title: Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor.
abstract: BACKGROUND
  Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.
  
  
  RESULTS
  COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).
  
  
  CONCLUSIONS
  Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden; Department of Chemistry, Umeå University, Umeå, Sweden.
authors.forename: Sandra
authors.initials: S
authors.lastname: Gouveia-Figueira
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Jessica
authors.initials: J
authors.lastname: Karlsson
authors.affiliation: Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Deplano
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Sanaz
authors.initials: S
authors.lastname: Hashemian
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Mona
authors.initials: M
authors.lastname: Svensson
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Marcus
authors.initials: M
authors.lastname: Fredriksson Sundbom
authors.affiliation: Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari, Italy.
authors.forename: Cenzo
authors.initials: C
authors.lastname: Congiu
authors.affiliation: Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Onnis
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fowler
mesh_terms: D000577:Amides / Q000737:chemistry / Q000494:pharmacology*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D001095:Arachidonic Acids / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D002219:Carbamates / Q000494:pharmacology; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D063388:Endocannabinoids / Q000378:metabolism; D005480:Flurbiprofen / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007371:Interferon-gamma / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D050356:Lipid Metabolism / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D053284:Polyunsaturated Alkamides / Q000378:metabolism; D011453:Prostaglandins / Q000378:metabolism; D000067996:RAW 264.7 Cells; D013237:Stereoisomerism; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000577:Amides; D000581:Amidohydrolases; D016718:Arachidonic Acid; D001095:Arachidonic Acids; D001549:Benzamides; D002219:Carbamates; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D016861:Cyclooxygenase Inhibitors; D063388:Endocannabinoids; D005480:Flurbiprofen; D007371:Interferon-gamma; D015759:Ionomycin; D007527:Isoenzymes; D008070:Lipopolysaccharides; D053284:Polyunsaturated Alkamides; D011453:Prostaglandins; D000099252:Fatty Acid Amide Hydrolases; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C078814:anandamide