In the recent years, considerable progress has been made in understanding the role of the ECS in the modulation of pain processing. The system includes cannabinoid receptors 1 (CB₁) and 2 (CB₂), endogenous agonists called endocannabinoids: AEA and 2-arachidonyl glycerol (2-AG) and enzymes involved in their biosynthesis and degradation. Endocannabinoids are produced in injured tissues to suppress sensitization and inflammation by activation of CB₁ and CB₂ (Piomelli and Sasso, 2014). The key enzyme responsible for the catabolism of AEA is FAAH. However, AEA can also be metabolized by COX-2 and lipoxygenase 12/15 (LOX12/15). COX-2 metabolizes AEA to prostaglandins (PG), which are involved in inflammatory response (Kozak et al., 2002; Yang et al., 2005; Fowler, 2007). CB₁ receptors expressed in the peripheral and central nervous system (CNS) are responsible for the conducting of pain signaling (Richardson et al., 1998). CB₂ receptors expressed in immune cells (also microglia), regulate the neuro-immune interactions and interfere with the inflammatory hyperalgesia, thus also playing an important role in nociception (Malek et al., 2015b; Mecha et al., 2015). Endocannabinoids have been shown to behave as analgesics in models of both acute nociception and chronic pain such as inflammation (Pernía-Andrade et al., 2009) and painful neuropathy (Toth et al., 2010; La Porta et al., 2013). The main limitation of exogenous cannabinoid receptor agonists is the number of CNS side effects, including dysphoria, movement disorders, dizziness, memory loss, drug abuse potential, and dependence (Thaler et al., 2011). An alternative approach that may prevent the occurrence of these side effects is the stimulation of endogenous mechanisms that regulate ECS activity (Lichtman and Chapman, 2011; Lomazzo et al., 2015).

Endogenous cannabinoids are synthesized “on demand” in areas of cellular stress and therefore do not produce CB₁ agonist side effects (Di Marzo and Petrosino, 2007). Unfortunately, locally released endocannabinoids have a very short half-life due to efficient enzymatic degradation (Deutsch and Chin, 1993; Ahn et al., 2008). Therefore, a subsequent grip point for pain therapies is FAAH, which is responsible for the degradation of AEA and other related fatty acid amides including N-palmitoylethanolamine (PEA), oleoylethanolamine (OEA), N-arachidonoyl glycine (NAGly; Bisogno et al., 2002; Khanna and Alexander, 2011). A series of in vivo experiments proved that genetic or pharmacological inactivation of FAAH results in elevation of endocannabinoids in the spinal cord and brain stem (Lichtman et al., 2004; De Lago et al., 2005). FAAH inhibition, rather than its deletion, may offer a distinctive strategy for the treatment of chronic pain, because no genotypic differences in pain behavior were evident in chronic pain models (Lichtman et al., 2004; Kinsey et al., 2009). FAAH inhibition was shown to cause anti-nociceptive, anti-inflammatory or anti-edemic effects in numerous acute (Holt et al., 2005), chronic (Jayamanne et al., 2006), and neuropathic pain animal models (Jhaveri et al., 2006; Kinsey et al., 2009; Guindon et al., 2013). Nevertheless, FAAH inhibitors (like URB957) are not optimal due to some limitations. Tissue-specific changes in the sensitivity to URB597 in neuropathic pain in rats, which may arise as a result of changes in FAAH activity, metabolic pathways, and tissue pH were reported (Chang et al., 2006; Paylor et al., 2006). Due to the differential effects of URB597 in carrageenan-induced inflammation and spinal nerve ligation models, efforts aimed at optimizing the clinical efficacy of FAAH inhibitors should be revised and redesigned (Di Marzo, 2012; Okine et al., 2012). Similarly, despite many promising preclinical results in various chronic pain models (Ahn et al., 2011), one of the most potent FAAH inhibitors, PF-04457845, has failed to show efficacy in humans in a randomized, placebo-controlled phase II clinical trial (Di Marzo, 2012; Huggins et al., 2012). Moreover, safety of FAAH inhibitors became questioned after “first-in-human” trial to test safety of BIA 10-2474 in healthy volunteers, which concluded with one person dead and five more hospitalized. The probable cause of the failure is unjustified dose, 80 times higher than that presumed to induce total FAAH inhibition, used in the study¹.

TRPV1 has emerged as a promising target for the development of new analgesic and anti-inflammatory drugs. TRPV1 is a non-selective ion channel that is highly associated with pain nociception and linked to ECS through the common agonist AEA (Zygmunt et al., 1999; Van der Stelt et al., 2005; Lizanecz et al., 2006). This polymodal pain transducer is known to be expressed in peripheral sensory afferents (Singh Tahim et al., 2005; Ikeda-Miyagawa et al., 2015), spinal cord (Kanai et al., 2006; Horvath et al., 2008), and some brain stem nuclei involved in nociception, including periaqueductal gray matter (PAG) and cingulate cortex (Roberts et al., 2004; Cristino et al., 2006; Starowicz et al., 2007).

A growing body of evidence suggests that TRPV1 is essential in driving nociceptive response (Davis et al., 2000; Immke and Gavva, 2006; Horvath et al., 2008). TRPV1-knockout mice exhibited attenuated inflammation-induced hyperalgesia, while receptor blockade and desensitization caused analgesia (Karai et al., 2004; Bölcskei et al., 2005). Furthermore, enhanced TRPV1 expression and increased TRPV1 sensitivity to AEA during inflammation and neuropathic conditions has been described previously (De Petrocellis et al., 2001; Baamonde et al., 2005; Singh Tahim et al., 2005). Unfortunately, off-target effects of TRPV1 modulation on thermoregulation are widely described. TRPV1 activation results in hypothermia, whereas antagonizing TRPV1 causes hyperthermia, which accounted for the failure of AMG517 during a phase I clinical trial (Di Marzo et al., 2000; Swanson et al., 2005; Gavva et al., 2008). Nevertheless, TRPV1 antagonism has still emerged as an interesting strategy to alleviate pain, especially with simultaneous action on either FAAH and/or COX-2, which could allow lower doses to be used (Lee et al., 2015; Malek et al., 2015a, 2016).

The basis for the use of non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of pain and inflammation is COX inhibition. Cyclooxygenases are enzymes that catalyze the conversion of membrane phospholipids to prostanoids, which include PG, prostacyclins (PGI) essential for intestine and kidney functioning, and thromboxanes (TXA), responsible for platelet aggregation. The two COX isozymes are COX-1 and COX-2. COX-1 is characterized by constitutive expression in tissues and COX-2 is an induced isoform with low baseline expression in human tissues that can be induced during a response to extracellular and intracellular stimuli. The increase in COX-2 expression elevates proinflammatory cytokine, mitogen and growth factor levels (Smith et al., 1996), as well it plays a key role in the transmission of pain to the brain and spinal cord (Ito et al., 2001). Moreover, blocking COXs can influence the ECS by stopping the conversion of endocannabinoids into prostanoid-like derivatives, which may be of benefit for the treatment of chronic pain (Jhaveri et al., 2007; Hu et al., 2008). The inflammatory process can dramatically reduce the level of endocannabinoids, while its inhibition may contribute to increased analgesic action. Although widely used in the treatment of pain, NSAIDs do not always provide adequate relief and have the risk for serious complications, including death by internal hemorrhaging (5–10% of chronic NSAIDs therapy) and severe kidney damage (Rahme and Bernatsky, 2010; Altman, 2011). However, it should be noted that some of the NSAIDs have the ability to inhibit FAAH (Fowler et al., 1999; Seidel et al., 2003; Guindon et al., 2006). Therefore, interaction of NSAIDs with ECS may be a novel approach for the treatment of chronic pain.

Endogenous levels of AEA can activate but not desensitize TRPV1 in non-pathological conditions, leading to stimulation of pain pathways. Elevation of endocannabinoid tone by FAAH inhibitors may lead to further activation of this channel, implying that the development of combined FAAH/TRPV1 blockers may have essential therapeutic applications in directing AEA's action toward the analgesic CB_1/2 pathway. Lack of efficacy of FAAH inhibitors in patients (as described above) may be associated with activation of the alternative AEA biotransformation pathways after completely blocking the FAAH enzyme and the ability of AEA to induce pro-nociceptive signaling through TRPV1 receptors (Maione et al., 2006; Piscitelli and Di Marzo, 2012; Starowicz et al., 2013). The most promising aforementioned agent is the endogenous lipid signaling molecule N-arachidonoyl-5-hydroxytryptamine (AA-5-HT), which was originally described as an FAAH inhibitor and called an “unique dual FAAH/TRPV1 blocker” about a decade later (Maione et al., 2007). In the context of its inhibitory activity, similar to other FAAH blockers, AA-5-HT elevates AEA levels in the CNS and on the periphery (Capasso et al., 2005; Maione et al., 2007). Moreover, AA-5-HT is able to antagonize human and rat recombinant TRPV1 receptors with an affinity slightly stronger than capsaicin, though, it is not clear whether AA-5-HT is a competitive or non-competitive antagonist (Maione et al., 2007). AA-5-HT appears to be less effective at TRPV1 blockade when the pH of the cellular environment is reduced, meaning that it may be less effective under inflammatory conditions, although this hypothesis has not been tested in vivo yet (Fowler et al., 2003; Paylor et al., 2006). Nevertheless, studies involving a variety of animal models have been conducted to better understand the mechanism of action of AA-5-HT. A significant contribution was completed by Maione and colleagues, who indicated the significant analgesic impact exerted by the compound in acute and chronic pain models in rodents following systemic administration (Maione et al., 2007). A subsequent study showed that intra-PAG AA-5-HT also causes analgesia and that the effect is mimicked by co-injection of URB597 and the selective TRPV1 antagonist iodoresiniferatoxin (I-RTX). Furthermore, antagonizing the CB₁ and TRPV1 receptors abolished AA-5-HT-exerted analgesia, suggesting that these co-localized receptors are AA-5-HT molecular targets in the PAG and cortex. Moreover, single AA-5-HT dosages exhibited anti-allodynic actions more effectively than URB597 or I-RTX (De Novellis et al., 2008, 2011). In the same series of experiments, de Novellis et al. determined the role of AA-5-HT in cognitive and emotional functions (i.e., decision making, goal-directed behavior and working memory) in neuropathic pain. The authors reported that AA-5-HT is able to restore the balance between excitatory and inhibitory responses in prefrontal cortex neurons, highlighting the potential of dual-acting drugs for mitigating the central sequelae associated with neuropathic pain (De Novellis et al., 2011). AA-5-HT was also proven to dose-dependently relieve pain in a model of acute inflammation in mice after systemic administration (Costa et al., 2010). Its anti-nociceptive action was associated with an increase in AEA levels in both inflamed paws and spinal cord, though CB₁ and TRPV1 receptors were differentially involved; TRPV1 was responsible for the anti-inflammatory properties of AA-5-HT, while both CB₁ and TRPV1 receptors mediated its anti-hyperalgesic activity (Costa et al., 2010). Our most recent research indicates that CB₂ receptors are also involved in the anti-nociceptive actions of AA-5-HT, after intrathecal administration (Malek et al., 2016). Despite the fact that AA-5-HT was proven to be active it is unstable due to effective hydrolysis, therefore efforts to synthesize AA-5-HT analogs with improved modes of action have been undertaken (Ortar et al., 2007). The chemical synthesis led to discovery of piperazinyl carbamates and ureas that are characterized by higher stability than AA-5-HT, nevertheless none of them have shown higher efficacy than AA-5-HT (Ortar et al., 2007, 2013; Morera et al., 2009). Of the many synthesized compounds, OMDM-198 is best described and tested in vivo for its anti-nociceptive and analgesic properties. OMDM-198 exhibits anti-nociceptive, anti-hyperalgesic and anti-edemic actions in acute and inflammatory pain. It inhibits the second phase of formalin-induced nociceptive behavior in rodents and blocks carrageenan-induced thermal hyperalgesia and paw edema in mice (Maione et al., 2013). The anti-hyperalgesic effects of OMDM-198 were also evaluated in an animal model of osteoarthritic pain. It was shown that the effect of OMDM-198 depends on CB₁ receptor activation and TRPV1 receptor blockade (Malek et al., 2015a). Moreover, OMDM-198 required lower doses to achieve a comparable analgesic effect than the two single-target compounds used in the study: URB597 and SB-366,791 (TRPV1 antagonist; Malek et al., 2015a). These data suggest that the use of dual-acting compounds may be beneficial in a matter of side effects characteristic for TRPV1 antagonists (hyperthermia) and FAAH inhibitors (lack of efficacy in clinics), because of significant decrease of the working doses. It was shown that hyperthermia was not observed in animals upon AA-5-HT treatment (Maione et al., 2007; Costa et al., 2010; Malek et al., 2016). The same observations were made for OMDM-198 (unpublished data). The field of FAAH/TRPV1 blockers is still developing. A recently synthesized series of arylboronic acids characterized by high efficacy on FAAH and TRPV1 have been reported, though these hybrid compounds have not yet been evaluated in animal studies for pain (Morera et al., 2016).

Interactions between COX and FAAH enzymes that control endogenous lipid (including endocannabinoids) levels were reported (Guindon and Beaulieu, 2006; Jhaveri et al., 2008). AEA metabolism is of special interest because blocking FAAH enzyme activates AEA biotransformation by COX-2 pathway, leading to decreases in endocannabinoid tone. This may be another mechanism responsible for lack of analgesic action from selective FAAH inhibitors (Starowicz et al., 2013; Starowicz and Di Marzo, 2013). Indeed, co-administration of diclofenac (NSAID) and URB597 showed synergic analgesic effects in abdominal pain dependent on both CB₁ activation and blocking prostaglandin production (Naidu et al., 2010). Another study demonstrated an additive anti-nociceptive activity of PF-3845 and diclofenac in the animal models of inflammatory and neuropathic pain (Grim et al., 2014). Moreover, ketorolac (NSAID) was shown to have an additive anti-nociceptive effect with WIN-55,212-2 (non-selective cannabinoid agonist; Ulugöl et al., 2006). In these studies, AEA levels were shown to be elevated with simultaneous inhibition of PG synthesis. Studies have shown that common NSAIDs (ibuprofen and flurbiprofen) act also as FAAH inhibitors (Fowler et al., 1999; Seidel et al., 2003; Guindon et al., 2006). The synergistic effect of ibuprofen and AEA was proven in experimental models for acute and neuropathic pain (Guindon and Beaulieu, 2006). Those data demonstrate the rationale underlying design of novel compounds that can interact with both COXs and FAAH enzymes. Indeed, numerous novel compounds with promising analgesic characteristics were developed recently, i.e., ARN2508, which was tested and effective in suppressing intestinal inflammation (Sasso et al., 2015). It also causes no gastric damage, which is often a case of chronic NSAID treatment and the protective effect was dependent on FAAH inhibition. This suggests that COX/FAAH inhibitors shouldn't exhibit NSAIDs side effects present during chronic treatment, although clinical studies would be needed to verify this hypothesis. A panel of novel ARN2508 derivatives was synthesized, of which (S)-(+)-ARN2508 demonstrated increased OEA plasma levels and decreased PGI and TXA levels after intravenous administration (Migliore et al., 2016). The in vivo results suggest that this compound may be useful for FAAH-COX related pathologies, such as pain.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.