Abstract
BackgroundIncreased 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.Methodology/Principal FindingsCOX 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/SignificanceBoth 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.
Highlights
According to the textbooks, non-steroidal anti-inflammatory drugs (NSAIDs) produce their effects upon pain and inflammation as a result of the inhibition of cyclooxygenase (COX)derived prostaglandin production [1]
are anandamide (AEA) and 2-AG are effectively metabolised both by hydrolytic and other pathways [6]. With respect to the former, AEA is hydrolysed by both fatty acid amide hydrolase (FAAH) and N-acylethanolamine acid amidase (NAAA), whilst 2-AG is hydrolysed by monoacylglycerol lipase, α/β-hydrolase domain 6/12 and FAAH to form arachidonic acid [6]
COX-2 is a homodimeric enzyme, and the authors suggested that the selectivity was due to the fact that 2-AG oxygenation was blocked when the inhibitor had bound to one of the monomers, whereas blockade of arachidonic acid metabolism required binding to both monomers [9]
Summary
Non-steroidal anti-inflammatory drugs (NSAIDs) produce their effects upon pain and inflammation as a result of the inhibition of cyclooxygenase (COX)derived prostaglandin production [1]. The compound increases brain 2-AG levels, and produces potentially beneficial effects in models of anxiety [10] This group has investigated the (R)-enantiomers of the profens and found them to be potent inhibitors of AEA and 2-AG oxygenation without affecting arachidonic acid oxidation [11]. Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for painrelief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin2-yl)propanamide (Flu-AM1). The effects of the compound upon COX-2derived lipids in intact cells are not known
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