Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism

Abstract

Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB₁ cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB₁ receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

Figure 1

URB937 is a peripherally restricted FAAH inhibitor. (a) FAAH activity in liver (closed circles) and brain (closed squares) 1 h after injection of URB937 (0.03–100 mg-kg⁻¹, s.c.) in Swiss Webster mice. (b) Temporal distribution of URB937 in liver, brain and serum (inset) after a single injection in Swiss-webster mice (1 mg-kg⁻¹, i.p.). (c) Serum concentrations of URB937 after i.c.v. infusion in rats (0.01–0.1 mg-kg⁻¹). (d) Liver FAAH activity after intracerebroventricular (i.c.v.) infusion of vehicle (open bar) or URB937 (0.01–0.1 mg-kg⁻¹, closed bars) in rats. (e) Brain FAAH activity after systemic administration of vehicle (V), URB597 (1 mg-kg⁻¹, s.c.), or URB937 (shaded bar: 1 mg-kg⁻¹; closed bars: 25 mg-kg⁻¹, s.c.); URB937 was administered alone or in combination with drug-transport inhibitors, 2,6-dichloro-4-nitrophenol (DCNP, 40 mg-kg⁻¹, i.p.), Ko−143 (Ko, 10 mg-kg⁻¹, i.p.), verapamil (Ver, 50 mg-kg⁻¹, i.p.), probenecid (Pro, 150 mg-kg⁻¹, i.p.), and rifampicin (Rif, 50 mg-kg⁻¹, i.p.). (f) Effects of vehicle (open bars) or URB937 (1 mg-kg⁻¹, i.p., closed bars) on anandamide and palmitoylethanolamide (PEA) levels in liver, forebrain and hypothalamus of Swiss Webster mice. (g) Effects of URB937 on anandamide and PEA levels in liver of wild-type C57Bl/6 mice (+/+) and FAAH-deficient littermates (−/−). Results are expressed as mean ± s.e.m; n = 3; *P<0.05 and ***P<0.001 vs vehicle.

Figure 2

URB937 suppresses pain responses elicited by i.p. injections of acetic acid in Swiss Webster mice. (a) Behavioral score (number of writhing episodes in 20 min) assessed 1 h after administration of vehicle (V, open bars), URB937, URB597, or indomethacin (IDM) (each at 1 mg-kg⁻¹, s.c., closed bars). Also illustrated are the effects of vehicle and URB937 administered without acetic acid. (b) Statistical correlation between behavioral score and percent inhibition of liver FAAH activity after URB937 administration (1 mg-kg⁻¹, s.c.). (c) Effects of vehicle (open bars) or URB937 (1 mg-kg⁻¹, s.c., closed bars) on acetic acid-induced writhing in wild-type C57Bl/6 mice (+/+) and FAAH-deficient littermates (−/−). (d) CB₁ antagonists rimonabant (Rim) and AM251, and PPAR-α antagonist MK886 (each at 1 mg-kg⁻¹, s.c.) prevent the antinociceptive effects of URB937. CB₂ antagonist AM630 is ineffective. (e) Effects of vehicle (open bars) or URB937 (1 mg-kg⁻¹, s.c., closed bars) on acetic acid-induced writhing in wild-type C57Bl/6 mice (+/+) and PPAR-α-deficient mice (−/−). Results are expressed as mean±s.e.m.; n = 5−17. *P<0.05; **P<0.01 and ***P<0.001 vs vehicle.

Figure 3

URB937 suppresses pain behavior elicited by neural injury in mice. (a-c) Effects of single administration of vehicle (shaded bars) or URB937 (1 mg-kg⁻¹, i.p.; closed bars) on (a) mechanical hyperalgesia, (b) thermal hyperalgesia, and (c) mechanical allodynia produced by sciatic nerve ligation. BL, baseline (measured before nerve ligation); IL, ipsilateral (ligated) paw; CL, contralateral (non-ligated) paw. (d–f) Effects of repeated injections of vehicle or URB937 (1 mg-kg⁻¹, i.p, once-daily for 7 consecutive days) on (d) mechanical hyperalgesia, (e) thermal hyperalgesia, and (f) mechanical allodynia. (g–i) CB₁ antagonist rimonabant (R, 1 mg-kg⁻¹, i.p.), but not CB₂ antagonist AM630 (AM, 1 mg-kg⁻¹, i.p.), prevents the effects of URB937 on (g) mechanical hyperalgesia, (h) thermal hyperalgesia, and (i) mechanical allodynia. Results are expressed as mean±s.e.m.; n = 6 in each panel. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs baseline; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs vehicle.

Figure 4

URB937 attenuates pain behavior elicited by inflammation in mice. Effects of URB937 (1 mg-kg⁻¹, i.p.), administered alone or in combination with rimonabant (R, 1 mg-kg⁻¹, i.p.) or AM630 (AM, 1 mg-kg⁻¹, i.p.), on carrageenan-induced responses: (a) mechanical hyperalgesia; (b) thermal hyperalgesia; (c) mechanical allodynia; and (d) paw edema. Mechanical and thermal hyperalgesia were measured immediately before carrageenan injection (0 h) or 4 h and 24 h after injection. Mechanical allodynia was measured 0 h and 24 h after carrageenan. Results are expressed as mean±s.e.m.; n = 6. ^*P<0.05, ^**P < 0.01 and ^***P<0.001 vs vehicle; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs URB937.

Figure 5

URB937 attenuates formalin-induced pain behavior and spinal cord Fos protein expression in rats. (a) URB937 (1 mg-kg⁻¹, i.p.), injected together with formalin, produced time-dependent changes in composite pain score relative to vehicle, rimonabant (Rim, 2 mg-kg⁻¹, i.p.) or a combination of URB937 and rimonabant (F_14,22 = 1.86, P = 0.039). (b) URB937 decreased area under the curve (AUC) of pain behavior during Phase 2 of the formalin response (10–60 min; F_1,3 = 3.05, P = 0.050).3 Results are expressed as mean±s.e.m.; n = 5–7. ^*P<0.05, all groups vs URB937; ^#P<0.05, URB937 plus rimonabant vs vehicle. (c–e) Representative sections of lumbar spinal cord showing formalin-induced Fos-positive cells after injection of (c) vehicle; (d) URB937 (1 mg-kg⁻¹, i.p.); or (d) URB937 plus rimonabant (2 mg-kg⁻¹, i.p.). Calibration bar, 0.5 mm. (f) Quantitative analysis of the effects of vehicle (open bars), URB937 (closed bars), rimonabant (Rim), and URB937 plus rimonabant on number of Fos-positive cells in superficial dorsal horn (lamina I, II), nucleus proprius (lamina III, IV), neck region of the dorsal horn (lamina V, VI), and ventral horn. Results are expressed as mean±s.e.m.; n = 5–7. ^*P<0.05, all groups vs URB937; ^#P<0.05, URB937 plus rimonabant, or rimonabant alone vs URB937; ^&P<0.05, vehicle, or rimonabant alone vs URB937.