The prefrontal cortical endocannabinoid system modulates fear-pain interactions in a subregion-specific manner.

The emotional processing and coordination of top-down responses to noxious and conditioned aversive stimuli involves the medial prefrontal cortex (mPFC). Evidence suggests that subregions of the mPFC [infralimbic (IfL), prelimbic (PrL) and anterior cingulate (ACC) cortices] differentially alter the expression of contextually induced fear and nociceptive behaviour. We investigated the role of the endocannabinoid system in the IfL, PrL and ACC in formalin-evoked nociceptive behaviour, fear-conditioned analgesia (FCA) and conditioned fear in the presence of nociceptive tone. FCA was modelled in male Lister-hooded rats by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. The effects of intra-mPFC administration of AM251 [cannabinoid type 1 (CB1 ) receptor antagonist/inverse agonist], URB597 [fatty acid amide hydrolase (FAAH) inhibitor] or URB597+AM251 on FCA and freezing behaviour were assessed. AM251 attenuated FCA when injected into the IfL or PrL and reduced contextually induced freezing behaviour when injected intra-IfL but not intra-PrL or intra-ACC. Intra-ACC administration of AM251 alone or in combination with URB597 had no effect on FCA or freezing. URB597 attenuated FCA and freezing behaviour when injected intra-IfL, prolonged the expression of FCA when injected intra-PrL and had no effect on these behaviours when injected intra-ACC. These results suggest important and differing roles for FAAH substrates or CB1 receptors in the PrL, IfL and ACC in the expression of FCA and conditioned fear in the presence of nociceptive tone. This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

FAAH inhibition attenuates TLR3-mediated hyperthermia, nociceptive- and anxiety-like behaviour in female rats

Aberrant activation of toll-like receptor (TLR)s results in persistent and prolonged neuroinflammation and has been implicated in the pathogenesis and exacerbation of psychiatric and neurodegenerative disorders. TLR3 coordinates the innate immune response to viral infection and recent data have demonstrated that inhibiting fatty acid amide hydrolase (FAAH), the enzyme that primarily metabolizes anandamide, modulates TLR3-mediated neuroinflammation. However, the physiological and behavioural consequences of such modulation are unknown. The present study examined the effect of URB597, a selective FAAH inhibitor, on neuroinflammation, physiological and behavioural alterations following administration of the TLR3 agonist and viral mimetic poly I:C to female rats. URB597 attenuated TLR3-mediated fever, mechanical and cold allodynia, and anxiety-like behaviour in the elevated plus maze and open field arena. There was no effect of URB597 on TLR3-mediated decreases in body weight and no effect in the sucrose preference or forced swim tests. URB597 attenuated the TLR3-mediated increase in the expression of CD11b and CD68, markers of microglia/macrophage activation. In summary, these data demonstrate that enhancing FAAH substrate levels suppresses TLR3-mediated microglia/macrophage activation and associated changes in fever, nociceptive responding and anxiety-related behaviour. These data provide further support for FAAH as a novel therapeutic target for neuroinflammatory disorders.

Inhibition of Fatty Acid Amide Hydrolase (FAAH) by Macamides.

The pentane extract of the Peruvian plant, Lepidium meyenii (Maca), has been demonstrated to possess neuroprotective activity in previous in vitro and in vivo studies (Pino-Figueroa et al. in Ann N Y Acad Sci 1199:77-85, 2010; Pino-Figueroa et al. in Am J Neuroprot Neuroregener 3:87-92, 2011). This extract contains a number of macamides that may act on the endocannabinoid system (Pino-Figueroa et al. in Ann N Y Acad Sci 1199:77-85, 2010; Pino-Figueroa et al., 2011; Dini et al. in Food Chem 49:347-349, 1994). The aim of this study was to characterize the inhibitory activity of four of these maccamides (N-benzylstearamide, N-benzyloleamide, N-benzyloctadeca-9Z,12Z-dienamide, and N-benzyloctadeca-9Z,12Z,15Z-trienamide) on fatty acid amide hydrolase (FAAH), an enzyme that is responsible for endocannabinoid degradation in the nervous system (Kumar et al. in Anaesthesia 56:1059-1068, 2001). The four compounds were tested at concentrations between 1 and 100μM, utilizing an FAAH inhibitor screening assay. The results demonstrated concentration-dependent FAAH inhibitory activities for the four macamides tested. N-Benzyloctadeca-9Z,12Z-dienamide demonstrated the highest FAAH inhibitory activity whereas N-benzylstearamide had the lowest inhibitory activity. In addition, N-benzylstearamide, N-benzyloleamide, and N-benzyloctadeca-9Z,12Z-dienamide demonstrated time-dependent inhibition when tested after a pre-incubation period, indicating that the mechanism of inhibition for these compounds most likely is irreversible. Of interest, unsaturation in the fatty acid moiety resulted in greater FAAH inhibitory activity. LC/MS/MS analysis demonstrated that FAAH was able to hydrolyze N-benzyloctadeca-9Z,12Z-dienamide, suggesting that N-benzyloctadeca-9Z,12Z-dienamide is also a slow substrate for FAAH. These results provide useful information about the mechanism of action of Lepidium meyenii and may help with the development of new compounds with FAAH inhibitory or modulatory activity.

N-Docosahexaenoylethanolamine (synaptamide): Carbon-14 radiolabeling and metabolic studies

N-Docosahexaenoylethanolamine (synaptamide) is structurally similar to the endocannabinoid N-arachidonoylethanolamine (anandamide), but incorporates the omega-3 22:6 fatty acid docosahexaenoic acid (DHA) in place of the omega-6 20:4 fatty acid arachidonic acid (AA). Some brain membrane lipid effects may be mediated via synaptamide. In competition experiments with mouse brain homogenate in vitro, we found that synaptamide was an order-of-magnitude poorer inhibitor of radioactive anandamide hydrolysis than was anandamide itself. Also, enzyme-mediated hydrolysis of synaptamide was observed to occur at a slower rate than for anandamide. We have synthesized synaptamide radiolabeled with carbon-14 in both the ethanolamine ([α,β-14C2]synaptamide) and in the DHA ([1-14C]synaptamide) moieties. The brain penetration, distribution, and metabolism of radiolabeled synaptamide were studied in mice in vivo relative to anandamide, DHA, and AA. Brain uptake of labeled synaptamide was greater than for labeled DHA, consistent with previous studies of labeled anandamide and AA in our laboratory. After administering either isotopomer of radiolabeled synaptamide, radiolabeled phospholipids were found in mouse brain. Pretreatment of mice with PF3845, a potent, specific inhibitor of fatty acid amide hydrolase (FAAH), eliminated formation of labeled phospholipids measured after 15min, suggesting that synaptamide is hydrolyzed nearly exclusively by FAAH, though it is a poorer substrate for FAAH than anandamide.

Suppression of acute and anticipatory nausea by peripherally restricted fatty acid amide hydrolase inhibitor in animal models: role of PPARα and CB1 receptors.

Effective treatments of nausea are limited. In this study we evaluated the ability of the peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor, URB937, to suppress acute and anticipatory nausea in rats and examined the pharmacological mechanism of this effect. We investigated the potential of URB937 (administered i.p.) to reduce the establishment of lithium chloride-induced conditioned gaping (model of acute nausea) and to reduce the expression of contextually-elicited conditioned gaping (model of anticipatory nausea) in rats. The role of CB1 receptors, CB2 receptors and PPARα in the anti-nausea effect of URB937 was examined. The potential of URB937 to suppress FAAH activity in tissue collected from the area postrema (AP), prefrontal cortex (PFC), liver and duodenum and to elevate levels of FAAH substrates - anandamide (AEA), N-oleoylethanolamide (OEO) and N-palmitoylethanolamide (PEA) - in the AP was also evaluated. URB937 reduced acute nausea by a PPARα-dependent mechanism and reduced anticipatory nausea by a CB1 receptor-dependent mechanism. The PPARα agonist, GW7647, similarly attenuated acute nausea. URB937 reduced FAAH activity in the liver and the duodenum but not in the PFC. In addition, URB937 reduced FAAH activity and elevated levels of fatty-acid ethanolamides in the AP, a brain region that is not protected by the blood-brain barrier. The anti-nausea action of URB937 may occur in the AP and may involve PPARα to suppress acute nausea and CB1 receptors to suppress anticipatory nausea.

Targeting Fatty-Acid Amide Hydrolase with Prodrugs for CNS-Selective Therapy.

The blood-brain barrier (BBB) can be a substantial impediment to achieving therapeutic levels of drugs in the CNS. Certain chemical functionality such as the carboxylic acid is a general liability for BBB permeability preventing significant CNS distribution of a drug from a systemic dose. Here, we report a strategy for CNS-selective distribution of the carboxylic acid containing thyromimetic sobetirome using prodrugs targeted to fatty-acid amide hydrolase (FAAH), which is expressed in the brain. Two amide prodrugs of sobetirome were shown to be efficient substrates of FAAH with Vmax/KM values comparable to the natural endocannabinoid FAAH substrate anandamide. In mice, a systemic dose of sobetirome prodrug leads to a substantial ∼60-fold increase in brain distribution (Kp) of sobetirome compared to an equimolar systemic dose of the parent drug. The increased delivery of sobetirome to the brain from the prodrug was diminished by both pharmacological inhibition and genetic deletion of FAAH in vivo. The increased brain exposure of sobetirome arising from the prodrug corresponds to ∼30-fold increased potency in brain target engagement compared to the parent drug. These results suggest that FAAH-targeted prodrugs can considerably increase drug exposure to the CNS with a concomitant decrease in systemic drug levels generating a desirable distribution profile for CNS acting drugs.

Targeting fatty acid amide hydrolase and transient receptor potential vanilloid-1 simultaneously to modulate colonic motility and visceral sensation in the mouse: A pharmacological intervention with N-arachidonoyl-serotonin (AA-5-HT).

Endocannabinoid anandamide (AEA) inhibits intestinal motility and visceral pain, but it may also be proalgesic through transient receptor potential vanilloid-1 (TRPV1). AEA is degraded by fatty acid amide hydrolase (FAAH). This study explored whether dual inhibition of FAAH and TRPV1 reduces diarrhea and abdominal pain. Immunostaining was performed on myenteric plexus of the mouse colon. The effects of the dual FAAH/TRPV1 inhibitor AA-5-HT on electrically induced contractility, excitatory junction potential (EJP) and fast (f) and slow (s) inhibitory junction potentials (IJP) in the mouse colon, colonic propulsion and visceromotor response (VMR) to rectal distension were studied. The colonic levels of endocannabinoids and fatty acid amides were measured. CB1-positive neurons exhibited TRPV1; only some TRPV1 positive neurons did not express CB1. CB1 and FAAH did not colocalize. AA-5-HT (100nM-10μM) decreased colonic contractility by ~60%; this effect was abolished by TRPV1 antagonist 5'-IRTX, but not by CB1 antagonist, SR141716. AA-5-HT (1μM-10μM) inhibited EJP by ~30% and IJPs by ~50%. The effects of AA-5-HT on junction potentials were reversed by SR141716 and 5`-IRTX. AA-5-HT (20mg/kg; i.p.) inhibited colonic propulsion by ~30%; SR141716 but not 5`-IRTX reversed this effect. AA-5-HT decreased VMR by ~50%-60%; these effects were not blocked by SR141716 or 5`-IRTX. AA-5-HT increased AEA in the colon. The effects of AA-5-HT on visceral sensation and colonic motility are differentially mediated by CB1, TRPV1 and non-CB1/TRPV1 mechanisms, possibly reflecting the distinct neuromodulatory roles of endocannabinoid and endovanilloid FAAH substrates in the mouse intestine.

Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells.

Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified.

Potent multitarget FAAH-COX inhibitors: Design and structure-activity relationship studies

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by inhibiting cyclooxygenase (COX)-1 and COX-2. Though widely prescribed for pain and inflammation, these agents have limited utility in chronic diseases due to serious mechanism-based adverse events such as gastrointestinal damage. Concomitant blockade of fatty acid amide hydrolase (FAAH) enhances the therapeutic effects of the NSAIDs while attenuating their propensity to cause gastrointestinal injury. This favorable interaction is attributed to the accumulation of protective FAAH substrates, such as the endocannabinoid anandamide, and suggests that agents simultaneously targeting COX and FAAH might provide an innovative strategy to combat pain and inflammation with reduced side effects. Here, we describe the rational design and structure-active relationship (SAR) properties of the first class of potent multitarget FAAH-COX inhibitors. A focused SAR exploration around the prototype 10r (ARN2508) led to the identification of achiral (18b) as well as racemic (29a-c and 29e) analogs. Absolute configurational assignment and pharmacological evaluation of single enantiomers of 10r are also presented. (S)-(+)-10r is the first highly potent and selective chiral inhibitor of FAAH-COX with marked in vivo activity, and represents a promising lead to discover novel analgesics and anti-inflammatory drugs.