Combination inhibitors of FAAH TRPV and or COX may have

Combination inhibitors of FAAH, TRPV1, and/or COX2 may have the advantage of effective pain relief with a high therapeutic index. For example, arachidonoyl serotonin (AA-5-HT) inhibits both FAAH (IC=1–12μM) and TRPV1 (IC=37–270nM against 100nM capsaicin in HEK-293 cells)., In mice, AA-5-HT had greater efficacy at relieving carrageenan-induced hyperalgesia than either a high-potency, FAAH-selective inhibitor or a TRPV1-selective inhibitor. Similarly, AA-5-HT was more effective in an animal model of anxiety than selective FAAH or TRPV1 inhibitors. Dual inhibitors of FAAH and TRPV1 that are more stable and drug-like than AA-5-HT have been pursued by others., Dual inhibition of COX2 and FAAH has also been explored, with early indications that greater analgesia can be achieved with fewer adverse effects than targeting each alone. NSAIDs treat pain by inhibiting COX, which catalyzes the first steps in the conversion of arachidonic Ki16198 (AA) into prostanoids associated with pain and inflammation. Most NSAIDs reversibly bind the COX active site, mimicking the unsaturated fatty chain and carboxylic acid head group of AA. Based on their ability to bind the AA site on COX enzymes, NSAIDs were hypothesized to be able to also effectively mimic the AA portion of AA-5-HT at its binding sites on FAAH and TRPV1. Evidence to support this hypothesis includes the ability of some NSAIDs to weakly inhibit FAAH, as well as inhibition of FAAH by some analogues of ibuprofen., In this work, a series of NSAIDs were conjugated to serotonin by forming an amide bond between the serotonin amine and the carboxylic acid group of the NSAIDs. The resulting NSAID-5-HT analogues were tested for their ability to inhibit FAAH, TRPV1, and COX2. Serotonin conjugates were prepared as shown in by treating a stirred solution of the NSAID in DMF with hydroxybenzotriazole (HOBt) and -(3-dimethylaminopropyl)-′-ethylcarbodiimide hydrochloride (EDC) at 0°C. The solution was then brought to room temperature, followed by addition of serotonin-HCl and triethylamine. After stirring overnight, products were extracted into ethyl acetate, subjected to aqueous workup, and purified by flash chromatography. The serotonin conjugates shown in were synthesized and tested as inhibitors against FAAH, TRPV1, and COX2. To assist in identifying structure-related trends in activity, the NSAID components were selected from each of the major structural classes of carboxylic acid-containing NSAIDs: salicylates (salicylate-5-HT and ASA-5-HT), arylacetic acids (Diclofenac-5-HT), heteroarylacetic acids (Indomethacin-5-HT), -arylanthranilic acids (Flufenamate-5-HT), 2-arylpropionic acids (Flurbiprofen-5-HT, Ibuprofen-5-HT, Naproxen-5-HT, Fenoprofen-5-HT, and Ketoprofen-5-HT), and a cyclized heteroarylpropionic acid (Ketrolac-5-HT). The results from inhibition assays of these NSAIDs conjugated with serotonin are shown in . AA-5-HT was purchased and used as a positive control. AA-5-HT is a synthetic compound originally identified in a screen for novel FAAH inhibitors. Subsequent work showed AA-5-HT is also an antagonist of TRPV-1 and it was reported to be the first dual inhibitor of FAAH and TRPV1 to appear in the literature. As the prototype for dual FAAH/TRPV1 inhibition, AA-5-HT has been the reference compound of choice for drug discovery efforts in this area., All the NSAID-5-HT analogues and AA-5-HT significantly inhibited COX2 using an inhibitor concentration of 10μM in activity screens. At 10μM, AA-5-HT was among the least potent inhibitors of COX2 while, consistent with observations made by other investigators who have studied COX inhibition by NSAID amides, indomethacin-5-HT was most potent. Fenoprofen-5-HT and Naproxen-5-HT appear to be able to inhibit both TRPV1 and COX2 with approximately the same potency as AA-5-HT, but do not inhibit FAAH, even at concentrations of 50μM. Only Ibuprofen-5-HT and Flurbiprofen-5-HT seem to inhibit all three targets with potencies similar to AA-5-HT. None of the parent NSAIDs, which are known COX inhibitors, showed significant inhibition of FAAH in DMSO at 10μM (data not shown). The parent NSAIDs were also less active than their serotonin counterparts at reducing TRPV1 activity. Flufenamic acid and diclofenac sodium at 50μM reduced residual TRPV1 activity to 66% and 61%, respectively, and indomethacin at 250μM reduced TRPV1 activity to 82% (). None of the other NSAIDs showed activity at these concentrations.