Primary sensory afferents and their neighboring host-defense cells are a rich source of lipid-derived mediators that contribute to the sensation of pain caused by tissue damage and inflammation. But an increasing number of lipid molecules have been shown to act in an opposite way, to suppress the inflammatory process, restore homeostasis in damaged tissues and attenuate pain sensitivity by regulating neural pathways that transmit nociceptive signals from the periphery of the body to the central nervous system.

The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, which remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme, fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), which lacks amidase activity but binds anandamide with low micromolar affinity and facilitates its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, block these effects. Additionally, we identify a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, which prevents anandamide internalization in vitro, interrupts anandamide deactivation in vivo, and exerts profound analgesic effects in rodent models of nociceptive and inflammatory pain, which are mediated by CB1 cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.

The chemokine fractalkine (FKN) is a critical mediator of spinal neuronal-microglial communication in chronic pain. Mature FKN is enzymatically cleaved from neuronal membranes and activation of its receptor, CX3CR1, which is expressed by microglia, induces phosphorylation of p38 MAPK. We used CX3CR1 knockout (KO) mice to examine pain behaviour in the absence of FKN signalling. Naive CX3CR1 KO mice had normal responses to acute noxious stimuli. However, KO mice showed deficits in inflammatory and neuropathic nociceptive responses. After intraplantar zymosan, KO mice did not display thermal hyperalgesia, whereas mechanical allodynia developed fully. In the partial sciatic nerve ligation model of neuropathic pain, both mechanical allodynia and thermal hyperalgesia were less severe in KO mice than in wild-types (WT). Dorsal horn Iba1 immunostaining and phosphorylation of p38 MAPK increased after injury in WT controls but not in KO animals. In WT mice, inflammation and nerve injury increased spinal cord CX3CR1 and FKN expression. FKN protein was also increased in KO mice following inflammation but not after neuropathy, suggesting the FKN/CX3CR1 system is differently affected in the two pain models. Loss of FKN/CX3CR1 neuroimmune communication attenuates hyperalgesia and allodynia in a modality-dependent fashion highlighting the complex nature of microglial response in pathological pain models.

Fatty-acid amide hydrolase (FAAH) catalyzes the intracellular hydrolysis of the endocannabinoid anandamide and other bioactive lipid amides. In the present study, we conducted a comparative characterization of the effects of the newly identified brain-impermeant FAAH inhibitor, URB937 ([3-(3-carbamoylphenyl)-4-hydroxy-phenyl] N-cyclohexylcarbamate), in various rodent models of acute and persistent pain. When administered by the oral route in mice, URB937 was highly active (median effective dose, ED50, to inhibit liver FAAH activity: 0.3 mg-kg−1) and had a bioavailability of 5.3%. The antinociceptive effects of oral URB937 were investigated in mouse models of acute inflammation (carrageenan), peripheral nerve injury (chronic sciatic nerve ligation) and arthritis (complete Freund’s adjuvant). In all models, URB937 was as effective or more effective than standard analgesic and anti-inflammatory drugs (indomethacin, gabapentin, dexamethasone) and reversed pain-related responses (mechanical hyperalgesia, thermal hyperalgesia, and mechanical allodynia) in a dose-dependent manner. ED50 values ranged from 0.2 to 10 mg-kg−1, depending on model and readout. Importantly, URB937 was significantly more effective than two global FAAH inhibitors, URB597 and PF-04457845, in the complete Freund’s adjuvant model. The effects of a combination of URB937 with the non-steroidal anti-inflammatory agent, indomethacin, were examined in the carrageenan and chronic sciatic nerve ligation models. Isobolographic analyses showed that the two compounds interacted synergistically to attenuate pain-related behaviors. Furthermore, URB937 reduced the number and severity of gastric lesions produced by indomethacin, while exerting no ulcerogenic effect when administered alone. The results indicate that the peripheral FAAH inhibitor URB937 is more effective than globally active FAAH inhibitors at inhibiting inflammatory pain. Our findings further suggest that FAAH and cyclooxygenase inhibitors interact functionally in peripheral tissues, to either enhance or hinder each other’s actions.

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Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. In the present study, we examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The anti-nociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B-radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the pro-inflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α.

Among the growing family of ribosomally synthesized, post-translationally modified peptides, particularly intriguing are class III lanthipeptides containing the triamino acid labionin. In the course of a screening program aimed at finding bacterial cell wall inhibitors, we discovered a new lanthipeptide produced by an Actinoplanes sp. The molecule, designated NAI-112, consists of 22 amino acids and contains an N-terminal labionin and a C-terminal methyl-labionin. Unique among lanthipeptides, it carries a 6-deoxyhexose moiety N-linked to a tryptophan residue. Consistently, the corresponding gene cluster encodes, in addition to the LanKC enzyme characteristic of this lanthipeptide class, a glycosyl transferase. Despite possessing weak antibacterial activity, NAI-112 is effective in experimental models of nociceptive pain, reducing pain symptoms in mice in both the formalin and the chronic constriction injury tests. Thus, NAI-112 represents, after the labyrinthopeptins, the second example of a lanthipeptide effective against nociceptive pain.

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