Effect of Fatty Acid Amide Hydrolase Inhibitor URB597 on Orofacial Pain Perception in Rats

Abstract

1. Introduction

Orofacial pain is the pain caused by damage to the branches of the trigeminal nerve or neurogenic inflammation that spreads to the orofacial area [1,2]. It accounts for approximately 10% to 26% of the adult population; in 7–11% of this population, it is classified as chronic and constitutes a serious medical and social problem [3,4]. Although the exact etiopathogenesis of most orofacial pain complaints has not been fully elucidated, recent findings suggest that the endoopioid (EOS) and endocannabinoid (ECS) systems have similar pharmacological characteristics, are frequently co-localized and exert a strong analgesic effect mainly through activation of the µ-opioid (MOR) and cannabinoid (CBR) receptors [5,6,7,8].

The cannabinoid (CB) receptors play a key role in peripheral, spinal and supraspinal nociception, including the ascending and descending pain pathways [9] (Scheme 1).

At the periphery, cannabinoid type-1 receptors (CB1R) located in nociceptive terminals inhibit nociceptive transmission, whereas cannabinoid type-2 receptors (CB2R) ones reduce the release of pronociceptive agents [10,11]. Moreover, CB1Rs are expressed in the dorsal root ganglia (DRG) and in the spinal cord dorsal horn (DH), where they inhibit neurotransmitter release and pain transmission [8,12]. At the supraspinal level, CB1Rs inhibit ascending nociceptive transmission and activate the inhibitory descending pathway of pain through the inhibition of gamma aminobutyric acid (GABA) release in the periaqueductal central gray (PAG) and rostral ventromedial medulla (RVM) [13,14].

Pain information from the orofacial area reaches the trigeminal ganglion (TG) and the nucleus trigeminalis caudalis (NTC) through the primary afferents of the trigeminal nerve [15,16,17,18]. The activated NTC transmits pain signals to higher brain centers, including the mesencephalon, thalamus, hypothalamus and the somatosensory cortex. Increased activation of neurons in the TG trigeminal ganglia may lead to reduced levels of endocannabinoid anandamide (AEA), which may in turn lead to an increase in the calcitonin gene peptide (CGRP) and Substance P (SP) release [16,19,20,21]. Low levels of the two main endocannabinoids, AEA and 2-arachidonoylglycerol (2-AG), have been correlated with headaches and provide clinical evidence for the theory of endocannabinoid deficiency, which underlies the pathophysiology of orofacial pain [22,23,24,25,26]. It was previously observed that endocannabinoids inhibit, through a CB1R-dependent retrograde mechanism, the release of neuropeptides and neurotransmitters controlling nociceptive inputs [16,19,21] and that their levels are high in the regions known to be involved in transmission and modulation of pain signals including PAG, RVM and NTC [27,28,29]. Inactivation of endocannabinoid signaling occurs through enzymatic hydrolysis mediated by two major enzymes, hydrolases fatty acid amide hydrolase (FAAH) and the monoacylglycerol lipase (MAGL), responsible for the degradation of AEA and 2-AG, respectively [30]. The action of FAAH inhibitors is based on extending the biological effects of AEA, and also endocannabinoid-like congeners, by increasing their levels in the peripheral (PNS) and central nervous system (CNS). Recent pharmacological studies have shown that inhibition of FAAH increases ECS activity and causes analgesia and reduces inflammation in animal models of migraine [21,31,32], but little is known about its effects in orofacial pain. Recently collected data suggest a strong contribution of peripheral mechanisms in pain suppression [22,31,33]. One way to explore the therapeutic potential of peripheral-endocannabinoid-mediated analgesia is to use CNS-penetrant inhibitors of the anandamide-degrading enzyme FAAH [21,31,34]. Since the currently recommended pharmacological treatments for orofacial pain are of low effectiveness and often cause many troublesome side effects, we decided to investigate the effect of centrally and peripherally administered FAAH inhibitor URB597 on the mRNA expression levels of genes encoding selected neuropeptides and on AEA, 2-AG levels and their receptors in the brain structures important for the generation and propagation of pain signals specific to orofacial pain. A better understanding of the genetic disorders associated with orofacial pain can improve prognosis and prognostication and help in the development of modern, effective treatments for this pain.

2. Results

3. Discussion

The ECS plays an important role in the transmission of trigeminal pain by releasing various neurotransmitters from nerve endings that induce peripheral and central sensitization [20,21]. Previous studies have shown that FAAH inhibitors that act by increasing endocannabinoid levels and extending their duration of action without eliciting the psychoactive effects related to direct CB1 receptor activation may offer a promising treatment option for headaches [21,22,25,31,34]. This strategy of increasing endocannabinoids by inhibiting their degrading enzymes has the benefit of activating cannabinoid receptors at the sites of nociceptive pathways with high endocannabinoid turnover rather than global activation of CB1 receptors, which can result in side effects [22,29,35]. In order to learn about the involvement of the ECS in the pathophysiology of orofacial pain, we decided to investigate the influence of central and peripheral administration of the FAAH inhibitor URB597 on the trigemino-hypoglossal reflex induced by tooth pulp stimulation.

We have shown that cerebral ventricles AEA, URB597 and endomorphin-2 (EM-2) perfusion lowered the amplitude of the ETJ reflex because the cell receptors of the neurons that conduct these impulses in the trigemino-hypoglossal reflex arc are probably saturated with endogenous cannabinoids and opioids. It can also be assumed that these compounds, when given intracerebroventricularly (i.c.v.), quickly penetrated the ventricular lining and exerted an effect on the adjacent structures, producing an analgesic effect, as the elements of the trigemino-hypoglossal reflex arc are located near the cerebral ventricles. In contrast, 2-AG showed lower efficacy and had no significant effect on the ETJ amplitude in this experiment. A plausible explanation for this negative result is that a more robust noxious stimulus may be needed for 2-AG to be mobilized. This finding is not clear and requires further investigation.

Since the ECS may control the release of mediators involved in headache [21,24], as a further test in this study we examined the effect of peripherally administered FAAH inhibitor URB597 and tooth pulp stimulation on mRNA levels of selected mediators, including endocannabinoids and their receptors, in the brain structures involved in the processing of pain signals from the orofacial area in rats. We observed that tooth pulp stimulation caused a significant increase in the expression of the SP, CGRP and EM-2 genes in the midbrain, thalamus and hypothalamus. On the other hand, peripheral administration of URB597 one hour before stimulation decreased the mRNA level of these endogenous neuropeptides compared to the control and stimulated all examined brain structures. This finding suggests that URB597 increases the AEA levels in the brain, which in turn leads to decreased activation of the neuropeptidergic pathways.

The biological effects of endocannabinoids and opioids in humans and animals are mainly related to the distribution of their specific receptors in the CNS [7,36,37,38] (Scheme 2).

Therefore, as another test of this hypothesis, we investigated the effect of tooth pulp stimulation and peripheral administration of URB597 on the expression of CB1R, CB2R and MOR in the midbrain, thalamus and hypothalamus of rats. We observed upregulation of CB1R in each of the brain regions after administration of URB597, while in the hypothalamus, we additionally noted downregulation of CB1R after stimulation with tooth pulp. CB1 receptors were likely activated by the increased levels of AEA in the midbrain, thalamus and hypothalamus produced by administering URB597 before stimulation. These observations suggest that the analgesic effect of URB597 requires the CB1R, confirming the role of this receptor in the response to orofacial pain. In another study, it was found that CB1 receptors expressed in peripheral trigeminal nerve endings can contribute to antinociception by reducing the probability of spike generation and decreased neurotransmitter release [21,39].

On the other hand, stimulation of the tooth pulp and URB597 caused changes in the expression of CB2R mRNA only in the hypothalamus, where downregulation of CB2R expression in response to URB597 administration compared to the control was observed. This may be because the signaling of CB1 and CB2 receptors is pleiotropic and it is difficult to activate these receptors directly without affecting other receptors, such as TRPV1 [40,41,42], PPARy [43,44], GABA A [45,46,47], Adenosine A3 [48] and GPR55 [43,49], by binding to endocannabinoids, which can exert physiological effects. Moreover, CB receptors form both homo- and heterodimers and undergo allosteric modulation, which correlates with the receptor functionality and anatomical localization [50,51,52].

Since endocannabinoids interact with opioids and vice versa to enhance the analgesic effect [6,10,50], in the present study we also analyzed the effect of tooth pulp stimulation and URB597 administration on MOR mRNA expression in the midbrain, thalamus and hypothalamus. We observed that tooth pulp stimulation caused upregulation of MOR mRNA expression, and URB597 administration lowered its expression in all brain structures. These observations suggest that the stimulation of the tooth pulp may lead to increased axonal transport of MOR receptors to the sensory fiber terminals through mechanisms that directly or indirectly increase in their synthesis in the brain, as well as confirm the ability of URB597 to modulate the pronociceptive effects.

At the next stage, we investigated whether nociceptive stimulation or i.p. administration of URB597 would impact AEA and 2-AG concentration in the mesencephalon, thalamus and hypothalamus. We observed that tooth pulp stimulation had no effect on the mobilization of AEA and 2-AG compared to controls in any of the brain structures, while URB597 caused a significant increase in AEA concentration in the midbrain, thalamus and hypothalamus, i.e., in the regions where this lipid messenger is produced de novo on demand [53]. These findings are consistent with other reports that URB597 increases brain AEA levels in rats and mice [21,35,54,55,56,57].

On the other hand, URB597 had no significant effect on the concentration of 2-AG in the examined brain structures. Inhibition of FAAH activity by URB597 resulted in an increase in AEA and CB1 receptor levels, as well as a decrease in CB2 receptor expression. Interestingly, 2-AG was less effective in our experiments despite the fact that it is present in higher concentrations in the brain than AEA and can induce stimulation equally through CB1 and CB2 receptors [58]. It may be due to lower levels of 2-AG access to the presynaptic CB1 receptors, as well as to the fact that the 2-AG-degrading enzyme is closer to the target of the CB1R than AEA. It should also be taken into account that the production and synthesis of AEA and 2-AG can occur independently in the cell and can often cause contradictory effects [59,60,61], or that the FAAH/MAGL activity profile is different in the TG associated with orofacial pain and the DRG involved in transmission of this pain. Additionally, the measurements of headache sensitivity after DAGL inhibition suggest that reduced 2-AG signaling in the cortex and PAG, but not in the NTC or TG, drives the initiation of headache [26]. Our research shows that trigeminal neurons do not always respond in the same way as spinal neurons with respect to somatosensory modulation, as was also confirmed in the studies by Akerman et al. [62] and Levine et al. [26]. Furthermore, our findings are consistent with the theory that AEA and 2-AG can independently regulate pain perception as they have different experimental model-specific and animal-species-specific regulatory mechanisms [63,64]. This may further complicate the interpretation of our results and necessitates further research into its function in orofacial pain.

There is evidence to suggest that dysregulation of endocannabinoid signaling may contribute to the etiology and pathophysiology of migraine headache [21,62,65,66,67,68]. In an animal model of nitroglycerin-induced migraine, systemic administration of the FAAH inhibitor, URB597 attenuated nitroglycerin-induced mechanical hyperalgesia in mice and rats, indicating elevation of the AEA level by the FAAH inhibitors and a reduction in migraine pain [21,66]. The action of the FAAH inhibitor was antagonized by a CB1 receptor antagonist rimonabant [66]. The administration of the FAAH inhibitor also reduced nitroglycerin-induced neuronal c-Fos protein expression in the brainstem trigeminal nuclei, which was blocked by a CB1 receptor antagonist [21,66,69]. Additionally, knockout of FAAH but not MAGL in mice attenuated nitroglycerin-induced mechanical hyperalgesia, indicating a crucial role of AEA in attenuation of pain [66]. In another study, the brain-impermeable FAAH inhibitor URB937 was effective at reducing acute and chronic trigeminal hyperalgesia induced by nitroglycerin, suggesting that an increase in AEA levels and activation of CB1 receptors in peripheral nerves and tissues may reduce migraine pain and produce fewer side effects as compared with those acting in the CNS [68,70].

Taken together, our results suggest that peripheral and central inhibition of FAAH by URB597 demonstrated analgesic effect in a rat model orofacial pain. These data confirm the ability of URB597 to modulate the analgesic effects induced by tooth pulp stimulation in the orofacial area innervated by the trigeminal nerve by elevating AEA and CB1R levels. Simultaneously, the transcription of several mediator genes involved in the pathophysiology of this pain was attenuated in the mesencephalon, thalamus and hypothalamus. Our results provide indirect support for the hypothesis that AEA and 2-AG in the periphery differentially suppress pain transmission initiated by tooth pulp stimulation, and the effects are likely to be dependent upon the specific endocannabinoid elevated. This peripheral FAAH inhibition may exert tight regulatory control over pain processing and may offer a more effective pain management strategy than global inhibition of FAAH that circumvents the limitations of orthosteric agonists at the cannabinoid receptors. The results support a role of AEA-mediated endocannabinoid signaling in migraine and suggest that FAAH may offer a new therapeutic option for the prevention of orofacial pain. However, clinical research in this area is still at an early stage, and some initial setbacks introduce a note of caution. Therefore, further experimental research is needed to confirm this speculation.

4. Materials and Methods

5. Conclusions

In the paper, we demonstrate that URB597 significantly increased the brain AEA levels and produced CB1R-mediated antinociception and reduced the gene expression of some mediators involved in orofacial pain. Modulation of the ECS by inhibition of FAAH with URB597 administered both i.c.v. and i.p. is effective at inhibiting orofacial pain and is a promising therapeutic approach to the treatment of this pain.

Acknowledgments

Abbreviations

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ijms23094665/s1.

Author Contributions

Conceptualization, M.Z. (Marek Zubrzycki) and M.S.; methodology, M.Z. (Marek Zubrzycki), M.Z. (Maria Zubrzycka), J.S. and H.J.; investigation, M.Z. (Marek Zubrzycki) and G.W. (rat brain dissections); writing, M.Z. (Marek Zubrzycki) and M.Z. (Maria Zubrzycka); visualization, M.Z. (Marek Zubrzycki), G.W. and M.S.; final revision and editing, M.Z. (Marek Zubrzycki) and M.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by grant no. 503/1-079-01/503-11-001-19-00 from the Medical University of Lodz.

Institutional Review Board Statement

All procedures were approved by the Local Ethical Committee for Animal Experiments at the Medical University of Lodz (42/ŁB 103/2018; 42/ŁB 103-A-DLZ/2020; 43/ŁB 103-B-NZP/2020) and performed in agreement with the guidelines of the European Community Directive 2010/63/EU of 22 September 2010.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated and analyzed during this study are included in this published article (and its Supplementary Information Files).

Conflicts of Interest

The authors declare that they have no conflict of interest.

Footnotes

References