Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

G protein signaling-biased mu opioid receptor agonists that produce sustained G protein activation are noncompetitive agonists.

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity, and biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor, which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein-biased mu opioid receptor (MOR) agonists SR-17018 and SR-14968 stabilize the MOR in a wash-resistant yet antagonist-reversible G protein-signaling state. Furthermore, we demonstrate that these structurally related biased agonists are noncompetitive for radiolabeled MOR antagonist binding, and while they stimulate G protein signaling in mouse brains, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR-17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long-lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.

Probing the CB1 Cannabinoid Receptor Binding Pocket with AM6538, a High-Affinity Irreversible Antagonist.

Cannabinoid receptor 1 (CB1) is a potential therapeutic target for the treatment of pain, obesity and obesity-related metabolic disorders, and addiction. The crystal structure of human CB1 has been determined in complex with the stabilizing antagonist AM6538. In the present study, we characterize AM6538 as a tight-binding/irreversible antagonist of CB1, as well as two derivatives of AM6538 (AM4112 and AM6542) as slowly dissociating CB1 antagonists across binding simulations and cellular signaling assays. The long-lasting nature of AM6538 was explored in vivo wherein AM6538 continues to block CP55,940-mediated behaviors in mice up to 5 days after a single injection. In contrast, the effects of SR141716A abate in mice 2 days after injection. These studies demonstrate the functional outcome of CB1 antagonist modification and open the path for development of long-lasting CB1 antagonists.

Investigation of Diacylglycerol Lipase Alpha Inhibition in the Mouse Lipopolysaccharide Inflammatory Pain Model.

Diacylglycerol lipase (DAGL) α and ß, the major biosynthetic enzymes of the endogenous cannabinoid (endocannabinoid) 2-arachidonylglycerol (2-AG), are highly expressed in the nervous system and immune system, respectively. Genetic deletion or pharmacological inhibition of DAGL-ß protects against lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages and reverses LPS-induced allodynia in mice. To gain insight into the contribution of DAGL-α in LPS-induced allodynia, we tested global knockout mice as well as DO34, a dual DAGL-α/ß inhibitor. Intraperitoneal administration of DO34 (30 mg/kg) significantly decreased whole-brain levels of 2-AG (∼83%), anandamide (∼42%), and arachidonic acid (∼58%). DO34 dose-dependently reversed mechanical and cold allodynia, and these antinociceptive effects did not undergo tolerance after 6 days of repeated administration. In contrast, DO34 lacked acute thermal antinociceptive, motor, and hypothermal pharmacological effects in naive mice. As previously reported, DAGL-ß (-/-) mice displayed a protective phenotype from LPS-induced allodynia. However, DAGL-α (-/-) mice showed full allodynic responses, similar to their wild-type littermates. Interestingly, DO34 (30 mg/kg) fully reversed LPS-induced allodynia in DAGL-α (+/+) and (-/-) mice, but did not affect the antinociceptive phenotype of DAGL-ß (-/-) mice in this model, indicating a DAGL-α-independent site of action. These findings suggest that DAGL-α and DAGL-ß play distinct roles in LPS-induced nociception. Whereas DAGL-α appears to be dispensable for the development and expression of LPS-induced nociception, DAGL-ß inhibition represents a promising strategy to treat inflammatory pain.

The Selective Monoacylglycerol Lipase Inhibitor MJN110 Produces Opioid-Sparing Effects in a Mouse Neuropathic Pain Model.

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required µ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice.

Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-​3-​pyridinyl-​4-​[[3-​[[5-​(trifluoromethyl)-​2-​pyridinyl]oxy]phenyl]methyl]-​1-​piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ(9)-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition combined with partial MAGL inhibition reduces neuropathic and inflammatory pain states with minimal cannabimimetic effects.

Δ9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice.

A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and nonreinforced behaviors.

Hyperactivity in Mice Induced by Opioid Agonists with Partial Intrinsic Efficacy and Biased Agonism Administered Alone and in Combination with Morphine.

Opioid analgesics such as morphine and fentanyl induce mu-opioid receptor (MOR)-mediated hyperactivity in mice. Herein, we show that morphine, fentanyl, SR-17018, and oliceridine have submaximal intrinsic efficacy in the mouse striatum using 35S-GTPγS binding assays. While all of the agonists act as partial agonists for stimulating G protein coupling in striatum, morphine, fentanyl, and oliceridine are fully efficacious in stimulating locomotor activity; meanwhile, the noncompetitive biased agonists SR-17018 and SR-15099 produce submaximal hyperactivity. Moreover, the combination of SR-17018 and morphine attenuates hyperactivity while antinociceptive efficacy is increased. The combination of oliceridine with morphine increases hyperactivity, which is maintained over time. These findings provide evidence that noncompetitive agonists at MOR can be used to suppress morphine-induced hyperactivity while enhancing antinociceptive efficacy; moreover, they demonstrate that intrinsic efficacy measured at the receptor level is not directly proportional to drug efficacy in the locomotor activity assay.

Comparison of morphine, oxycodone and the biased MOR agonist SR-17018 for tolerance and efficacy in mouse models of pain.

The mu opioid receptor-selective agonist, SR-17018, preferentially activates GTPγS binding over ßarrestin2 recruitment in cellular assays, thereby demonstrating signaling bias. In mice, SR-17018 stimulates GTPγS binding in brainstem and produces antinociception with potencies similar to morphine. However, it produces much less respiratory suppression and mice do not develop antinociceptive tolerance in the hot plate assay upon repeated dosing. Herein we evaluate the effects of acute and repeated dosing of SR-17018, oxycodone and morphine in additional models of pain-related behaviors. In the mouse warm water tail immersion assay, an assessment of spinal reflex to thermal nociception, repeated administration of SR-17018 produces tolerance as does morphine and oxycodone. SR-17018 retains efficacy in a formalin-induced inflammatory pain model upon repeated dosing, while oxycodone does not. In a chemotherapeutic-induced neuropathy pain model SR-17018 is more potent and efficacious than morphine or oxycodone, moreover, this efficacy is retained upon repeated dosing of SR-17018. These findings demonstrate that, with the exception of the tail flick test, SR-17018 retains efficacy upon chronic treatment across several pain models.

Novel Functionalized Cannabinoid Receptor Probes: Development of Exceptionally Potent Agonists.

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitro and in vivo assays and was designated as "megagonist," a property attributed to its tight binding profile. By acting both centrally and peripherally, 27a distinguishes itself from our previously reported "megagonist" AM841, whose functions are restricted to the periphery.

Toward Directing Opioid Receptor Signaling to Refine Opioid Therapeutics.

The mu opioid receptor (MOR) is a diversely regulated target for the alleviation of pain in the clinical setting. However, untoward side effects such as tolerance, dependence, respiratory suppression, constipation, and abuse liability detract from the general activation of these receptors. Studies in genetically modified rodent models suggest that activating G protein signaling pathways while avoiding phosphorylation of the receptor or recruitment of ß-arrestin scaffolding proteins could preserve the analgesic properties of MOR agonists while avoiding certain side effects. With the development of novel MOR "biased" agonists, which lead to preferential activation of G protein pathways over receptor phosphorylation, internalization, or interaction with other effectors, this hypothesis can be tested in a native, physiological setting. Overall, it is clear that the MOR is not a simple on-off switch and that the diverse means by which the receptor can be regulated may present an opportunity to refine therapeutics for the treatment of pain.

A G protein signaling-biased agonist at the µ-opioid receptor reverses morphine tolerance while preventing morphine withdrawal.

It has been demonstrated that opioid agonists that preferentially act at µ-opioid receptors to activate G protein signaling over ßarrestin2 recruitment produce antinociception with less respiratory suppression. However, most of the adverse effects associated with opioid therapeutics are realized after extended dosing. Therefore, we tested the onset of tolerance and dependence, and assessed for neurochemical changes associated with prolonged treatment with the biased agonist SR-17018. When chronically administered to mice, SR-17018 does not lead to hot plate antinociceptive tolerance, receptor desensitization in periaqueductal gray, nor a super-sensitization of adenylyl cyclase in the striatum, which are hallmarks of opioid neuronal adaptations that are seen with morphine. Interestingly, substitution with SR-17018 in morphine-tolerant mice restores morphine potency and efficacy, whereas the onset of opioid withdrawal is prevented. This is in contrast to buprenorphine, which can suppress withdrawal, but produces and maintains morphine antinociceptive tolerance. Biased agonists of this nature may therefore be useful for the treatment of opioid dependence while restoring opioid antinociceptive sensitivity.

Effectiveness comparisons of G-protein biased and unbiased mu opioid receptor ligands in warm water tail-withdrawal and drug discrimination in male and female rats.

One emerging strategy to address the opioid crisis is the development of mu opioid receptor (MOR) ligands that preferentially signal the G-protein vs. ß-arrestin pathway. The present study compared the relative potency and effectiveness of two G-protein biased (GPB)-MOR ligands TRV130 and SR-14968 to five unbiased MOR ligands (NAQ, nalbuphine, buprenorphine, morphine, and methadone) on therapeutic-related (e.g. antinociception) and abuse-related (e.g. discriminative stimulus effects) endpoints. Male and female rats were tested in a warm water tail-withdrawal procedure (50 °C) or trained to discriminate fentanyl (0.04 mg/kg, SC) from saline in a two-lever food-reinforced discrimination procedure. TRV130 and SR-14968 were approximately two-fold more potent to produce fentanyl stimulus effects vs. antinociception. Morphine, fentanyl, and methadone were significantly more potent in the fentanyl discrimination vs. tail withdrawal procedure. In addition, maximum antinociceptive and discriminative stimulus effects of fixed-proportion fentanyl/naltrexone mixtures (1:0.018, 1:0.054, 1:0.18, 1:0.3, and 1:0.54) were used to quantify 1) the relative in vivo efficacy of the two GPB-MOR agonists and five unbiased MOR ligands, and 2) potential species differences in MOR ligand effects between rats and monkeys. The efficacy-effect function generated from the fentanyl/naltrexone mixtures stratified the five unbiased ligands consistent with agonist-stimulated GTPγS binding (NAQ = nalbuphine < buprenorphine < morphine < methadone). However, TRV130 and SR-14968 produced greater antinociception and less fentanyl-like stimulus effects than was predicted. Furthermore, there was a significant positive correlation between rat and monkey antinociceptive effects. Overall, these results demonstrate GPB-MOR agonists produce undesirable abuse-related effects, albeit with slightly better potency and efficacy ratios compared to unbiased agonists. This article is part of the Special Issue entitled 'Opioid Neuropharmacology: Advances in treating pain and opioid addiction'.

The effect of quinine in two bottle choice procedures in C57BL6 mice: Opioid preference, somatic withdrawal, and pharmacokinetic outcomes.

Previous reports assessing morphine effects in two bottle choice (TBC) paradigms often use taste adulterants such as sweeteners (e.g., saccharin) and/or bitterants (e.g., quinine) to demonstrate morphine preference with C57BL6 mice. The effect of these additional components on the morphine preference of C57BL6 remains poorly understood. Thus, we sought to elucidate the interrelationship of morphine and quinine in the TBC paradigm. As expected, when morphine was included in the opposite bottle from quinine, a preference for the morphine solution was observed. Conversely, when quinine was included in each bottle, or when fentanyl without quinine was used, no preference was observed. All opioid-drinking mice displayed withdrawal signs, and morphine was detectable in plasma and brain. When these results were compared to previous results via conversion to quinine preference scores, quinine was revealed to determine largely the measured morphine preference. Thus, quinine is effective to drive morphine consumption and engender dependence but may confound the ability to measure oral abuse liability of morphine. Together, these results suggest future TBC procedures should consider the effect of quinine upon measured preference for compounds in the opposite bottle, and that excessively high quinine concentrations appear to influence preference more so than the opposite solute when using C57BL6 mice. Alternative conditions to assess oral abuse liability may be necessary to complement and confirm results from TBC experiments utilizing morphine or other opioids.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity.

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Pharmacological characterization of repeated administration of the first generation abused synthetic cannabinoid CP47,497.

A series of in vivo and in vitro assays were conducted to characterize the pharmacological effects of the first generation abused synthetic cannabinoid CP47,497, a racemic bicyclic cannabinoid that is similar in structure to the potent, high-efficacy synthetic cannabinoid CP55,940. CP47,497 was less efficacious than CP55,940 in activating G-proteins and dose-dependently produced common CB1 receptor-dependent pharmacological effects (i.e. catalepsy, hypothermia, antinociception, and hypolocomotion). CP47,497 also substituted for Δ9-tetrahydrocannabinol (THC) in the mouse drug discrimination, indicating that both drugs elicited a similar interceptive stimulus. The pharmacological effects of CP47,497 underwent tolerance following repeated administration and showed cross-tolerance following repeated THC administration, further suggesting a common cannabimimetic mechanism of action. Finally, the CB1 receptor antagonist rimonabant precipitated similar magnitudes of somatic withdrawal responses in mice treated repeatedly with THC or CP47,497. Taken together, these data verify the acute cannabimimetic effects of CP47,497, and indicate tolerance and dependence following repeated administration. The assays used here provide a straightforward approach to characterize the emerging next generation of abused synthetic cannabinoids.

Effects of acute and repeated dosing of the synthetic cannabinoid CP55,940 on intracranial self-stimulation in mice.

BACKGROUND: Synthetic cannabinoids have emerged as a significant public health concern. To increase the knowledge of how these molecules interact on brain reward processes, we investigated the effects of CP55,940, a high efficacy synthetic CB1 receptor agonist, in a frequency-rate intracranial self-stimulation (ICSS) procedure. METHODS: The impact of acute and repeated administration (seven days) of CP55,940 on operant responding for electrical brain stimulation of the medial forebrain bundle was investigated in C57BL/6J mice. RESULTS: CP55,940 attenuated ICSS in a dose-related fashion (ED50 (95% C.L.)=0.15 (0.12-0.18)mg/kg). This effect was blocked by the CB1 receptor antagonist rimonabant. Tolerance developed quickly, though not completely, to the rate-decreasing effects of CP55,940 (0.3mg/kg). Abrupt discontinuation of drug did not alter baseline responding for up to seven days. Moreover, rimonabant (10mg/kg) challenge did not alter ICSS responding in mice treated repeatedly with CP55,940. CONCLUSIONS: The finding that CP55,940 reduced ICSS in mice with no evidence of facilitation at any dose is consistent with synthetic cannabinoid effects on ICSS in rats. CP55,940-induced ICSS depression was mediated through a CB1 receptor mechanism. Additionally, tolerance and dependence following repeated CP55,940 administration were dissociable. Thus, CP55,940 does not produce reward-like effects in ICSS under these conditions.

Combined inhibition of FAAH and COX produces enhanced anti-allodynic effects in mouse neuropathic and inflammatory pain models.

UNLABELLED: Common pharmacological treatments of neuropathic and chronic inflammatory pain conditions generally lack efficacy and/or are associated with significant untoward side effects. However, recent preclinical data indicate that combined inhibition of cyclooxygenase (COX) and fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of the endocannabinoid N-arachidonoylethanolamine (anandamide; AEA), produces enhanced antinociceptive effects in a variety of murine models of pain. Accordingly, the primary objective of the present study was to investigate the consequences of co-administration of the COX inhibitor diclofenac and the highly selective FAAH inhibitor PF-3845 in models of neuropathic pain (i.e., chronic constrictive injury of the sciatic nerve (CCI)) and inflammatory pain induced by an intraplantar injection of carrageenan. Here, we report that combined administration of subthreshold doses of these drugs produced enhanced antinociceptive effects in CCI and carrageenan pain models, the latter of which was demonstrated to require both CB1 and CB2 receptors. The combined administration of subthreshold doses of these drugs also increased AEA levels and decreased prostaglandin levels in whole brain. Together, these data add to the growing research that dual blockade of FAAH and COX represents a potential therapeutic strategy for the treatment of neuropathic and inflammatory pain states. PERSPECTIVE: Tandem inhibition of FAAH and COX attenuates inflammatory and neuropathic pain states, which may avoid potentially harmful side effects of other therapeutic options, such as NSAIDs or opioids.