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New Tricks for Some Old Drugs

By: Carol A. Rouzer, VICB Communications
Published: October 19, 2011


Selective blockade of endocannabinoid metabolism by NSAIDs is a previously unrecognized mechanism of anti-inflammatory and analgesic action.

In addition to its well known psychotropic activity (euphoria, altered space, time, and sensory perception, loss of anxiety), marijuana also has analgesic and anti-inflammatory activities.  In 1964, tetrahydrocannabinol (THC) was identified as the primary psychoactive compound produced by the marijuana plant Cannabis sativa (Figure 1).  The discovery of the CB1 and CB2 cannabinoid receptors in 1988 and 1990, respectively, established the site of action of THC and provided a biochemical basis for marijuana’s pharmacologic effects.  The existence of specific receptors for THC also suggested that there should be an “endogenous cannabinoid” (endocannabinoid) that serves as the physiological ligand for those receptors.  Support for this hypothesis came in 1992 and 1995, with the discovery, respectively, of arachidonoyl ethanolamide (anadamide, AEA) and 2-arachidonoylglycerol (2-AG), endogenous compounds that bind to the CB1 and CB2 receptors and exert cannabinoid-like effects. 

Figure 1Cannabis sativa, the marijuana plant and the structure of tetrahydrocannabinol (THC), the primary psychoactive component produced by the plant.  Figure reproduced from Wikimedia Commons under the GNU Free Documentation License.


As their names suggest, both AEA and 2-AG are derived from the same precursor, arachidonic acid (AA), a 20-carbon long polyunsaturated fatty acid that is a normal component of cell membrane lipids (Figure 2).  AA has another interesting role in vertebrate physiology in that it serves as the precursor for a number of oxygenated metabolites, including the prostaglandins (PGs), leukotrienes, and lipoxins.  These compounds regulate a wide variety of physiological functions, among them fever, pain, and the inflammatory response.  In fact, inhibition of the conversion of AA to PGs by the enzyme cyclooxygenase serves as the primary mode of action of the commonly used nonsteriodal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen (Advil), naproxen (Alleve), and indomethacin (Indocin).  Thus, AA plays complex roles in the inflammatory response.  AA-derived PGs, produced by the action of cyclooxygenase, are pro-inflammatory, while the AA-derived endocannabinoids are anti-inflammatory.

Figure 2. Arachidonic acid (AA) is a 20-carbon long polyunsaturated fatty acid that serves as the precursor for prostaglandins (PGs) through the action of the enzyme cyclooxygenase-2 (COX-2).  The endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamide (AEA) are derivatives of AA.  Both of these compounds act by binding to the cannabinoid receptors (CB1 and/or CB2).  The activity of 2-AG and AEA at the cannabinoid receptors is stopped by the enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively, which hydrolyze the endocannabinoids to yield AA.  Both endocannabinoids can also be oxygenated by COX-2 to PG derivatives.  Oxygenation of 2-AG yields prostaglandin glyceryl esters (PG-Gs) and oxygenation of AEA yields prostaglandin ethanolamides (PG-EAs).  Here, PGE2, PGE2-G, and PGE2-EA are shown as an example of each of these classes of compounds.  Oxygenation of 2-AG and AEA also blocks their activity at the cannabinoid receptors.


The discovery that the endocannabinoids are derivatives of AA suggested the possibility that they could be subject to the same oxidative metabolic pathways as AA, and indeed, in many cases, they are.  In particular, both AEA and 2-AG serve as substrates for one of the two isoforms of cyclooxygenase, COX-2.  The products of COX-2-dependent oxygenation of 2-AG and AEA are glyceryl esters (PG-Gs) or ethanolamides (PG-EAs) of PGs, respectively (Figure 2).   Some of these compounds have been shown to have biological activities of their own, but this pathway also suggests that one function of COX-2 may be to metabolize, and thereby inactivate endocannabinoids.  If this is the case, then NSAIDs could act as anti-inflammatory agents by two mechanisms: blockade of the formation of pro-inflammatory PGs from AA, and blockade of the destruction of anti-inflammatory endocannabinoids.  Now, Vanderbilt Institute of Chemical Biology (VICB) investigator Larry Marnett and his lab, provide strong evidence that this novel mechanism of NSAID action, in fact, occurs [K.C. Duggan et al. (2011)Nat. Chem. Bio., published online September 25, DOI: 10.1038/NCHEMBIO.663] .

The Marnett lab tested a large number of traditional NSAIDs and found that all of them blocked the conversion of 2-AG to PG-Gs by COX-2.  Of particular interest was the finding that some NSAIDs, such as ibuprofen and naproxen, which are relatively weak reversible inhibitors of the oxygenation of AA, are strong irreversible inhibitors of 2-AG oxygenation.  Strong irreversible inhibitors of AA oxygenation had a similar activity against 2-AG.  Thus, NSAIDs clearly blocked the metabolism of endocannabinoids by COX-2, and in many cases, did so with greater potency than the blockade of AA metabolism. 

The profens are a group of aryl acetic acid-containing NSAIDs, including ibuprofen, flurbiprofen, and naproxen (Figure 3).  All of these compounds contain a methyl group alpha to the carboxylic acid, resulting in two enantiomeric forms.  In every case, the (S)-enantiomer inhibits the conversion of AA to PGs, while the (R)-enantiomer is considered inactive.  However, recent reports that (R)-flurbiprofen has anti-inflammatory activity in vivo led the Marnett lab to test the ability of the (R)-profens to inhibit the metabolism of 2-AG and AEA by COX-2.  In all three cases, they found a potent ability of these compounds to inhibit endocannabinoid oxygenation.  Thus, the (R)-profens are an extreme case of the enhanced ability of some NSAIDs to inhibit endocannabinoid oxygenation more efficiently than AA oxygenation.


Figure 3
. Structures of flurbiprofen, naproxen, and ibuprofen, showing the two enantiomeric forms of each molecule.


X-ray crystal structures of COX-2 complexed with (R)-flurbiprofen and (R)-naproxen showed that both compounds bind in the enzyme active site in a conformation very similar to that of the (S)-enantiomer.  However, in both cases, the position of the alpha-methyl group in the (R)-enantiomer causes the movement of key amino acids in the protein, resulting in a weaker binding interaction than is seen with the (S)-enantiomer (Figure 4).  COX-2 is a dimer of two identical subunits.  Differential binding of substrates and inhibitors to the two subunits provided a model to explain the higher sensitivity to inhibition of endocannabinoid oxygenation as compared to AA oxygenation.

Figure 4. Stereoview of the structure of (R)-naproxen (green) overlaid with (S)-naproxen (white) in the COX-2 active site.  Movement of Arg120 and Tyr355 to accommodate the methyl group of (R)-naproxen may weaken the binding interaction.  Reproduced by permission from Macmillan Publishers, Ltd. from Duggan et al. [(2011) Nat. Chem. Biol. published online Sep. 25, DOI:10.1038/NCHEMBIO.663].  Copyright 2011.


The analgesic effects of endocannabinoids are believed to occur at central pain pathways, so the investigators tested the ability of (R)-profens to block endocannabinoid oxygenation in cultures of dorsal root ganglion cells (Figure 5).  These cultures, prepared from mouse embryos, contain a mixture of neurons and glial cells.  Treatment of the cells with inflammatory stimuli induced COX-2 expression in the cells, leading to the production of PGs, PG-Gs, and PG-EAs.  Treatment with all three (R)-profens blocked PG-G and PG-EA synthesis, but not the synthesis of PGs.  Furthermore, (R)-profen treatment led to increased levels of 2-AG and AEA, but not AA in the stimulated cultures

Figure 5. Photomicrograph of a culture of dorsal root ganglion cells stained for neurons (green), nuclei (blue), and the expression of COX-2 (red).  Reproduced by permission from Macmillan Publishers, Ltd. from Duggan et al. [(2011) Nat. Chem. Biol. published online Sep. 25, DOI:10.1038/NCHEMBIO.663].  Copyright 2011.


These results confirm that (R)-profens can selectively inhibit endocannabinoid oxygenation by COX-2, leading to higher intracellular levels of anti-inflammatory endocannabinoids.  The findings suggest the exciting possibility that these compounds may be useful anti-inflammatory and/or analgesic agents without the gastrointestinal side effects of traditional NSAIDs.  The (R)-profens will also be a useful tool to further explore the physiological role of endocannabinoid oxygenation in vivo.

 

 

 


                                                            

 



 

 


 

 


 

 
     

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