Potential analgesic effects of a novel N-acylethanolamine acid amidase inhibitor F96 through PPAR-α

Abstract

Pharmacological blockade of N-acylethanolamine acid amidase (NAAA) activity is an available approach for inflammation and pain control through restoring the ability of endogenous PEA. But the recently reported NAAA inhibitors suffer from the chemical and biological unstable properties, which restrict functions of NAAA inhibition in vivo. It is still unrevealed whether systematic inhibition of NAAA could modulate PEA-mediated pain signalings. Here we reported an oxazolidinone imide compound 3-(6-phenylhexanoyl) oxazolidin-2-one (F96), which potently and selectively inhibited NAAA activity (IC50 = 270 nM). Intraperitoneal (i.p.) injection of F96 (3-30 mg/kg) dose-dependently reduced ear edema and restored PEA levels of ear tissues in 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced ear edema models. Furthermore, F96 inhibited acetic acid-induced writhing and increased spared nerve injury induced tactile allodynia thresholds in a dose-dependent manner. Pharmacological effects of F96 (10 mg/kg, i.p.) on various animal models were abolished in PPAR-α(-/-) mice, and were prevented by PPAR-α antagonist MK886 but not by canabinoid receptor type 1 (CB1) antagonist Rimonabant nor canabinoid receptor type 2 (CB2) antagonist SR144528. Zebrafish embryos experiments showed better security and lower toxicity for F96 than ibuprofen. These results revealed that F96 might be useful in treating inflammatory and neuropathic pain by NAAA inhibition depending on PPAR-α receptors.

Figure 1. Characterization of the NAAA inhibitor F96.

(a) Structure of compound F96. (b) Concentration-dependent inhibition of extracted recombinant rat NAAA (rNAAA) activity by F96. (c) Concentration-dependent inhibition of extracted recombinant rat FAAH (rFAAH) activity by F96.

Figure 2. Effects of F96 on TPA induced ear edema and ear tissues levels of fatty acid ethanolamides.

(a) H&E-stained sections of normal mouse ear (Control), TPA-induced irritant dermatitis (Vehicle) and various doses (30, 10, 3 mg/kg) of F96 treated. (b) F96 (30, 10, 3 mg/kg) dose-dependently reduced ear weight. (c) F96 (10 mg/kg) reduced ear weigh in wild type 129 s mice but not in PPAR-α knockout mice. (d–e) F96 normalized PEA and OEA levels in a dose-dependent manner after TPA application for 3 h in C57BL/6J mice. **p < 0.01 and ***p < 0.001 vs Control vehicle-treated group. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs TPA+F96-treated group. n = 8–10.

Figure 3. F96 suppressed pain responses elicited by intraperitoneal injections of acetic acid in mice.

(a) Number of writhing (assessed episodes in 20 min after acetic acid injection) reduced after indomethacin (10 mg/kg, i.p., Gray bars) and F96 (10 mg/kg, i.p., closed bars) administration. PPAR-α antagonist MK886 (2 mg/kg, i.p.) prevented the anti-nociceptive effects of F96. CB₁ antagonist Rimonabant (1 mg/kg, i.p.) and CB₂ antagonist SR144528 (1 mg/kg, i.p.) did not abolish the analgesic effects of F96. (b) Effects of vehicle (white bars) or F96 (10 mg/kg, i.p., black bars) on acetic acid induced writhing in wild-type 129 s mice (+/+) and PPAR-α knockout mice (−/−).

Figure 4. Analgesic effects of F96 in the SNI models.

Time-course effect of vehicle (5% PEG/5% Tween-80 in saline, 10 mL/kg, i.p., open squares), F96 (10 mg/kg, i.p., open triangle) and gabapentin (40 mg/kg, i.p., open inverted triangle) on mechanical allodynia in SNI mice at (a) 3^rd day post-surgery, and (b) the 7^th day post-surgery. Mechanical allodynia were measured 1, 4 and 12 h after treatment. Sham, sham-operated mice (open cycles). (c) Dose-response of F96 (open triangles) on mechanical allodynia in SNI mice 7^th day post surgery. Withdraw threshold was assessed 1 h after F96 treatment. Sham, sham-operated mice (open cycle). (d) PPAR-α antagonist MK886 (2 mg/kg, i.p.) prevented the anti-allodynia effects of F96 (10 mg/kg, i.p., closed bars). CB₁ antagonist Rimonabant (1 mg/kg, i.p.) and CB₂ antagonist SR144528 (1 mg/kg, i.p.) were ineffective. (e) F96 (10 mg/kg, i.p.) increased mechanical paw withdraw threshold in wild-type 129 s mice, but not in PPAR-α knockout mice. Results are expressed as mean ± SEM (n = 6–10 each group).

Figure 5. Primary toxicity evaluation of F96 in the zebrafish models.

The larvae hatching rate, total mortality, average heartbeats, and body length of the embryos undergoing (a) 2/4 celled stages and (b) 24 h post-fertilization and exposing to different solutions on 72 h. Vehicle, zebrafish medium (open cycles). Control, zebrafish medium containing 0.1% DMSO (open rhombus). Ibuprofen, zebrafish medium containing different concentrations of ibuprofen (black cycles). F96, zebrafish medium containing different concentrations of ibuprofen (open square).

Figure 6. Open field tests.

F96 (30, 100, 300 mg/kg, i.p.) did not affect the total distance (a), center distance moved in the arena (b) all the time. High dose of F96 (100, 300 mg/kg, i.p.) slightly increased the time spent in the center (c) only on 6 h after administration. *p < 0.05, compared with vehicle group.