1. The inhibition of N-acylethanolamine acid amidase activity produces antinociceptive effect INTRODUCTION N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lisosomal enzyme that deactivates bioactive fatty acid ethanolamides (FAEs) with preference to N- palmitoylethanolamide (PEA), which is one of the most studied acylethanolamine. PEA has potent anti-inflammatory and analgesic effects via stimulation of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-α) (Lo Verme et al., 2005). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo (Solorzano et al., 2009), which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. In the present study, we examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. O. Sasso, A. Reggiani and D. Piomelli Fondazione Istituto Italiano di Tecnologia, Department of Drug Discovery and Development Via Morego, 30 16163 Genova, Italy CONCLUSIONS  The present results show that the compound ARN077, a potent and selective NAAA inhibitor recently identified by our group protects endogenous FAEs from NAAA-catalyzed degradation in inflamed tissues of live mice.  The results further show that ARN077 attenuates hyperalgesic and allodynic states elicited, in mice and rats, by local inflammation or nerve damage.  The anti-nociceptive effects of ARN077 are prevented by the selective PPAR-α antagonist GW6471. We interpret these findings to indicate that ARN077 modulates nociceptive responses in mice and rats by interrupting NAAA-mediated FAE degradation and restoring intrinsic FAE signaling at PPAR-α.  The results identify ARN077 as a potent inhibitor of intracellular NAAA activity and NAAA as a novel key enzyme involved in the regulation of peripheral pain initiation. RESULTS Intraplantar administration of carrageenan resulted in the development of paw edema and heat hyperalgesia. Both responses were markedly attenuated, in a dose- and time- dependent manner, by a single topical dosing with ARN077 (1- 30% weight/volume, in petrolatum/5% lauric acid) applied 90 min after carrageenan (Fig. 1 A, B). The anti-inflammatory and anti-hyperalgesic effects of topical ARN077 (30%) were prevented by the selective PPAR-α antagonist GW6471 (administered by intraplantar injection 30 min before ARN077; 1 µg per mouse), but not by the CB1 inverse agonist AM251 or the CB2 antagonist AM630 (administered by intraplantar injection 30 min before ARN077; 1 µg per mouse) (Fig. 2 A, B). Significant hyperalgesia was achieved in rats by exposing the glabrous skin of the right hind paw to UVB radiation (30 min, 250 mJ/cm 2 ). Topical application of ARN077 (3-30%, 24 h after irradiation) reduced UVB-induced heat hyperalgesia in a dose- dependent manner (Fig. 3 A). Pre-treatment with the CB1 inverse agonist AM251 and the CB2 antagonist AM630 (each at 60 μg/60 μl/paw failed to reverse the anti-hyperalgesic effect of ARN077, which was completely prevented by the pharmacological blockade of PPAR-α (Fig. 3 B). A single application of ARN077 (1-30%) was sufficient to reduce significantly both hyperalgesia and allodynia in CCI model. Surgical ligation was accompanied by a substantial decrease in PEA content in sciatic nerve tissue (Fig. 5 A). This effect was prevented by treatment with ARN077 (10%), which normalized the levels of PEA in ligated, but not sham-operated mice (Fig. 5). Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Email: oscar.sasso@iit.it Topical application of ARN077 in carrageenan model Antagonists in carrageenan model Effects of ARN077 in UVB model FAEs level in CCI model Effects of ARN077 in CCI model