Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism

Journal name:
Nature Neuroscience
Volume:
13,
Pages:
1265–1270
Year published:
DOI:
doi:10.1038/nn.2632
Received
Accepted
Published online

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.

At a glance

Figures

  1. URB937 is a peripherally restricted FAAH inhibitor.
    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 per kg, subcutaneous) 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 per kg, intraperitoneal). (c) Serum concentrations of URB937 after intracerebroventricular (icv) infusion in rats (0.01–0.1 mg per kg). (d) Liver FAAH activity after icv infusion of vehicle (white bar) or URB937 (0.01–0.1 mg per kg, black bars) in rats. (e) Brain FAAH activity after systemic administration of vehicle (V), URB597 (597, 1 mg per kg, subcutaneous) or URB937 (937, gray bar, 1 mg per kg; black bars, 25 mg per kg, subcutaneous); URB937 was administered alone or in combination with drug-transport inhibitors, 2,6-dichloro-4-nitrophenol (DCNP, 40 mg per kg, intraperitoneal), Ko-143 (Ko, 10 mg per kg, intraperitoneal), verapamil (ver, 50 mg per kg, intraperitoneal), probenecid (pro, 150 mg per kg, intraperitoneal) and rifampicin (rif, 50 mg per kg, intraperitoneal). (f) Effects of vehicle (white bars) or URB937 (1 mg per kg, intraperitoneal, black bars) on anandamide and PEA levels in liver, forebrain and hypothalamus (hypoth) 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 versus vehicle.

  2. URB937 suppressed pain responses elicited by intraperitoneal injections of acetic acid in Swiss Webster mice.
    Figure 2: URB937 suppressed pain responses elicited by intraperitoneal 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, URB937, URB597 or indomethacin (IDM) (each at 1 mg per kg, subcutaneous). 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 per kg, subcutaneous). (c) Effects of vehicle (white bars) or URB937 (1 mg per kg, subcutaneous, black 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 per kg, subcutaneous) prevented the antinociceptive effects of URB937. CB2 antagonist AM630 was ineffective. (e) Effects of vehicle (white bars) or URB937 (1 mg per kg, subcutaneous, black 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 versus vehicle.

  3. URB937 suppressed pain behavior elicited by neural injury in mice.
    Figure 3: URB937 suppressed pain behavior elicited by neural injury in mice.

    (ac) Effects of single administration of vehicle (gray) or URB937 (1 mg per kg, intraperitoneal; black) on mechanical hyperalgesia (a), thermal hyperalgesia (b) and mechanical allodynia (c) produced by sciatic nerve ligation. BL, baseline (measured before nerve ligation); CL, contralateral (nonligated) paw; IL, ipsilateral (ligated) paw. (df) Effects of repeated injections of vehicle or URB937 (1 mg per kg, intraperitoneal, once a day for 7 consecutive days) on mechanical hyperalgesia (d), thermal hyperalgesia (e) and mechanical allodynia (f). (gi) CB1 antagonist rimonabant (R, 1 mg per kg, intraperitoneal), but not CB2 antagonist AM630 (AM, 1 mg per kg, intraperitoneal), prevented the effects of URB937 on mechanical hyperalgesia (g), thermal hyperalgesia (h) and mechanical allodynia (i). Results are expressed as mean ± s.e.m. (n = 6 in each panel). *P < 0.05, **P < 0.01 and ***P < 0.001 versus baseline; #P < 0.05, ##P < 0.01 and ###P < 0.001 versus vehicle.

  4. URB937 attenuated pain behavior elicited by inflammation in mice.
    Figure 4: URB937 attenuated pain behavior elicited by inflammation in mice.

    (ad) Effects of URB937 (1 mg per kg, intraperitoneal), administered alone or in combination with rimonabant (R, 1 mg per kg, intraperitoneal) or AM630 (AM, 1 mg per kg, intraperitoneal) on carrageenan-induced responses to mechanical hyperalgesia (a), thermal hyperalgesia (b), mechanical allodynia (c) and paw edema (d). 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 versus vehicle; #P < 0.05, ##P < 0.01 and ###P < 0.001 versus URB937.

  5. URB937 attenuated formalin-induced pain behavior and spinal cord Fos protein expression in rats.
    Figure 5: URB937 attenuated formalin-induced pain behavior and spinal cord Fos protein expression in rats.

    (a) URB937 (1 mg per kg, intraperitoneal), injected 1 h prior to formalin, produced time-dependent changes in composite pain score relative to vehicle, rimonabant (Rim, 2 mg per kg, intraperitoneal) or a combination of URB937 and rimonabant (F14,22 = 1.86, P = 0.039). (b) URB937 decreased the area under the curve (AUC) of pain behavior during phase 2 of the formalin response (10–60 min, F1,3 = 3.05, P = 0.050). Results are expressed as mean ± s.e.m. (n = 5–7). *P < 0.05, all groups versus URB937; #P < 0.05, URB937 plus rimonabant versus vehicle. (ce) Representative sections of lumbar spinal cord showing formalin-induced Fos-positive cells after injection of vehicle (c), URB937 (1 mg per kg, intraperitoneal, d); or URB937 plus rimonabant (2 mg per kg, intraperitoneal; e). Scale bar represents 0.5 mm. (f) Quantitative analysis of the effects of vehicle (white bars), URB937 (black bars), rimonabant (Rim) and URB937 plus rimonabant on the numbers 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 versus URB937; #P < 0.05, URB937 plus rimonabant or rimonabant alone versus URB937; **P < 0.05, vehicle or rimonabant alone versus URB937.

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Author information

  1. These authors contributed equally to this work.

    • Jason R Clapper,
    • Guillermo Moreno-Sanz &
    • Roberto Russo

Affiliations

  1. Department of Pharmacology, University of California Irvine, Irvine, California, USA.

    • Jason R Clapper,
    • Guillermo Moreno-Sanz,
    • Ana Guijarro &
    • Daniele Piomelli
  2. Department of Biochemistry and Molecular Biology III, Complutense University, Ciudad Universitaria, Madrid, Spain.

    • Guillermo Moreno-Sanz
  3. Department of Experimental Pharmacology, University of Naples “Federico II,” Naples, Italy.

    • Roberto Russo &
    • Antonio Calignano
  4. Pharmaceutical Department, University of Parma, Campus Universitario, Parma, Italy.

    • Federica Vacondio &
    • Marco Mor
  5. Department of Health and Drug Sciences, University of Urbino “Carlo Bo,” Urbino, Italy.

    • Andrea Duranti,
    • Andrea Tontini,
    • Silvano Sanchini &
    • Giorgio Tarzia
  6. Neuroscience and Behavior Program, Department of Psychology, University of Georgia, Athens, Georgia, USA.

    • Natale R Sciolino &
    • Andrea G Hohmann
  7. Program in Neuroscience, Biomedical Health Science Institute, University of Georgia, Athens, Georgia, USA.

    • Jessica M Spradley &
    • Andrea G Hohmann
  8. Drug Discovery and Development, Italian Institute of Technology, Genoa, Italy.

    • Daniele Piomelli

Contributions

J.R.C., G.M.-S., R.R., A.G., N.R.S., J.M.S., F.V., A.D., A.T. and S.S. participated in the design, performance and interpretation of the experiments and chemical syntheses. A.G.H., A.C., M.M., G.T. and D.P. participated in the design and interpretation of the experiments and chemical syntheses. D.P. oversaw the project and wrote the manuscript with help from J.R.C., G.M.-S., A.G.H., A.D., M.M. and G.T.

Competing financial interests

A patent application covering URB937 and allied compounds has been filed on behalf of the inventors (D.P., J.R.C., G.M.-S., A.D., A.T., M.M. and G.T.) by the University of California, Irvine, the Italian Institute of Technology, and the Universities of Urbino and Parma.

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