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JPET 143487 Synergy Between Enzyme Inhibitors of Fatty Acid JPET Fast Forward. Published on January 9, 2009 as DOI:10.1124/jpet.108.143487 JPET 143487 Synergy between enzyme inhibitors of fatty acid amide hydrolase and cyclooxygenase in visceral nociception* Pattipati S. Naidu, Lamont Booker, Benjamin F. Cravatt, and Aron H. Lichtman Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613. (P.S.N., L.B., and A.H.L.) The Skaggs Institute for Chemical Biology and Departments of Cell Biology and Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, CA 92037. (B.F.C.) 1 Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics. JPET 143487 Running Title: Synergy between FAAH and COX inhibitors Corresponding Author: Aron H. Lichtman P.O. Box 980613 Richmond, VA 23298-0613 Telephone: 804-828-8480 Facsimile: 804-828-2117 E-mail: [email protected] Number of text pages: 16 Number of figures: 6 Number of tables: 1 Number of references: 38 Number of words in the Abstract: 247 Number of words in the Introduction: 720 Number of words in the Discussion: 1463 Abbreviations: CB1, cannabinoid receptor 1; CB2 cannabinoid receptor 2; CNS, central nervous system; FAAH, fatty acid amide hydrolase, COX, cyclooxygenase; NSAIDs, Nonsteroidal anti- inflammatory drugs; URB597, 3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate Recommended section assignment: Behavioral pharmacology 2 JPET 143487 Abstract The present study investigated whether inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide catabolism, produces antinociception in the acetic acid- induced abdominal stretching model of visceral nociception. Genetic deletion or pharmacological inhibition of FAAH reduced acetic acid-induced abdominal stretching. FAAH- NS transgenic mice that express this enzyme exclusively on neurons displayed the antinociceptive phenotype, indicating the involvement of peripheral fatty acid amides. The CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the antinociceptive phenotype of FAAH (-/-) mice, as well as the analgesic effects of URB597 or OL-135, respective irreversible and reversible FAAH inhibitors, administered to C57BL/6 mice. The opioid receptor antagonist, naltrexone, did not block the analgesic effects of either FAAH -1 inhibitor. URB597 (ED50 (95% CI) = 2.1 (1.5-2.9) mg kg ) and the nonselective COX inhibitor -1 diclofenac sodium (ED50 (95% CI) = 9.8 (8.2-11.7) mg kg ) dose-dependently inhibited acetic acid-induced abdominal stretching. Combinations of URB597 and diclofenac yielded synergistic analgesic interactions according to isobolographic analysis. Importantly, FAAH (-/-) mice, as well as URB597-treated mice displayed significant reductions in the severity of gastric irritation caused by diclofenac. URB597 lost its gastro-protective effects in CB1 (-/-) mice, while it maintained its efficacy in CB2 (-/-) mice; indicating a CB1 mechanism of action. Taken together, the results of the present study suggest that FAAH represents a promising target for the treatment of visceral pain and combination of FAAH inhibitors and NSAIDs may have great utility to treat visceral pain, with reduced gastric toxicity. 3 JPET 143487 Introduction Visceral pain is a major cause of consulting in gastroenterology and the principal symptom of functional bowel disorders. This symptom is often associated with gut hypersensitivity to distension. The endogenous cannabinoid system possesses attractive targets for drugs that could potentially treat visceral and other types of pain. These targets include cannabinoid (i.e., CB1 and CB2) receptors, as well as fatty acid amide hydrolase (FAAH), the enzyme responsible for degradation of the endogenous cannabinoid, anandamide, and other fatty acid amides (FAAs) (Walker and Hohmann, 2005). Indeed, direct-acting cannabinoid receptor agonists, such as Δ9- tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, as well as the irreversible FAAH inhibitor cyclohexylcarbamic acid 3-carbamoyl biphenyl-3-yl ester (URB597) inhibited visceral nociception, as assessed in the phenyl-p-quinone (PPQ) model (Haller et al., 2006). The antinociceptive effects of both of these compounds were blocked by the CB1 receptor antagonist, rimonabant, indicating a CB1 receptor mechanism of action. Although direct-acting cannabinoid receptor agonists possess analgesic properties similar to that of opioids, but without respiratory depressant effects, their psychomimetic side effects have dampened enthusiasm for their development as therapeutic agents. In contrast, increasing endogenous cannabinoid levels by blocking FAAH represents an attractive alternate approach to elicit antinociception, but without eliciting cannabimimetic effects (Cravatt et al., 2001; Gobbi et al., 2005). Deletion of the FAAH gene leads to increased levels of anandamide, accompanied with CB1 receptor-mediated hypoalgesic phenotypes in models of acute and inflammatory pain (Cravatt et al., 2001). Similarly, wild type mice treated with FAAH inhibitors, such as URB597 (Kathuria et al., 2003) or the reversible FAAH inhibitor, 1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenyl heptane (OL-135 (Lichtman et al., 2004a)) elicited hypoalgesic effects in acute models of pain 4 JPET 143487 that were accompanied with elevations of anandamide in the CNS. Additionally, these FAAH inhibitors reduced hypersensitivity to thermal and mechanical hypersensitivity in neuropathic pain models (Chang et al., 2006; Russo et al., 2007). Nonsteroidal anti-inflammatory drugs (NSAIDs), the mainstays in acute and chronic pain management, produce their beneficial actions by inhibiting cyclooxygenases (COX): constitutive COX-1 and inducible COX-2 (Warner and Mitchell, 2004), which are responsible for the biosynthesis of the pro-inflammatory prostaglandins in the periphery (Vinegar et al., 1976) and CNS (Samad et al., 2001). However, the gastrointestinal adverse effects of nonselective COX inhibitors remain a major clinical concern (Wallace, 1996). On the other hand, FAAH deletion or inhibition leads to protective effects in animal models of colitis (Massa et al., 2004; Storr et al., 2008). Of relevance, co-administration of locally applied anandamide and COX inhibitors produced synergistic antinociceptive effects in the rat formalin test (Guindon et al., 2006) and also reduced mechanical and thermal hyperalgesia in the rat partial sciatic nerve ligation model (Guindon and Beaulieu, 2006). In addition, co-administration of the NSAID, ketorolac, and the mixed CB1/CB2 receptor agonist, WIN55,212-2, elicited additive analgesic effects in the acetic acid abdominal stretching test, a preclinical model of visceral nociception (Ulugol et al., 2006). However, the cannabimimetic properties of WIN55,212-2 that include hypomotility, catalepsy, and hypothermia, as well as THC-like subjective effects in the drug discrimination paradigm (Compton et al., 1992) limit the development of this compound for therapeutic applications. Conversely, FAAH blockade does not elicit any of these THC-like side effects (Cravatt et al., 2001; Kathuria et al., 2003; Lichtman et al., 2004a; Gobbi et al., 2005). 5 JPET 143487 Thus, simultaneous inhibition of FAAH and COX might offer an attractive therapeutic approach that maintains analgesic efficacy, while minimizing untoward side effects associated with direct-acting cannabinoid receptor agonists, as well as the NSAIDs. Consequently, there were four goals of the present study. First, we examined whether FAAH (-/-) mice or wild type mice treated with inhibitors of FAAH would display decreased nociceptive behavior in the acetic acid-induced abdominal stretching test. Second, we sought to determine the receptor mechanism of action underlying the antinociceptive phenotype of FAAH-compromised mice. Mice were evaluated with the respective CB1 and CB2 receptor antagonists, rimonabant and SR144528. In addition, because the antinociceptive effects of FAAH inhibitors has been suggested to include an opioid receptor mechanism of action (Chang et al., 2006), we also evaluated whether naltrexone would block the antinociceptive effects of URB597 and OL-135. Third, we used an isobolographic approach to determine whether URB597 given in combination with diclofenac, a non-selective COX inhibitor, would elicit additive or synergistic antinociceptive effects. Fourth, we evaluated whether FAAH inhibition would offer gastro-protective effects against NSAID- induced gastric ulcers. 6 JPET 143487 Methods Subjects The subjects consisted of male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME), FAAH (-/-) mice backcrossed for at least 13 generations, CB1 (-/-) mice backcrossed for at least 13 generations, and CB2 (-/-) mice backcrossed for five generations onto a C57Bl/6J background. In addition, male and female FAAH-NS (nervous system FAAH restricted; (Cravatt et al., 2004)) mice backcrossed onto a C57Bl/6J background for at least 11 generations were used to determine whether the FAAH (-/-) antinociceptive phenotype was due to the deletion of this enzyme from neuronal tissue. All subjects weighed between 20 and 30 g and were housed four mice per cage in a temperature-controlled (20-22oC) facility, with food and water available ad libitum. All animal protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee and were in concordance with the National Institutes of Health guide for the care and use of Laboratory animals
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