The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice

Abstract

Background and purpose: Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ(9) -tetrahydrocannabinol (THC).

Experimental approach: Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.

Key results: FAAH (-/-) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role.

Conclusions and implications: AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.

Linked articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Figure 1

Intraplantar injection of LPS is more potent in eliciting tactile allodynia than in producing paw oedema. (A) Decreased mechanical paw withdrawal thresholds (PWTs) were reduced in the LPS-injected paw 24 h after injection. LPS injection, at doses of 2.5 µg or 25 µg per paw, increased sensitivity to tactile stimulation that was significantly different from PWT in the saline-injected paw of the same mice. (B) Paw oedema was significantly increased in hind paws injected with the high dose of LPS (i.e. 25 µg). Control paw represents the saline-injected paw of LPS treated mice. Values represent the mean (±SEM) mechanical PWT. n= 6–9 mice per group. **P < 0.01; ***P < 0.001 versus saline-treated paw.

Figure 2

Systemic administration of gabapentin or THC reduces the tactile allodynia caused by i.pl. LPS (2.5 µg). (A) Gabapentin (30 mg·kg⁻¹) reversed tactile allodynia induced by LPS. (B) THC (5 or 10 mg·kg⁻¹) reversed LPS-induced allodynia in the treated paw. Control paw represents the saline-injected paw of LPS-treated mice. Values represent the mean (±SEM) mechanical paw withdrawal threshold (PWT). n= 6–10 mice per group. *P < 0.05; **P < 0.01 versus vehicle in the LPS-treated paw.

Figure 3

Deletion of FAAH within the nervous system reduces LPS-induced allodynia. (A) Control FAAH (+/−) mice displayed profound allodynic responses to i.pl. LPS (2.5 µg), whereas FAAH knockout mice (−/−) showed an anti-allodynic phenotype. FAAH-NS mice that express FAAH exclusively in neuronal tissue displayed a wild-type allodynic response to LPS. (B) The anti-allodynic phenotype in FAAH (−/−) mice was suppressed by the pretreatment of rimonabant (3 mg·kg⁻¹; Rim) and SR144528 (3 mg·kg⁻¹; SR2). Control paw represents the saline-injected paw of FAAH (+/−) LPS-treated mice. All values represent the mean (±SEM) mechanical paw withdrawal threshold (PWT). n= 6–10 mice per group. **P < 0.01 versus FAAH (+/−) mice or FAAH-NS mice (panel A); ##P < 0.01 versus LPS-treated FAAH (−/−) mice that received vehicle. Rim: rimonabant; SR2: SR144528.

Figure 4

FAAH inhibitors reduce LPS-induced allodynia. (A) Three different FAAH inhibitors dose-dependently reversed tactile allodynia 24 h after i.pl. LPS administration. URB597 (10 mg·kg⁻¹), PF-3845 (10 mg·kg⁻¹) and OL-135 (30 mg·kg⁻¹), reversed the allodynic response produced by i.pl. injection of LPS (2.5 µg). Open symbols represent the control saline-injected paw of each respective treatment group. (B) Intraplantar administration of PF-3845 (1, 3, or 10 µg) partially reversed LPS-induced allodynia. (C) PF-3845 administered to the saline-treated, control paw did not reduce allodynic responses in the LPS-treated paw. Control paw represents the saline-injected paw of LPS only treated mice. Values represent the mean (±SEM) mechanical paw withdrawal threshold (PWT). n= 7–12 mice per group. **P < 0.01 versus vehicle treatment in the LPS-treated paw; ##P < 0.01 versus saline-treated paw.

Figure 5

Blocking FAAH in the nervous system mediates the FAAH (−/−) anti-allodynic phenotypic in response to i.pl. LPS. FAAH (−/−) mice displayed an anti-allodynic phenotype that was not present in either the FAAH (+/−) mice or FAAH-NS (i.e. neural specific knock-in) mice. Pretreatment with PF-3845 (10 mg·kg⁻¹) restored the anti-allodynic phenotype in FAAH (+/−) mice and FAAH-NS mice. Control paw represents the saline-injected paw of FAAH (+/−) LPS only treated mice. All values represent the mean (±SEM) mechanical paw withdrawal threshold (PWT). n= 8–10 mice per group. **P < 0.01 versus the vehicle-treated FAAH (+/−) or FAAH-NS mice; #P < 0.05, ##P < 0.01 versus vehicle-treated mice for each respective genotype.

Figure 6

PF-3845 reduces LPS-induced allodynia through a cannabinoid receptor mechanism of action. (A) PF-3845 (10 mg·kg⁻¹) reduced LPS-induced allodynia in CB₁ (+/+) mice, but not in CB₁ (−/−) mice. Control paw represents the saline-injected paw of CB₁ (+/+) LPS only treated mice. (B) PF-3845 (10 mg·kg⁻¹) reduced LPS-induced allodynia in CB₂ (+/+) mice, but not in CB₂ (−/−) mice. Control paw represents the saline-injected paw of CB₂ (+/+) LPS only treated mice. (C) The anti-allodynic effects of PF-3845 (10 mg·kg⁻¹) in the LPS model were not blocked by the opioid receptor antagonist naltrexone (1 mg·kg⁻¹), the TRPV1 receptor antagonist IRTX (0.5 mg·kg⁻¹) or the PPARα antagonist MK886 (3 mg·kg⁻¹). Data shown in (C) represent two combined experiments contracted into a single figure. Control paw represents the saline-injected paw of LPS only treated mice. n= 6–10 mice per group. Values represent the mean (±SEM) mechanical paw withdrawal threshold (PWT). **P < 0.01 versus vehicle-treated mice in the LPS-injected paw; ##P < 0.01 versus PF-3845-treated CB₁ (+/+) or CB₂ (+/+) mice.