Inhibition of fatty acid binding proteins elevates brain anandamide levels and produces analgesia

Abstract

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

Figure 1. Structures and binding affinities of FABP inhibitors.

(A) Chemical structures of FABP inhibitors. (B) Superimposed models of SBFI26, SBFI50, and SBFI60 in the binding pockets of FABP3, FABP5, and FABP7 reveal similar binding geometries. Amino acids lining the binding pockets are shown with teal, gold, and grey representing FABP3, FABP5, and FABP7, respectively. (C) Correlation between binding affinities and energy scores of SBFI26, SBFI50, and SBFI60 for FABP3, FABP5, and FABP7. Linear regression revealed an r² value of 0.744. The binding affinities and energy scores are derived from Table 1 and Table 2.

Figure 2. FABP inhibitors reduce nociception in models of inflammatory and visceral pain.

(A) Effects of SBFI26, SBFI50, SBFI60, and SBFI62 (20 mg/kg, i.p.) upon carrageenan-induced thermal hyperalgesia (left panel) and paw edema (right panel) in mice. *, p<0.05; **, p<0.01 versus carrageenan injected animals (black bar) (n = 6). (B) Effect of FABP inhibitors (20 mg/kg, i.p.) upon the first (left panel) and second phases (right panel) of formalin-induced nociception in mice. *, p<0.05 versus vehicle control (n = 6). (C) SBFI26 reduces acetic acid-induced writhing in mice. **, p<0.01 (n = 6). (D) Dose-response of SBFI26-mediated inhibition of acetic acid writhing in mice. **, p<0.01 (n = 6). (E) The antinociceptive effects of SBFI26 are reversed by the cannabinoid receptor 1 antagonist rimonabant (SR1, 3 mg/kg) and the peroxisome proliferator-activated receptor alpha antagonist GW6471 (4 mg/kg). In contrast, the cannabinoid receptor 2 antagonist SR144518 (SR2, 3 mg/kg) and the opioid antagonist naloxone (2 mg/kg) were without effect. Rimonabant also reversed the antinociceptive effects of the FAAH inhibitor PF-3845 (blue bars). When administered alone, the antagonists did not modulate nociception (green bars). *, p<0.05; **, p<0.01 versus vehicle control. ##, p<0.01; ###, p<0.001 versus SBFI26 treated mice (n = 6–9).

Figure 3. SBFI26 pharmacokinetics and its effect upon brain endocannabinoid levels.

(A) Time course of SBFI26 levels in mouse plasma (left panel) and brain (right panel) following a single injection (20 mg/kg, i.p.) (n = 5). (B) SBFI26 (20 mg/kg, i.p.) elevates brain levels of the AEA. Tissues were harvested 90 min after SBFI26 administration. **, p<0.01 (n = 6). (C) Effect of FABP inhibitors (20 mg/kg, i.p.) upon AEA and 2-OG hydrolysis by mouse brain and liver homogenates following a single injection (n = 3). (D) SBFI26 (20 mg/kg, i.p.) does not alter locomotor activity in mice. Locomotor activity was measured 90 min after SBFI26 administration (n = 6). (E) SBFI26 (20 mg/kg, i.p.) does not reduce rectal temperature in mice. Left graph: scatterplot of rectal temperatures at baseline and 90 min following injection of vehicle (black circles) or SBFI26 (red circles). Right graph: change in rectal temperature between baseline and 90 min following administration of vehicle (white bar) or SBFI26 (red bar). Statistical analysis was performed by paired t-test (p = 0.205) (n = 6).

Figure 4. SBFI26 blocks thermal hyperalgesia in a rat model of neuropathic pain.

(A) Thermal latencies and (B) mechanical thresholds in rats subjected to CCI were measured following injection of 20 mg/kg SBFI26. SBFI26 was injected at the 0 hr time point and evoked behaviors were measured at 1 and 4 hrs post-administration. The results are expressed as fraction of the pre-injury baseline measurement per animal (mean +/− SEM). Pre-injury thermal latency was 23.5 +/− 0.7 s and mechanical threshold was 29.9 +/− 0.6 g (mean +/− SEM). Results were analyzed by repeated measures ANOVA followed by Tukey's post-hoc analysis. *, p<0.05 versus 0 hr time point (n = 6).

Figure 5. Activation of PPARα by FABP inhibitors.

HeLa cells transfected with a PPARα reporter were incubated with the indicated compounds for 6 hours and luciferase and β-galactosidase activities were subsequently measured. Luciferase activity was normalized to β-galactosidase. The blue line represents baseline PPARα activation in the absence of agonists (n = 3).

Figure 6. Cytotoxicity of SBFI26 and BMS309403 in HeLa cells.

Cells were incubated with the indicated compounds and viability was assessed 24₅₀ values of 31.1±4.1 μM and 11.3±0.9 μM, respectively (n = 3).