Antinociceptive effects of the N-acylethanolamine acid amidase inhibitor ARN077 in rodent pain models

Abstract

Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, 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. The present study 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. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α.

Figure 1

Effects of ARN077 on rat NAAA activity in vitro. (A) Concentration-dependent inhibition of recombinant rat NAAA activity by ARN077. (B) Time-course of the NAAA reaction in the presence of ARN077 (0.1 µM); (C) Michaelis-Menten analysis of the NAAA reaction in the presence of vehicle (squares; DMSO, 1%), 50 nM ARN077 (triangles) or 300 nM ARN077 (circles). (D) Effects of dialysis (24 h, 0–4°C) on NAAA activity by ARN077 (0.3 µM). Results are expressed as mean ± SEM (n = 6). ** p<0.01.

Figure 2

Effects of topical application of ARN077 on TPA-induced edema and skin FAE levels. ARN077 (10% in acetone) reduced tissue weight (A) normalized PEA and OEA, but not AEA, content in mouse ear skin (B, C and D) 6 h after co-application with TPA (3% in 10 l). Results are expressed as mean ± SEM (n = 5 each group). ** p<0.01 and *** p<0.001 vs. TPA-ARN077-treated group; ^## p<0.01 vs. TPA vehicle-treated group.

Figure 3

Effects of topical application of ARN077 on carrageenan-induced hyperalgesia and edema. ARN077 (1–30% in petrolatum/5% lauric acid) reduced paw edema (A) and heat hyperalgesia (B) measured immediately before (0 h) and 1.5 h after carrageenan injection, and at various times after ARN077 application. Results are expressed as mean ± SEM (n = 6, each group). ** p<0.01 and *** p<0.001 vs. vehicle.

Figure 4

Effects of intraplantar injection of ARN077 on carrageenan-induced hyperalgesia and edema. ARN077 (0.005–50 g/mouse, dissolved in 80% sterile saline/10% PEG-400/10% TWEEN 80) reduced paw edema (A) and heat hyperalgesia (B), measured immediately before (0 h) and 1.5 h after carrageenan injection, and at various times after ARN077 application. Results are expressed as mean ± SEM (n = 6, each group). * p<0.05, ** p< 0.01 and *** p<0.001 vs. vehicle.

Figure 5

The PPAR- antagonist GW6471 blocks the anti-hyperalgesic and anti-inflammatory effects of topical ARN077 in the carrageenan model. Intraplantar administration of GW6471 (1 g, 1 h after carrageenan injection) prevented the effects of topical ARN077 (1–30% in petrolatum/5% lauric acid) on paw edema (A) and heat hyperalgesia (B). Intraplantar administration of the CB₁ inverse agonist AM251 or the CB₂ antagonist AM630 had no such effect. Edema and hyperalgesia were measured immediately before (0 h) and 1.5 h after carrageenan injection, and at the time of maximal effect of ARN077 (4 h). Results are expressed as mean ± SEM (n = 6 each group). *** p<0.001 vs. vehicle and ^### p<0.001 vs. ARN077.

Figure 6

Effects of ARN077 in wild-type and PPAR- -deficient mice. ARN077 (30%) reduced paw edema (A) and heat hyperalgesia (B) in wild-type C57BL/6J mice, but not in PPAR- -null mice. Edema and hyperalgesia were measured immediately before (0 h) and 1.5 h after carrageenan injection, and at the peak time of 4 h after ARN077 application. Open symbols, wild-type C57BL/6J mice; Closed symbols, PPAR- -deficient mice. Results are expressed as mean ± SEM (n = 6 each group). *** p<0.001 vs. wild-type controls.

Figure 7

Antihyperalgesic effects of ARN077 in the chronic nerve constriction model. Topical ARN077 (1–30%) reduced both heat hyperalgesia (A) and mechanical allodynia (B). The effect of gabapentin (50 mg-kg⁻¹, oral gavage) is shown for comparison. ARN077 was administered once daily 3, 7, and 14 days following the ligation. Hyperalgesia and allodynia were measured 1 h after treatment. Results are expressed as mean ± SEM (n = 6, each group). ** p< 0.01 and *** p<0.001 vs. vehicle; ^### p<0.001 vs. sham-operated mice.

Figure 8

Effects of topical application of ARN077 (10%) on sciatic nerve FAE levels in the chronic nerve constriction model. ARN077 (10%) normalized PEA content in mouse ligated nerve (A) and did not change OEA and anandamide content (B, C) 7 days after sciatic nerve ligation. Sciatic nerves are collected 1 hour after topical application of ARN077 on plantar surface of paw. Results are expressed as mean ± SEM (n = 5 each group). ** p<0.01 vs. vehicle-CCI; ^# p<0.05 vs. sham.

Figure 9

Anti-hyperalgesic effects of ARN077 in the UVB model. (A) Topical application of ARN077 (3, 10 and 30%) reduced heat hyperalgesia in a dose-dependent manner. (B) The anti-hyperalgesic effects of ARN077 were blocked by intraplantar administration of the selective PPAR- antagonist GW6471, but not by a selective CB₁ (AM251) or CB₂ (AM630) antagonist (60 µg/60 µl). Results are expressed as mean ± SEM (n = 6, each group). ** p<0.01 and *** p<0.001 vs. vehicle; ^### p<0.001 vs ARN077.