Regulation of inflammatory pain by inhibition of fatty acid amide hydrolase

Abstract

Although cannabinoids are efficacious in laboratory animal models of inflammatory pain, their established cannabimimetic actions diminish enthusiasm for their therapeutic development. Conversely, fatty acid amide hydrolase (FAAH), the chief catabolic enzyme regulating the endogenous cannabinoid N-arachidonoylethanolamine (anandamide), has emerged as an attractive target for treating pain and other conditions. Here, we tested WIN 55212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone], a cannabinoid receptor agonist, and genetic deletion or pharmacological inhibition of FAAH in the lipopolysaccharide (LPS) mouse model of inflammatory pain. WIN 55212-2 significantly reduced edema and hot-plate hyperalgesia caused by LPS infusion into the hind paws, although the mice also displayed analgesia and other central nervous system effects. FAAH(-/-) mice exhibited reduced paw edema and hyperalgesia in this model without apparent cannabimimetic effects. Transgenic mice expressing FAAH exclusively on neurons continued to display the antiedematous, but not the antihyperalgesic, phenotype. The CB(2) cannabinoid receptor (CB(2)) antagonist SR144528 [N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] blocked this non-neuronal, anti-inflammatory phenotype, and the CB(1) cannabinoid receptor (CB(1)) antagonist rimonabant [SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] blocked the antihyperalgesic phenotype. The FAAH inhibitor URB597 [cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester] attenuated the development of LPS-induced paw edema and reversed LPS-induced hyperalgesia through the respective CB(2) and CB(1) mechanisms of action. However, the transient receptor potential vanilloid type 1 antagonist capsazepine did not affect either the antihyperalgesic or antiedematous effects of URB597. Finally, URB597 attenuated levels of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha in LPS-treated paws. These findings demonstrate that simultaneous elevations in non-neuronal and neuronal endocannabinoid signaling are possible through inhibition of a single enzymatic target, thereby offering a potentially powerful strategy for treating chronic inflammatory pain syndromes that operate at multiple levels of anatomical integration.

Fig. 1.

LPS treatment elicited significant paw edema and hyperalgesia in mice. A, intraplantar LPS (25 μg/50 μl per paw) significantly increased paw edema 3 h after injection, peak effects occurred at 24 h, and the edema was sustained for 72 h after LPS injection. B, a similar pattern of results was observed in the hot-plate test, but responses returned to baseline values by 48 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus mice given intraplantar injections of saline at the corresponding time points.

Fig. 2.

A, pretreatment with dexamethasone (DEX; 2 mg/kg i.p.) or WIN 55212-2 (WIN; 2 mg/kg i.p.) administered 1 h before and 6 and 23 h after intraplantar LPS significantly prevented the development of paw edema. *, p < 0.05 versus mice treated with vehicle (VEH) at the corresponding time points. B, administration of DEX (2 mg/kg i.p.) significantly reversed LPS-induced hyperalgesia in the hot-plate test. Subjects were given a single injection of DEX or vehicle 23 h after LPS and assessed in the hot-plate test at 24 h. **, p < 0.01 versus mice given intraplantar saline injections; ††, p < 0.01 versus LPS-treated mice given intraperitoneal injections of saline. C, cumulative dose-response curve of WIN in mice given intraplantar injections of LPS. Low doses of WIN reversed the hyperalgesic effects of LPS treatment, but high doses showed significant analgesic effects in the hot-plate test. *, p < 0.05 versus preinjection latencies; †, p < 0.05; †††, p < 0.001 versus LPS-injected mice given vehicle. n = 8 to 10 mice per group. Data are depicted as means ± S.E.M.

Fig. 3.

Comparison between FAAH(+/+) and FAAH(−/−) mice in the LPS model of inflammatory pain. LPS elicited less edema (A) and thermal hyperalgesia (B) in FAAH(−/−) mice than in FAAH(+/+) mice. *, p < 0.05; **, p < 0.01 versus corresponding FAAH(+/+) mice at each respective time point. n = 8 to 10 mice per group. Data are depicted as means ± S.E.M.

Fig. 4.

Dissociation between the antiedema and antihyperalgesic phenotypes in FAAH-deficient mice. A, FAAH(−/−) mice and FAAH-NS mice, which express FAAH exclusively in the nervous system, showed a significant reduction in LPS-induced paw edema compared with the FAAH(+/−) control mice, but did not differ between each other. B, LPS-induced hyperalgesia was reduced in FAAH(−/−) mice compared with FAAH(+/−) mice. However, hot-plate latencies did not differ between FAAH-NS and FAAH(+/−) mice. *, p < 0.05; ***, p < 0.001 versus control FAAH(+/−) mice. n = nine mice per group. Data are depicted as means ± S.E.M.

Fig. 5.

Evaluation of cannabinoid receptors in the FAAH(−/−) antiedema and antihyperalgesic phenotypes. A, pretreatment with the CB₂ SR144528 (SR2; 3 mg/kg i.p.), but not the CB₁ rimonabant (SR1; 3 mg/kg i.p.) administered 1 h before and 6 and 23 h after intraplantar LPS significantly prevented the FAAH anti-inflammatory phenotype in LPS-treated mice. B, SR1 and SR2 normalized hot-plate responses in FAAH(−/−) mice. *, p < 0.05 compared with LPS-treated wild-type mice; †, p < 0.05 versus FAAH(−/−) LPS-treated mice. n = nine mice per group. Data are depicted as means ± S.E.M. Veh, vehicle.

Fig. 6.

Effects of a single injection of the FAAH inhibitor URB597 (URB; intraperitoneally) on LPS-induced edema and hyperalgesia. URB or vehicle was administered 23 h after LPS. Paw thickness values and hot-plate latencies were assessed at 24 h. A single injection of URB did not reverse LPS-induced edema (A), but significantly attenuated LPS-induced hyperalgesia (B). *, p < 0.05; **, p < 0.01 compared with LPS-treated mice that received intraperitoneal vehicle. n = 6 to 10 mice per group. Data are depicted as means ± S.E.M.

Fig. 7.

The antihyperalgesic and antiedematous effects of triple dosing of URB597 (URB) in the LPS model are mediated through respective CB₁ and CB₂ mechanisms of action. A, URB reduced LPS-induced paw edema in both CB₁(−/−) and CB₁(+/+) mice. B, URB reduced LPS-induced hyperalgesia in CB₁(+/+) mice, but not in CB₁(−/−) mice. C, URB reduced paw edema in CB₂(+/+) mice, but not in CB₂(−/−) mice. D, URB reduced hyperalgesia in both CB₂(+/+) and CB₂(−/−) mice. URB (10 mg/kg i.p.) or vehicle was administered 1 h before and 6 and 23 h after LPS. Paw thickness values and hot-plate latencies were assessed at 24 h. *, p < 0.05, compared with corresponding LPS control group; †, p < 0.05 versus LPS-treated CB₁(+/+) or CB₂(+/+) control mice. n = six to eight mice per group. Data are depicted as means ± S.E.M.

Fig. 8.

No apparent role of TRPV1 was found in the antihyperalgesic and antiedematous effects of URB597 (URB) in the LPS model. The TRPV1 antagonist capsazepine (CPZ) failed to prevent either the antiedematous (A) or the antihyperalgesic (B) effects of URB. Subjects were given an intraperitoneal injection of URB (10 mg/kg) 1 h before and 6 and 23 h after intraplantar LPS. The mean ± S.E.M. hot-plate latency before LPS administration was 13.4 ± 0.3 s. Subjects received CPZ (5 mg/kg i.p.) 10 min before each injection of URB597. *, p < 0.05; **, p < 0.001 versus vehicle-vehicle (Veh-Veh). n = 10 mice per group. Data are depicted as means ± S.E.M.

Fig. 9.

Dexamethasone (DEX) and URB597 (URB) reduce proinflammatory cytokines. URB (10 mg/kg i.p.), DEX (2 mg/kg i.p.), or vehicle (VEH) was administered 1 h before and 6 and 23 h after LPS. The mice were humanely euthanized at 24 h, and paws were harvested. Pretreatment with URB or DEX significantly attenuated LPS-induced IL-1β expression (A) and TNF-α (B). ***, p < 0.0001 compared with the vehicle plus saline (VEH+SAL) group. †, p < 0.05; ††, p < 0.01; †††, p < 0.001 compared with VEH+LPS group. n = 12 mice per group. Data are depicted as means ± S.E.M.