Inhibition of fatty acid amide hydrolase, a key endocannabinoid metabolizing enzyme, by analogues of ibuprofen and indomethacin

Abstract

There is evidence in the literature that the nonsteroidal anti-inflammatory drugs indomethacin and ibuprofen can interact with the cannabinoid system both in vitro and in vivo. In the present study, a series of analogues of ibuprofen and indomethacin have been investigated with respect to their ability to inhibit fatty acid amide hydrolase, the enzyme responsible for the hydrolysis of the endogenous cannabinoid anandamide. Of the fourteen compounds tested, the 6-methyl-pyridin-2-yl analogue of ibuprofen (“ibu-am5”) was selected for further study. This compound inhibited rat brain anandamide hydrolysis in a non-competitive manner, with IC₅₀ values of 4.7 and 2.5 μM being found at pH 6 and 8, respectively. By comparison, the IC₅₀ values for ibuprofen were 130 and 750 μM at pH 6 and 8, respectively. There was no measurable N-acylethanolamine hydrolyzing acid amidase activity in rat brain membrane preparations. In intact C6 glioma cells, ibu-am5 inhibited the hydrolysis of anandamide with an IC₅₀ value of 1.2 μM. There was little difference in the potencies of ibu-am5 and ibuprofen towards cyclooxygenase-1 and -2 enzymes, and neither compound inhibited the activity of monoacylglycerol lipase. Ibu-am5 inhibited the binding of [³H]-CP55,940 to rat brain CB₁ and human CB₂ cannabinoid receptors more potently than ibuprofen, but the increase in potency was less than the corresponding increase in potency seen for inhibition of FAAH activity. It is concluded that ibu-am5 is an analogue of ibuprofen with a greater potency towards fatty acid amide hydrolase but with a similar cyclooxygenase inhibitory profile, and may be useful for the study of the therapeutic potential of combined fatty acid amide hydrolase–cyclooxygenase inhibitors.

Introduction

There is evidence indicating that the cannabinoid system can contribute to the in vivo pharmacological effects of nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, the effects of spinally administered indomethacin in the formalin test of inflammatory pain are blocked by the CB₁ cannabinoid receptor antagonist AM251, and are not seen in CB₁-receptor knockout mice (Gühring et al, 2002). A similar result was seen for intrathecally administered flurbiprofen (Ates et al., 2003). Locally administered ibuprofen produces synergistic effects with the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA) in the formalin test, the synergy being prevented by AM251 (Guindon et al., 2006a).

The mechanisms involved in these effects are as yet unclear. Guindon et al. (2006b) reported that the combination of locally administered ibuprofen and anandamide produced increases in the paw levels of AEA and the related N-acylethanolamines palmitoylethanolamide and oleoylethanolamide over and above those seen with either compound per se. Similar results (and a similar synergistic effect) were seen with the cyclooxygenase-2 (COX-2) inhibitor rofecoxib (Guindon et al., 2006b). Gühring et al. (2002) and Ates et al. (2003) suggested that indomethacin and flurbiprofen may allow for an increased synthesis of endocannabinoids from arachidonic acid by blocking the cyclooxygenase (COX) catalyzed removal of arachidonic acid.

A key pathway for removal of anandamide and other N-acylethanolamines such as palmitoylethanolamide (PEA) is the enzyme fatty acid amide hydrolase (FAAH). Loss of activity of this enzyme, either by genetic deletion or by the use of selective inhibitors such as URB597 leads to increased levels of N-acylethanolamines and potentially beneficial effects in models of inflammation and inflammatory pain (see e.g. Lichtman et al., 2004, Holt et al., 2005, Jayamanne et al., 2006). In 1996, Paria et al. reported that indomethacin reduced the rate of anandamide hydrolysis in the mouse uterus (Paria et al., 1996). More systematic studies have since found that many NSAIDs, including indomethacin and ibuprofen, inhibit the activity of FAAH (Fowler et al., 1997, Fowler et al., 1999, Fowler et al., 2003), particularly at low pH (Holt et al., 2001, Holt and Fowler, 2003, Fowler et al., 2003), such as is seen in inflamed tissue (Häbler, 1929, Andersson et al., 1999). AEA is also a substrate for COX-2 (see Kozak and Marnett, 2002), and so it is possible that the effects of NSAIDs upon inflammation and inflammatory pain may not only be due to a decreased production of prostaglandin, but also a reduced removal of AEA (and other anti-inflammatory N-acylethanolamines) by COX-2 and/or FAAH (Holt et al., 2005, Guindon et al., 2006b). Certainly, when it is considered that AEA and PEA can also interact with other targets that are involved in the inflammation and pain, such as TRPV1 receptors (Zygmunt et al., 1999) and the peroxisome proliferator-activated receptor γ and α isoforms (Bouaboula et al., 2005, Lo Verme et al., 2005), and that the relative roles played by CB- and TRPV1-meditated responses to AEA are different in normal and inflamed tissue (Singh Tahim et al., 2005), the situation becomes very complicated indeed.

The pharmacological properties of FAAH inhibitors, together with the potential role of FAAH in the action of NSAIDs raise the possibility that compounds with dual actions upon FAAH and COX-2 may be useful novel compounds for the treatment of inflammation and inflammatory pain. An obvious template for the identification of such compounds is the NSAIDs themselves. However, structure–activity relationships of analogues of clinically used NSAIDs with respect to FAAH inhibition have not been examined in the literature, in contrast to the situation for the relative COX-1 and -2 potencies of such compounds (see e.g. Kalgutar et al., 2000a, Kalgutar et al., 2000b). In consequence, in the present study, a series of ibuprofen and indomethacin analogues have been investigated with respect to their ability to inhibit FAAH. The ibuprofen analogues have not previously been characterized biochemically, but have been shown to produce analgesic effects comparable to ibuprofen in the acetic acid writhing model, and in some cases to possess considerably less ulcerogenic properties (Cocco et al., 2003). Five of the indomethacin analogues tested show selectivity for COX-2 vs. COX-1 (Kalgutar et al., 2000a, Kalgutar et al., 2000b) but have not been tested vis-à-vis the endocannabinoid system. Finally, indomethacin morpholinamide has been reported to be a CB₂ receptor-selective inverse agonist (Gallant et al., 1996, New and Wong, 2003).