Inhibition of fatty acid amide hydrolase produces analgesia by multiple mechanisms

Abstract

1 The reversible fatty acid amide hydrolase (FAAH) inhibitor OL135 reverses mechanical allodynia in the spinal nerve ligation (SNL) and mild thermal injury (MTI) models in the rat. The purpose of this study was to investigate the role of the cannabinoid and opioid systems in mediating this analgesic effect. 2 Elevated brain concentrations of anandamide (350 pmol g(-1) of tissue vs 60 pmol g(-1) in vehicle-treated controls) were found in brains of rats given OL135 (20 mg kg(-1)) i.p. 15 min prior to 20 mg kg(-1) i.p. anandamide. 3 Predosing rats with OL135 (2-60 mg kg(-1) i.p.) 30 min before administration of an irreversible FAAH inhibitor (URB597: 0.3 mg kg(-1) intracardiac) was found to protect brain FAAH from irreversible inactivation. The level of enzyme protection was correlated with the OL135 concentrations in the same brains. 4 OL135 (100 mg kg(-1) i.p.) reduced by 50% of the maximum possible efficacy (MPE) mechanical allodynia induced by MTI in FAAH(+/+)mice (von Frey filament measurement) 30 min after dosing, but was without effect in FAAH(-/-) mice. 5 OL135 given i.p. resulted in a dose-responsive reversal of mechanical allodynia in both MTI and SNL models in the rat with an ED(50) between 6 and 9 mg kg(-1). The plasma concentration at the ED(50) in both models was 0.7 microM (240 ng ml(-1)). 6 In the rat SNL model, coadministration of the selective CB(2) receptor antagonist SR144528 (5 mg kg(-1) i.p.), with 20 mg kg(-1) OL135 blocked the OL135-induced reversal of mechanical allodynia, but the selective CB(1) antagonist SR141716A (5 mg kg(-1) i.p.) was without effect. 7 In the rat MTI model neither SR141716A or SR144528 (both at 5 mg kg(-1) i.p.), or a combination of both antagonists coadministered with OL135 (20 mg kg(-1)) blocked reversal of mechanical allodynia assessed 30 min after dosing. 8 In both the MTI model and SNL models in rats, naloxone (1 mg kg(-1), i.p. 30 min after OL135) reversed the analgesia (to 15% of control levels in the MTI model, to zero in the SNL) produced by OL135.

Figure 1

OL135 treatment increases endogenous anandamide in rats with SNL neuropathic lesions. Rats with a previously induced SNL neuropathic lesion on the ipsilateral (IL; left) side were dosed with 20 mg kg⁻¹ OL135 i.p. and 40 min later brains (a) or lumbar spinal cord segments (b) were collected for assay of anandamide levels. OL135 treatment produces a small but significant enhancement in anandamide levels in brain and in lumbar spinal cord segments. No statistically significant difference was observed between spinal cord anandamide levels contra and ipsilateral to the SNL ligation. n=6 per group.

Figure 2

OL135 treatment inhibits degradation of exogenously administered anandamide. Rats were dosed initially with OL135 (varying doses i.p.) and subsequently with 20 mg kg⁻¹ anandamide. After 30 min, brains were collected and assayed for anandamide. Significantly elevated levels of anandamide were found after 20 mg kg⁻¹ OL135, but not at lower doses. n=6 per group.

Figure 3

OL135 protects FAAH enzyme activity against an irreversible probe inhibitor in vivo. (a) Rats were injected i.p. with vehicle or OL135 at varying doses. After 30 min, they were deeply anaesthetised and received an intracardiac injection of URB597, an irreversible probe compound (1 mg kg⁻¹) or its vehicle. Excess probe was washed out by a rapid (30 s) intracardiac infusion of 60 ml cold PBS. Brains were removed and assayed for FAAH activity. FAAH activities of control rats were expressed as 100% and those treated with probe alone as 0%. One-way ANOVA with Neumann–Keul multiple comparisons (n=4–5 per group). *P<0.05 vs 0–20 mg kg⁻¹ OL135+URB597; ^**P<0.01 vs 60 and 0–6 mg kg⁻¹ OL135+URB597; ^***P<0.001 vs 0, 2, 20 and 60 mg kg⁻¹ OL135+URB597. (b) Values for OL135 concentrations determined in the same brain samples as used for the FAAH protection assay shown in (a) were correlated with the level of FAAH protection observed (r=0.89 using Prism curve fitting software).

Figure 4

OL135 analgesia is dependent upon the presence of FAAH. (a) FAAH knockout mice or C57Bl/6 wild-type mice were subjected to a mild thermal injury. When mechanical allodynia was fully established, OL135 (100 mg kg⁻¹ ip) was administered and mechanical allodynia was assessed at various time points, by measuring the median paw withdrawal threshold (PWT) in grams (g) using calibrated von Frey filaments. In the wild-type mice, OL135 produced a significant reversal of mechanical allodynia at 30 min post dosing, but the effect was absent in the knockout mice (two-way ANOVA with Bonferroni's post-tests, ^***P<0.001, df=1, F=27.25). (b) FAAH knockout mice were similarly responsive to 100 mg kg⁻¹ i.p. of ibuprofen, a reference analgesic not dependent upon FAAH.

Figure 5

OL135 reverses mechanical allodynia in two rat models of pain. (a) Rats were subjected to a mild thermal injury as described, and allodynia was measured using von Frey hairs. OL135 treatment (20 mg kg⁻¹ i.p.) resulted in a reduction of the allodynia that is comparable to 1 mg kg⁻¹ morphine which was maximal at 30 min post treatment and still significant at 60 min post treatment compared with the vehicle (two-way ANOVA with Bonferroni's post-tests, ^***P<0.001, df=2, F=30.97). (b) Rats with a fully developed SNL neuropathic lesion were dosed with either morphine (3 mg kg⁻¹ i.p.) or OL135 (20 mg kg⁻¹ i.p.). OL135 reversed the mechanical allodynia with an efficacy similar to that of morphine, and maximal effect at 30 min, which was still significant at 60 min compared to vehicle (two-way ANOVA with Bonferroni's post-tests, ^*** P<0.001, df=2, F=31.04).

Figure 6

OL135 reverses mechanical allodynia in a dose-dependent manner. (a) Rats subjected to MTI were given 2, 5, 10, 20, and 50 mg kg⁻¹ OL135 and their allodynic thresholds at 30 min were analysed by nonlinear regression. (b) Rats subjected to SNL were dosed with 2, 5, 10, 20, and 50 mg kg⁻¹ OL135 and their allodynic thresholds at 30 min were analysed by nonlinear regression. (c) The plasma concentration of OL135 at 30 min achieved by doses of 2, 5, 10, 20, and 50 mg kg⁻¹ were determined in cohort rats and correlated with efficacy in the MTI and SNL models. For comparison between the two models, the median PWTs were normalised to generate a percent maximum possible efficacy (MPE) value using the following transformation: %MPE=(PWT(t)−PWT(baseline))/[PWT(pre)−PWT(baseline)), where PWT(t)=PWT at time t post-treatment, PWT(baseline)=PWT at baseline, and PWT(pre)=PWT of naïve rats prior to initiation of pain model; r=0.97 for MTI and 0.86 for SNL.

Figure 7

FAAH inhibition produces analgesia by different mechanisms in different pain models. Rats were subjected either to a mild thermal injury or an SNL neuropathic lesion. When the tactile allodynia was fully developed, they were dosed with OL135 (20 mg kg⁻¹) and either SR141716A (CB₁ antagonist), SR144528 (CB₂ antagonist), a combination of SR141716A and SR144528, or a vehicle. Neither antagonist nor a combination thereof was able to reverse significantly the antiallodynic effect of OL135 in the MTI model. The CB₂ antagonist, but not the CB₁ antagonist, almost completely reversed the effect of OL135 in the neuropathic rats (one-way ANOVA with Bonferroni's post-tests, ^***P<0.001, df=3, F=17.31).

Figure 8

FAAH inhibition produces analgesia by an opioid-dependent mechanism in both MTI and spinal nerve ligation models. Rats were subjected to a mild thermal injury or spinal nerve ligation. When the tactile allodynia was fully developed, they were given OL135 (20 mg kg⁻¹) and after 10 min, either saline or naloxone (1 mg kg⁻¹). Allodynia was measured using von Frey hairs 30 min after administration of naloxone. Naloxone completely abolished the antiallodynic effect of OL135 in the MTI (one-way ANOVA with Bonferroni's post-tests, ^***P<0.001, df=3, F=14.87) and in the SNL models (one-way ANOVA with Bonferroni's post-tests, ^**P<0.01, df=3, F=7.831). For comparative purposes, administration of either naloxone or vehicle in the absence of OL135 did not produce elevated tactile thresholds.