Metabolism of N‐Acylethanolamines: To Phase II and Back Again


N‐acylethanolamines (NAEs) are a family of endogenous signalling molecules involved in various effects of the body including pain, inflammation, appetite and sleep. NAEs are mainly degraded by fatty acid amide hydrolase (FAAH) and N‐acylethanolamine acid amidase (NAAA). FAAH inhibitors have shown promising results in preclinical studies of pain, inflammation and anxiety, mediating effects mainly via increased cannabinoid receptor activity. However, FAAH inhibitors have failed in clinical pain trials, and in a recent phase I trial, an irreversible compound caused one death and sustained impairments in healthy volunteers. The latter is most likely due to off‐target effects of that compound, rather than an FAAH‐mediated effect, and design of dual‐action FAAH‐NAAA, ‐TRPV1 or ‐cyclooxygenase‐2 inhibitory compounds may solve the pain efficacy issue. NAAA inhibitors are still in preclinical testing and show a promising anti‐inflammatory profile mainly due to increased palmitoylethanolamide and oleoylethanolamide levels.

Key Concepts

  • N‐acylethanolamines as a family of lipids with diverse biological activities involving different receptor pathways.
  • Fatty acid amide hydrolase and N‐acylethanolamine acid amidase as the main enzymes responsible for hydrolysis of N‐acylethanolamines in mammals.
  • A pharmacological strategy for treatment of anxiety, inflammation and/or pain by potentiation of N‐acylethanolamine signalling through inhibition of metabolic enzymes.
  • The predictive validity of animal tests for pain.
  • The importance of ‘off‐target’ actions of a drug on its safety.
  • The concept of dual action inhibitors as opposed to two drugs.

Keywords: N‐acylethanolamines; fatty acid amide hydrolase; degradation; N‐acylethanolamine acid amide hydrolase; cyclooxygenase‐2; TRPV1; dual target; inhibitor

Figure 1. Structure and plasma concentrations in healthy humans of the most common NAEs (N‐acylethanolamines). Blood samples were collected from healthy volunteers, lipids were extracted and analysed by ultra performance liquid chromatography coupled to tandem mass spectrometry. Abbreviations: PEA, palmitoylethanolamide; SEA, stearoylethanolamide; OEA, oleoylethanolamide; LEA, linoleoyl ethanolamide; AEA, anandamide. The NAEs are followed by (N:n), which indicates the number of carbon atoms and double bonds, respectively, in the side chain. Thus, AEA has 20 carbon atoms and four double bonds in the arachidonoyl side chain. Data are adapted from Hellström et al. 2016.
Figure 2. Structures of irreversible (top five compounds) and reversible (OL-135, MK-4409) FAAH (fatty acid amide hydrolase) inhibitors. Structures were collected using the MolGrabber function built into the ChemDoodle programme v 8.1.0 for the Macintosh (iChemLabs, LLC, Somerset, NJ, USA). BIA10‐2474 is an irreversible inhibitor of FAAH, but has been separated from the others in view of its catastrophic clinical trial.
Figure 3. Structures of systemically active NAAA (N‐acylethanolamine acid amide hydrolase) inhibitors. Note that while selectivity versus FAAH has been established, the selectivity of the compounds vis‐a‐vis other off‐targets is uncertain.
Figure 4. Derivation of dual‐action COX‐FAAH inhibitors from flurbiprofen.


Ahn K, Johnson DS, Mileni M, et al. (2009) Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chemistry and Biology 16: 411–420.

Ahn K, Smith SE, Liimatta MB, et al. (2011) Mechanistic and pharmacological characterization of PF‐04457845: a highly potent and selective fatty acid amide hydrolase inhibitor that reduces inflammatory and noninflammatory pain. Journal of Pharmacology and Experimental Therapeutics 338: 114–124.

Alhouayek M, Bottemanne P, Subramanian KV, et al. (2015) N‐Acylethanolamine‐hydrolyzing acid amidase inhibition increases colon N‐palmitoylethanolamine levels and counteracts murine colitis. FASEB Journal 29: 650–661.

Bracey MH, Hanson MA, Masuda KR, et al. (2002) Structural adaptations in membrane enzyme that terminates endocannabinoid signaling. Science 298: 1793–1796.

Chobanian HR, Guo Y, Liu P, et al. (2014) Discovery of MK‐4409, a novel oxazole FAAH inhibitor for the treatment of inflammatory and neuropathic pain. ACS Medicinal Chemistry Letters 5: 717–721.

Clapper J, Moreno‐Sanz G, Russo R, et al. (2010) Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism. Nature Neuroscience 13: 1265–1270.

Cravatt BF, Demarest K, Patricellis MP, et al. (2001) Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proceedings of the National Academy of Sciences 98: 9371–9376.

Cravatt BF, Saghatelian A, Hawkins EG, et al. (2004) Functional disassociation of the central and peripheral fatty acid amide signaling systems. Proceedings of the National Academy of Sciences 101: 10821–10826.

Cyclia (2016) Cyclica Predicts Mechanism of Neurotoxicity for BIA 10‐2474, an Experimental Drug Causing Death in Clinical Trial [news release]. Retrieved from‐10‐2474/ (URL checked 26 June 2017).

Deutsch DG and Chin SA (1993) Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochemical Pharmacology 46: 791–796.

Devane WA, Hanus L, Breuer A, et al. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258: 1946–1949.

Duranti A, Tontini A, Antoinetti F, et al. (2012) N‐(2‐oxo‐3‐oxetanyl)carbamic acid esters as N‐acylethanolamine acid amidase inhibitors: synthesis and structure‐activity and structure–property relationships. Journal of Medicinal Chemistry 55: 4824–4836.

Fidelix TS, Macedo CR, Maxwell LJ, et al. (2014) Diacerein for osteoarthritis. Cochrane Database of Systematic Reviews. Art. No.: CD005117.

Gabrielsson L, Mattsson S and Fowler CJ (2016) Palmitoylethanolamide for the treatment of pain: pharmacokinetics, safety and efficacy. British Journal of Clinical Pharmacology 82: 932–942.

Gouveia‐Figueira S, Karlsson J, Deplano A, et al. (2015) Characterisation of (R)‐2‐(2‐fluorobiphenyl‐4‐yl)‐n‐(3‐methylpyridin‐2‐yl)propanamide as a dual fatty acid amide hydrolase: cyclooxygenase inhibitor. PLoS One 10: e139212.

Haller J, Goldberg SR, Pelcer KG, et al. (2013) The effects of anandamide signaling enhanced by the FAAH inhibitor URB597 on coping styles in rats. Psychopharmacology 230: 353–362.

Hellström F, Gouveia‐Figueira S, Nording ML, et al. (2016) Association between plasma concentrations of linoleic acid‐derived oxylipins and the perceived pain scores in an exploratory study in women with chronic neck pain. BMC Musculoskeletal Disorders 17: 103.

Hill MN, Kumar SA, Filipski SB, et al. (2014) Disruption of fatty acid amide hydrolase activity prevents the effects of chronic stress on anxiety and amygdalar microstructure. Molecular Psychiatry 18: 1125–1135.

Holt S, Comelli F, Costa B, et al. (2005) Inhibitors of fatty acid amide hydrolase reduce carrageenan‐induced hind paw inflammation in pentobarbital‐treated mice: comparison with indomethacin and possible involvement of cannabinoid receptors. British Journal of Pharmacology 146: 467–476.

Huggins JP, Smart TS, Langman S, et al. (2012) An efficient randomized, placebo‐controlled clinical trial with the irreversible fatty acid amide hydrolase‐1 inhibitor PF‐04457845, which modulates endocannabinoids but fails to induce effective analgesia in patients with pain due to osteoarthritis of the knee. Pain 153: 1837–1846.

Jhaveri MD, Richardson D, Kendall DA, et al. (2006) Analgesic effects of fatty acid amide hydrolase inhibition in a rat model of neuropathic pain. Journal of Neuroscience 26: 13318–13327.

Kaczocha M, Glaser ST, Chae J, et al. (2010) Lipid droplets are novel sites of N‐acylethanolamine inactivation by fatty acid amide hydrolase‐2. Journal of Biological Chemistry 285: 2796–2806.

Karlsson J, Morgillo CM, Deplano A, et al. (2015) Interaction of the N‐(3‐methylpyridin‐2‐yl)amide derivatives of flurbiprofen and ibuprofen with FAAH: enantiomeric selectivity and binding mode. PLoS One 10: e0142711.

Kathuria S, Gaetani S, Fegley D, et al. (2003) Modulation of anxiety through blockade of anandamide hydrolysis. Nature Medicine 9: 76–81.

Keith JM, Jones WM, Tichenor M, et al. (2015) Preclinical characterization of the FAAH inhibitor JNJ‐42165279. ACS Medicinal Chemistry Letters 6: 1204–1208.

Kerbrat A, Ferré JC, Fillatre P, et al. (2016) Acute Neurologic disorder from an inhibitor of fatty acid amide hydrolase. New England Journal of Medicine 375: 1717–1725.

Li GL, Winter H, Arends R, et al. (2011) Assessment of the pharmacology and tolerability of PF‐04457845, an irreversible inhibitor of fatty acid amide hydrolase‐1, in healthy subjects. British Journal of Clinical Pharmacology 73: 706–716.

Lichtman AH, Leung D, Shelton CC, et al. (2004) reversible inhibitors of fatty acid amide hydrolase that promotes analgesia: evidence for an unprecedented combination of potency and selectivity. Journal of Pharmacology and Experimental Therapeutics 311: 441–448.

Lo Verme J, Fu K, Astarita G, et al. (2005) The nuclear receptor peroxisome proliferator‐activated receptor‐alpha mediates the anti‐inflammatory actions of palmitoylethanolamide. Molecular Pharmacology 67: 15–19.

Malek N, Mrugala M, Makuch W, et al. (2015) A multi‐target approach for pain treatment: dual inhibition of fattya acid amide hydrolase and TRPV1 in a tar model of osteoarthritis. Pain 156: 890–903.

Mckinney MK and Cravatt BF (2003) Evidence for distinct roles in the catalysis for residues of the serine‐serine‐lysine catalytic triad of fatty acid amide hydrolase. Journal of Biological Chemistry 278: 37393–37399.

Naidu PS, Booker L, Cravatt BF, et al. (2009) Synergy between enzyme inhibitors of fatty acid amide hydrolase and cyclooxygenase in visceral nociception. Journal of Pharmacology and Experimental Therapeutics 329: 48–56.

Pawsey S, Wood M, Browne H, et al. (2016) Safety, tolerability and pharmacokinetics of FAAH inhibitor V158866: a double blind, randomised, placebo controlled phase I study in healthy volunteers. Drugs in R&D 16: 180–190.

Petrosino S, Ahmad A, Marcolongo G, et al. (2015) Diacerein is a potent and selective inhibitor of palmitoylethanolamide inactivation with analgesic activity in a rat model of acute inflammatory pain. Pharmacological Research 91: 9–14.

Ribeiro A, Pontis S, Mengatto L, et al. (2016) A potent systemically active N‐acylethanolamine acid amidase inhibitor that suppresses inflammation in human macrophage activation. ACS Chemical Biology 10: 1838–1846.

Sasso O, Moreno‐Sanz G, Martucci C, et al. (2013) Antinociceptive effects of the N‐acylethanolamine acid amidase inhibitor ARN077 in rodent pain models. Pain 154: 350–360.

Sasso O, Migliore M, Habrant D, et al. (2015) Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti‐inflammatory drug‐dependent gastrointestinal damage. FASEB Journal 29: 2616–2627.

Schmid P, Krebsbach R, Perry S, et al. (1995) Occurrence and postmortem generation of anandamide and other long‐chain N‐acylethanolamines in mammalian brain. FEBS Letters 375: 117–120.

Singh Tahim A, Sántha P and Nagy I (2005) Inflammatory mediators convert anandamide into a potent activator of the vanilloid type 1 transient receptor potential receptor in nociceptive primary sensory neurons. Neuroscience 136: 539–548.

Solorzano C, Zhu C, Battista N, et al. (2009) Selective N‐acylethanolamine‐hydrolyzing acid amidase inhibition reveals a key role for endogenous palmitoylethanolamide in inflammation. Proceedings of the National Academy of Sciences 106: 20966–20971.

Tchantchou F, Tucker LB, Fu AH, et al. (2014) The fatty acid amide hydrolase inhibitor PF‐3845 promotes neuronal survival, attenuates and improves functional recovery in mice with traumatic brain injury. Neuropharmacology 85: 427–439.

Tsuboi K, Zhao LY, Okamoto Y, et al. (2007) Predominant expression of lysosomal N‐acylethanolamine‐hydrolyzing acid amidase in macrophages revealed by immunochemical studies. Biochimica et Biophysica Acta 1771: 623–632.

U.S. Food and Drug Administration (2016) FDA Finds Drugs Under Investigation in the U.S. Related to French BIA 10‐2427 Drug Do Not Pose Similar Safety Risks [press release]. Retrieved from (URL checked 26 June 2017).

Van Esbroeck ACM, Janssen APA, Cognetta AB III, et al. (2017) Activity‐based protein profiling reveals off‐target proteins of the FAAH inhibitor BIA 10‐2427. Science 356: 1084–1087.

Vernalis plc (2015) Vernalis plc Completes Investment in its NCE Development [press release]. Retrieved from‐centre/latest‐releases/708‐vernalis‐plc‐completes‐investment‐in‐its‐nce‐development‐pipeline (URL checked 26 June 2017).

Wagenlehner FME, Van Till JWO, Houbiers JGA, et al. (2017) Fatty acid amide hydrolase inhibitor treatment in men with chronic prostatitis/chronic pelvic pain syndrome: an adaptive double blind, randomized controlled trial. Urology 103: 191–197.

Wei BQ, Mikkelsen TS, Mckinney MK, et al. (2006) A second fatty acid amide hydrolase with variable distribution among placental mammals. Biological Chemistry 231: 36569–36578.

Yang L, Li L, Chen L, et al. (2015) Potential analgesic effects of a novel N‐acylethanolamine acid amidase inhibitor F96 through PPAR‐α. Scientific Reports 5: 13565.

Zhao LY, Tsuboi K, Okamoto Y, et al. (2007) Proteolytic activation and glycosylation of N‐acylethanolamine‐hydrolyzing acid amidase, a lysosomal enzyme involved in the endocannabinoid metabolism. Biochimica et Biophysica Acta 1771: 1397–1405.

Further Reading

Bryden LA, Nicholson JR, Doods H, et al. (2015) Deficits in spontaneous burrowing behavior in the rat bilateral monosodium iodoacetate model of osteoarthritis: an objective measure of pain‐related behavior and analgesic efficacy. Osteoarthritis Cartilage 23: 1605–1612.

Fowler CJ (2015a) The potential of inhibitors of endocannabinoid metabolism for drug development: a critical review. Handbook of Experimental Pharmacology 231: 95–128.

Fowler CJ (2015b) The potential of inhibitors of endocannabinoid metabolism as anxiolytic and antidepressive drugs ‐ a practical view. European Neuropsychopharmacology 25: 749–762.

Kola I and Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery 3: 711–715.

Ligresti A, De Petrocellis L and Di Marzo V (2016) From phytocannabinoids to cannabinoid receptors and endocannabinoids: pleiotropic physiological and pathological roles through complex pharmacology. Physiological Reviews 96: 1593–1659.

Pacher P and Kunos G (2013) Modulating the endocannabinoid system in human health and disease ‐ successes and failures. FEBS Journal 280: 1918–1943.

Rice ASC, Cimino‐Brown D, Eisenach J, et al. (2008) Animal models and the prediction of efficacy in clinical trials of analgesic drugs: a critical appraisal and call for uniform reporting standards. Pain 139: 243–247.

Talevi A (2015) Multi‐target pharmacology: possibilities and limitation of the “skeleton key approach” from a medicinal chemist perspective. Frontiers in Pharmacology 6: 205. DOI: 10.3389/fphar.2015.00205.

Tuo W, Leleu‐Chavain N, Spencer J, et al. (2017) Therapeutic potential of fatty acid amide hydrolase, monoacylglycerol lipase, and N‐acylethanolamine acid amidase inhibitors. Journal of Medicinal Chemistry 60: 4–46.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Karlsson, Jessica, and Fowler, Christopher J(Nov 2017) Metabolism of N‐Acylethanolamines: To Phase II and Back Again. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0027664]