J Neural Transm DOI 10.1007/s00702-014-1251-x
NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
Attenuation of serotonin-induced itch responses by inhibition of endocannabinoid degradative enzymes, fatty acid amide hydrolase and monoacylglycerol lipase Nurcan Calimli Tosun • Ozgur Gunduz Ahmet Ulugol
•
Received: 17 December 2013 / Accepted: 25 May 2014 Ó Springer-Verlag Wien 2014
Abstract Itch and pain are two irritating sensations sharing a lot in common. Considering the antinociceptive effects of blockade of endocannabinoid degrading enzymes in pain states, we attempted to reduce scratching behavior by endocannabinoid modulation, i.e. by inhibiting fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), or cellular uptake of endocannabinoids. Scratching behavior was induced by intradermal injection of serotonin to Balb/c mice. URB597 (10 mg/kg, i.p.), a FAAH inhibitor, JZL184 (16 mg/kg, i.p.), a MAGL inhibitor, and AM404 (10 mg/kg, i.p.), an endocannabinoid transport inhibitor, were given to evaluate the effects of endocannabinoid modulation on scratching responses. Then, the CB1 receptor antagonist, AM251 (1 mg/kg, i.p.), and the CB2 receptor antagonist, SR144528 (1 mg/kg, i.p.), were administered to determine whether cannabinoid receptors mediate these effects. URB597 and JZL184, but not AM404, attenuated serotonin-induced scratches. The inhibitory effect of URB597 was reversed by SR144528, but cannabinoid receptor antagonists had no other effects on modulation by the inhibitors. We propose that augmenting the endocannabinoid tonus by inhibition of degradative enzymes, FAAH and MAGL, but not cellular uptake, may be a novel target for the development of antipruritic agents.
Submitted to ‘‘15th World Congress on Pain’’, Buenos Aires, Argentina, 6–11October 2014. N. C. Tosun O. Gunduz A. Ulugol (&) Department of Medical Pharmacology, Faculty of Medicine, Trakya University, 22030 Edirne, Turkey e-mail: [email protected]
Keywords AM404 Endocannabinoids FAAH MAGL Pruritus JZL184 URB597 List of Abbreviations 2-AG 2-Arachidonylglycerol AEA Anandamide (N-arachidonylethanolamide) CB Cannabinoid FAAH Fatty acid amide hydrolase MAGL Monoacylglycerol lipase TRPV-1 Tansient receptor potential vanilloid type-1
Introduction Itch is an unpleasant, but protective, sensation that provokes a desire to scratch. Itching has been shown to have much in common with pain sensation: primary afferent C fibers transmit itch sensation from the skin to superficial spinal dorsal horn, and then to thalamus and other central nervous system regions via spinothalamic pathways (Ikoma et al. 2011; Davidson and Giesler 2010). Similar to pain, glutamate is proposed as the principal excitatory transmitter for itch-related transmission in the spinal cord (Koga et al. 2011). Moreover, parallel to allodynia and hyperalgesia observed in neuropathic pain, comparable phenomena, called alloknesis and hyperknesis, are seen in patients with chronic pruritus (Ikoma et al. 2011). Pathologic itch, like abnormal pain, has been shown to occur as a consequence of loss of inhibitory interneurons in the dorsal spinal cord (Ross et al. 2010). These advances in itch research not only point to the similarities between itch and pain sensations, but also indicate that central nervous system is gaining attention for the development of
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antipruritic drugs (Ross 2011; Cevikbas et al. 2011; Kuraishi 2013). Recently, the endocannabinoid system has been drawing attention as an attractive target for pain management (Di Marzo 2008; Pertwee 2012; Guindon and Hohmann 2009). This system is constituted of CB1 and CB2 receptors, endogenous ligands known as endocannabinoids, and the processes taking part in the synthesis, release, transport and degradation of endocannabinoids (Pertwee 2012; Guindon and Hohmann 2009). Anandamide (N-arachidonylethanolamide, AEA) and 2-arachidonylglycerol (2-AG) are thoroughly identified endocannabinoids, metabolized predominantly by the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Uptake via endocannabinoid transport inhibitors also plays role in the termination of endocannabinoid action (Mitchell et al. 2007). Taken together, augmenting the endocannabinoid tonus by targeting FAAH/MAGL activity or inhibiting the uptake of endocannabinoids appears promising, due to low central unwanted effect potential and prolonged duration of action of endocannabinoids (Di Marzo 2008; Long et al. 2009; Pertwee 2012; Guindon and Hohmann 2009; Schlosburg et al. 2009b). Although a huge progress has been made in understanding the involvement of cannabinoids/endocannabinoids in pain sensation (Gunduz et al. 2011; Ulugol et al. 2004, 2006; Di Marzo 2008; Pertwee 2012; Dogrul et al. 2012), very little attempts have been made to comprehend the contribution of the cannabinoid system in the modulation of pruritus. The role of endocannabinoid system in the modulation of itch sensation is examined, and FAAH inhibition is suggested as a promising target to treat pruritus without eliciting apparent adverse effects (Schlosburg et al. 2009a). Here, we first assessed whether inhibition of the degradative enzymes, FAAH and MAGL, or prevention of endocannabinoid membrane transport reduces serotonininduced scratching behavior in mice. We then evaluated the contribution of cannabinoid CB1 and CB2 receptors in the anti-pruritic effects of endocannabinoid system modulators.
Materials and methods
Serotonin-induced scratching behavior Scratching behavior was produced by intradermal injection of 50 lg/50 lL of serotonin into the rostral part of the back of the mice, which was shaved 3 days before the experiments. After intradermal application, the animals were put back into an individual transparent acrylic cage and scratching of the injected site by the hind paws was videotaped for 30 min under quiet conditions. We played back the videotape to observe the scratching behavior. The mice generally showed several scratches per second and such behavior was counted as one bout of scratching. Prior to the experiments, animals were habituated to the acrylic cages and testing environment. Experimental protocol and drugs To determine the effects of endocannabinoid system modulation on serotonin-induced scratching behavior, URB597 (10 mg/kg, i.p.), a FAAH inhibitor, JZL184 (16 mg/kg, i.p.), a MAGL inhibitor, and AM404 (10 mg/kg, i.p.), an endocannabinoid transport inhibitor, were administered 1 h before serotonin injection. Then, the CB1 receptor antagonist, AM251 (1 mg/kg, i.p.), and the CB2 receptor antagonist, SR144528 (1 mg/kg, i.p.), were given 10 min before the inhibitors, to evaluate whether cannabinoid receptors mediate the antipruritic effects of endocannabinoid system modulators. The doses and treatment times of all drugs were selected from previous studies (Kinsey et al. 2009; Mitchell et al. 2007; Schlosburg et al. 2009a). URB597, JZL184, AM404 and SR144528 were purchased from Cayman, while serotonin hydrochloride and AM251 were obtained from Tocris. Serotonin hydrochloride was dissolved in saline, AM404 in ethanol, and the other drugs were dissolved in 20 % DMSO, 1 % Tween 80, 1 % ethanol, 78 % saline. Statistical analysis To determine statistical differences between the groups, Kruskal–Wallis, followed by Dunn test, was carried out. Values of P \ 0.05 were considered to be statistically significant. All data are expressed as mean ± standard error of the mean (SEM).
Animals and ethics Results Female Balb/c mice, weighing 20–30 g, were obtained from Center of the Laboratory Animals, Trakya University, and maintained under 12–12 h light–dark cycles at the temperature of 21 ± 2 °C; water and food were provided ad libitum. All experimental protocols of this study were approved by the local ‘‘Animal Care Ethics Committee’’.
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Effects of endocannabinoid system modulators on serotonin-induced scratching behavior As shown in Fig. 1, intradermal injection of 50 lg/50 lL of serotonin elicited intense scratching of the injected site
Attenuation of serotonin-induced itch responses
Fig. 1 Reduction of the number of serotonin-induced scratches by the FAAH inhibitor, URB597 (10 mg/kg, P \ 0.05), and the MAGL inhibitor, JZL184 (16 mg/kg, P \ 0.05), but not by the endocannabinoid transporter inhibitor, AM404 (10 mg/kg, n = 9 for each group)
Fig. 3 Effects of the CB1 receptor antagonist, AM251 (1 mg/kg), and the CB2 receptor antagonist, SR144528 (1 mg/kg), on attenuation of serotonin-induced scratches by the MAGL inhibitor, JZL184 (16 mg/kg, n = 9 for each group)
Table 1 Mechanism of action of the drugs studied Drugs
Mechanism of action
URB597
FAAH inhibitor
JZL184
MAGL inhibitor
AM404
endocannabinoid transporter inhibitor
AM251
CB1 receptor antagonist
SR144528
CB2 receptor antagonist
Discussion Fig. 2 Effects of the CB1 receptor antagonist, AM251 (1 mg/kg), and the CB2 receptor antagonist, SR144528 (1 mg/kg), on attenuation of serotonin-induced scratches by the FAAH inhibitor, URB597 (10 mg/kg, P \ 0.05, n = 9 for each group)
with the hind paws. The FAAH inhibitor, URB597 (10 mg/ kg, P \ 0.05, Fig. 1), and the MAGL inhibitor, JZL184 (16 mg/kg, P \ 0.05, Fig. 1) reduced the number of serotonin-induced scratches, whereas the endocannabinoid transporter inhibitor, AM404 (10 mg/kg, Fig. 1), did not produce any significant change in scratching behavior induced by serotonin. Effects of cannabinoid CB1 and CB2 receptor antagonists on the inhibitory action of FAAH and MAGL inhibitors on serotonin-induced scratches The inhibitory action of URB597, but not JZL184, was antagonized by the CB2 receptor antagonist, SR144528 (1 mg/kg, P \ 0.05, Fig. 2). On the other hand, the CB1 receptor antagonist, AM251 (1 mg/kg), had no effect on attenuation of serotonin-induced scratches neither by URB597 nor by JZL184 (Fig. 3).
Pruritus is a distressing symptom, reducing the quality of life, but generally controlled with difficulty by classic antipruritic agents, such as histamine H1 receptor antagonists. Taking into account the growing interest on the effect of modulation of endocannabinoids on pain sensation, we examined whether the same approach on endocannabinoids elicits anti-pruritic action. Elevating AEA and 2-AG levels through pharmacological inhibition of their catabolic enzymes or membrane transporters seems to be promising not only for reducing pain, but also itch sensation. Our findings demonstrate that, in line with previous reports, FAAH inhibition attenuate serotonin-induced scratching response in mice. In addition, we showed for the first time that, MAGL inhibition, but not antagonism of endocannabinoid transport, attenuate scratching behavior and this effect is mediated by cannabinoid CB2 receptors (Table 1). Blockade of FAAH by selective inhibitors leads to augmentation of endogenous AEA levels and produce analgesic effects in preclinical pain models, primarily via cannabinoid CB1, but also CB2 receptors (Kinsey et al. 2009; Russo et al. 2007; Jayamanne et al. 2006; Schlosburg et al. 2009b). Very little work has been performed in case of pruritus; FAAH suppression has been indicated to reduce scratching response through activation of cannabi-
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noid CB1 receptors (Schlosburg et al. 2009a). Furthermore, CB1 receptors are shown to mediate rimonabant-induced pruritic responses in mice (Schlosburg et al. 2011). Our results, on the other hand, indicate that FAAH inhibition by URB597 is reversed only by pharmacological antagonism of the CB2 receptor. URB597 reduced scratching in CB2 (-/-) mice to a lesser extend than CB (?/?) (Schlosburg et al. 2009a), and CB2 receptors are shown to mediate antinociception by endocannabinoids (Kinsey et al. 2009; Russo et al. 2007; Jayamanne et al. 2006; Schlosburg et al. 2009b). Thus, contribution of CB2 receptors to reduction of scratching behavior is expected. However, ineffectiveness of CB1 antagonists in our studies is surprising, since these receptors are suggested as the predominant receptor subtype in cannabinoid effect on both pain and inflammation, as well as itch (Kinsey et al. 2011; Schlosburg et al. 2011). Although there are similar observations indicating that inhibition or genetic deletion of FAAH have little effect on neuropathic pain behaviors (Lichtman et al. 2004; Jayamanne et al. 2006), differences in the sensation (itch vs. pain), itch model (serotonin vs. 48/80), animals (Balb/c vs. C57BL/6 J; female vs. male) and other methodologies may be the reason for this discrepancy. It is noteworthy that AEA also activates transient receptor potential vanilloid type-1 (TRPV-1) channels which may interfere with the analgesic effect of AEA, and simultaneous blockade of FAAH and TRPV-1 may be efficacious than using FAAH inhibitors alone (Maione et al. 2013; Starowicz and Di Marzo 2013). Prolonged elevation of 2-AG levels by MAGL inhibition is suggested to result in antinociception mediated by both CB1 and CB2 receptors (Guindon et al. 2007; Guindon and Hohmann 2009; Kinsey et al. 2009). To our knowledge, our findings indicating that selective MAGL inhibition attenuates serotonin-induced scratching behavior is the first data on the effect of MAGL inhibition on pruritus. Our results also suggest that this reduction in scratching response is changed neither with cannabinoid CB1 nor with CB2 antagonists. This is an unexpected finding, but it should be taken into consideration that other metabolic changes caused by MAGL inhibition, such as prevention of generation arachidonic acid, may mediate this anti-pruritic effect of MAGL inhibitors (Nomura et al. 2011). It will be relevant to mention here that dual FAAH/ MAGL inhibitors are developed, and can be useful against both pain and pruritus (Niphakis et al. 2012; Starowicz and Di Marzo 2013). Targeting the cellular uptake of AEA and 2-AG is another strategy to enhance extracellular endocannabinoid levels. While there are reports indicating that endocannabinoid transport inhibitors are eliciting analgesic effects in some pain models (Mitchell et al. 2007), it is generally accepted that antinociception produced by endocannabinoid
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transport inhibition is not as vigorous as that of FAAH or MAGL inhibition (Jhaveri et al. 2007). In our experiments, the endocannabinoid transport inhibitor AM404 had no effect on serotonin-induced scratches. In addition to the weak effect of transport inhibitors compared to degradative enzyme inhibitors, TRPV-1 activating effect of AM404 may also lie behind this result (Rawls et al. 2006). In earlier studies evidence was provided that selfgrooming, a behaviour that partially overlaps scratch responses to itch both in phenotype and central regulation, is connected to anxiety regulation and 5-HT2C receptors in rats (Graf et al. 2003). Furthermore, direct serotonin– endocannabinoid interactions have been described in the regulation of several behaviors and physiological functions including anxiety in humans and also rats. Among those 5-HT2C receptor–endocannabinoid receptor interactions were widely studied (Lazary et al. 2011). In addition, endocannabinoids and their receptors play a local role in the tone and permeability of blood vessels locally and also through central regulation. Similarly, serotonin also has local vascular effects, in addition to pain and inflammation. One should also pay attention to these, although they were not considered in the study design. Modulation of endocannabinoid system by inhibition of catabolic enzymes or cellular uptake of endocannabinoids is well known to be involved in pain processes, a very similar sensation to itch. FAAH inhibition was shown to attenuate scratching responses in a similar allergenic model. Our data extend these findings to include to antiscratching property of MAGL inhibition, but not antagonism of endocannabinoid transport. A limitation of this study is that TRPV-1 antagonists could be used, together either with FAAH or MAGL inhibitors, which will probably be tested in the near future. Another is that endocannabinoids levels could be measured directly which would help to strengthen our results and conclusions; taking into account these limitations, inhibition of catabolic enzymes, FAAH and MAGL, may represent a novel target for pruritus, with no apparent central side effects. Acknowledgments This work was supported by a grant from Trakya University Research Council (TUBAP-2012/187). The authors have no conflicts of interests to report.
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inhibitors and transient receptor potential channel modulators as ‘‘dual-target’’ analgesics. Pharmacol Res 76:98–105. doi:10. 1016/J.Phrs.07.003 Mitchell VA, Greenwood R, Jayamanne A, Vaughan CW (2007) Actions of the endocannabinoid transport inhibitor AM404 in neuropathic and inflammatory pain models. Clin Exp Pharmacol Physiol 34(11):1186–1190. doi:10.1111/J.1440-1681.2007. 04692.X Niphakis MJ, Johnson DS, Ballard TE, Stiff C, Cravatt BF (2012) O-Hydroxyacetamide carbamates as a highly potent and selective class of endocannabinoid hydrolase inhibitors. ACS Chem Neurosci 3(5):418–426. doi:10.1021/cn200089j Nomura DK, Morrison BE, Blankman JL, Long JZ, Kinsey SG, Marcondes MCG, Ward AM, Hahn YK, Lichtman AH, Conti B, Cravatt BF (2011) Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Science 334(6057):809–813. doi:10.1126/Science.1209200 Pertwee RG (2012) Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities. Philos Trans R Soc B 367(1607):3353–3363. doi:10.1098/Rstb.2011.0381 Rawls SM, Ding Z, Cowan A (2006) Role of TRPV1 and cannabinoid CB1 receptors in AM 404-evoked hypothermia in rats. Pharmacol Biochem Behav 83(4):508–516. doi:10.1016/J.Pbb.03.011 Ross SE (2011) Pain and itch: insights into the neural circuits of aversive somatosensation in health and disease. Current Opinion in Neurobiology 21 (6):880-887. doi:Doi 10.1016/J.Conb.2011. 10.012 Ross SE, Mardinly AR, McCord AE, Zurawski J, Cohen S, Jung C, Hu L, Mok SI, Shah A, Savner EM, Tolias C, Corfas R, Chen S, Inquimbert P, Xu Y, McInnes RR, Rice FL, Corfas G, Ma Q, Woolf CJ, Greenberg ME (2010) Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice. Neuron 65(6):886–898 Russo R, LoVerme J, La Rana G, Compton TR, Parrott J, Duranti A, Tontini A, Mor M, Tarzia G, Calignano A, Piomelli D (2007) The fatty acid amide hydrolase inhibitor URB597 (cyclohexylcarbamic acid 3 ‘-carbamoylbiphenyl-3-yl ester) reduces neuropathic pain after oral administration in mice. J Pharmacol Exp Ther 322(1):236–242. doi:10.1124/Jpet.107.119941 Schlosburg JE, Boger DL, Cravatt BF, Lichtman AH (2009a) Endocannabinoid modulation of scratching response in an acute allergenic model: a new prospective neural therapeutic target for pruritus. J Pharmacol Exp Ther 329(1):314–323. doi:10.1124/ Jpet.108.150136 Schlosburg JE, Kinsey SG, Lichtman AH (2009b) Targeting fatty acid amide hydrolase (FAAH) to treat pain and inflammation. AAPS J 11(1):39–44. doi:10.1208/S12248-008-9075-Y Schlosburg JE, O’Neal ST, Conrad DH, Lichtman AH (2011) CB1 receptors mediate rimonabant-induced pruritic responses in mice: investigation of locus of action. Psychopharmacology 216(3):323–331. doi:10.1007/S00213-011-2224-5 Starowicz K, Di Marzo V (2013) Non-psychotropic analgesic drugs from the endocannabinoid system: ‘‘Magic bullet’’ or ‘‘multipletarget’’ strategies? Eur J Pharmacol 716(1–3):41–53. doi:10. 1016/J.Ejphar.01.075 Ulugol A, Karadag HC, Ipci Y, Tamer M, Dokmeci I (2004) The effect of WIN 55,212-2, a cannabinoid agonist, on tactile allodynia in diabetic rats. Neurosci Lett 371(2–3):167–170 Ulugol A, Ozyigit F, Yesilyurt O, Dogrul A (2006) The additive antinociceptive interaction between WIN 55,212-2, a cannabinoid agonist, and ketorolac. Anesth Analg 102(2):443–447. doi:10.1213/01.Ane.0000194587.94260.1d
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NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
Attenuation of serotonin-induced itch responses by inhibition of endocannabinoid degradative enzymes, fatty acid amide hydrolase and monoacylglycerol lipase Nurcan Calimli Tosun • Ozgur Gunduz Ahmet Ulugol
•
Received: 17 December 2013 / Accepted: 25 May 2014 Ó Springer-Verlag Wien 2014
Abstract Itch and pain are two irritating sensations sharing a lot in common. Considering the antinociceptive effects of blockade of endocannabinoid degrading enzymes in pain states, we attempted to reduce scratching behavior by endocannabinoid modulation, i.e. by inhibiting fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), or cellular uptake of endocannabinoids. Scratching behavior was induced by intradermal injection of serotonin to Balb/c mice. URB597 (10 mg/kg, i.p.), a FAAH inhibitor, JZL184 (16 mg/kg, i.p.), a MAGL inhibitor, and AM404 (10 mg/kg, i.p.), an endocannabinoid transport inhibitor, were given to evaluate the effects of endocannabinoid modulation on scratching responses. Then, the CB1 receptor antagonist, AM251 (1 mg/kg, i.p.), and the CB2 receptor antagonist, SR144528 (1 mg/kg, i.p.), were administered to determine whether cannabinoid receptors mediate these effects. URB597 and JZL184, but not AM404, attenuated serotonin-induced scratches. The inhibitory effect of URB597 was reversed by SR144528, but cannabinoid receptor antagonists had no other effects on modulation by the inhibitors. We propose that augmenting the endocannabinoid tonus by inhibition of degradative enzymes, FAAH and MAGL, but not cellular uptake, may be a novel target for the development of antipruritic agents.
Submitted to ‘‘15th World Congress on Pain’’, Buenos Aires, Argentina, 6–11October 2014. N. C. Tosun O. Gunduz A. Ulugol (&) Department of Medical Pharmacology, Faculty of Medicine, Trakya University, 22030 Edirne, Turkey e-mail: [email protected]
Keywords AM404 Endocannabinoids FAAH MAGL Pruritus JZL184 URB597 List of Abbreviations 2-AG 2-Arachidonylglycerol AEA Anandamide (N-arachidonylethanolamide) CB Cannabinoid FAAH Fatty acid amide hydrolase MAGL Monoacylglycerol lipase TRPV-1 Tansient receptor potential vanilloid type-1
Introduction Itch is an unpleasant, but protective, sensation that provokes a desire to scratch. Itching has been shown to have much in common with pain sensation: primary afferent C fibers transmit itch sensation from the skin to superficial spinal dorsal horn, and then to thalamus and other central nervous system regions via spinothalamic pathways (Ikoma et al. 2011; Davidson and Giesler 2010). Similar to pain, glutamate is proposed as the principal excitatory transmitter for itch-related transmission in the spinal cord (Koga et al. 2011). Moreover, parallel to allodynia and hyperalgesia observed in neuropathic pain, comparable phenomena, called alloknesis and hyperknesis, are seen in patients with chronic pruritus (Ikoma et al. 2011). Pathologic itch, like abnormal pain, has been shown to occur as a consequence of loss of inhibitory interneurons in the dorsal spinal cord (Ross et al. 2010). These advances in itch research not only point to the similarities between itch and pain sensations, but also indicate that central nervous system is gaining attention for the development of
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antipruritic drugs (Ross 2011; Cevikbas et al. 2011; Kuraishi 2013). Recently, the endocannabinoid system has been drawing attention as an attractive target for pain management (Di Marzo 2008; Pertwee 2012; Guindon and Hohmann 2009). This system is constituted of CB1 and CB2 receptors, endogenous ligands known as endocannabinoids, and the processes taking part in the synthesis, release, transport and degradation of endocannabinoids (Pertwee 2012; Guindon and Hohmann 2009). Anandamide (N-arachidonylethanolamide, AEA) and 2-arachidonylglycerol (2-AG) are thoroughly identified endocannabinoids, metabolized predominantly by the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Uptake via endocannabinoid transport inhibitors also plays role in the termination of endocannabinoid action (Mitchell et al. 2007). Taken together, augmenting the endocannabinoid tonus by targeting FAAH/MAGL activity or inhibiting the uptake of endocannabinoids appears promising, due to low central unwanted effect potential and prolonged duration of action of endocannabinoids (Di Marzo 2008; Long et al. 2009; Pertwee 2012; Guindon and Hohmann 2009; Schlosburg et al. 2009b). Although a huge progress has been made in understanding the involvement of cannabinoids/endocannabinoids in pain sensation (Gunduz et al. 2011; Ulugol et al. 2004, 2006; Di Marzo 2008; Pertwee 2012; Dogrul et al. 2012), very little attempts have been made to comprehend the contribution of the cannabinoid system in the modulation of pruritus. The role of endocannabinoid system in the modulation of itch sensation is examined, and FAAH inhibition is suggested as a promising target to treat pruritus without eliciting apparent adverse effects (Schlosburg et al. 2009a). Here, we first assessed whether inhibition of the degradative enzymes, FAAH and MAGL, or prevention of endocannabinoid membrane transport reduces serotonininduced scratching behavior in mice. We then evaluated the contribution of cannabinoid CB1 and CB2 receptors in the anti-pruritic effects of endocannabinoid system modulators.
Materials and methods
Serotonin-induced scratching behavior Scratching behavior was produced by intradermal injection of 50 lg/50 lL of serotonin into the rostral part of the back of the mice, which was shaved 3 days before the experiments. After intradermal application, the animals were put back into an individual transparent acrylic cage and scratching of the injected site by the hind paws was videotaped for 30 min under quiet conditions. We played back the videotape to observe the scratching behavior. The mice generally showed several scratches per second and such behavior was counted as one bout of scratching. Prior to the experiments, animals were habituated to the acrylic cages and testing environment. Experimental protocol and drugs To determine the effects of endocannabinoid system modulation on serotonin-induced scratching behavior, URB597 (10 mg/kg, i.p.), a FAAH inhibitor, JZL184 (16 mg/kg, i.p.), a MAGL inhibitor, and AM404 (10 mg/kg, i.p.), an endocannabinoid transport inhibitor, were administered 1 h before serotonin injection. Then, the CB1 receptor antagonist, AM251 (1 mg/kg, i.p.), and the CB2 receptor antagonist, SR144528 (1 mg/kg, i.p.), were given 10 min before the inhibitors, to evaluate whether cannabinoid receptors mediate the antipruritic effects of endocannabinoid system modulators. The doses and treatment times of all drugs were selected from previous studies (Kinsey et al. 2009; Mitchell et al. 2007; Schlosburg et al. 2009a). URB597, JZL184, AM404 and SR144528 were purchased from Cayman, while serotonin hydrochloride and AM251 were obtained from Tocris. Serotonin hydrochloride was dissolved in saline, AM404 in ethanol, and the other drugs were dissolved in 20 % DMSO, 1 % Tween 80, 1 % ethanol, 78 % saline. Statistical analysis To determine statistical differences between the groups, Kruskal–Wallis, followed by Dunn test, was carried out. Values of P \ 0.05 were considered to be statistically significant. All data are expressed as mean ± standard error of the mean (SEM).
Animals and ethics Results Female Balb/c mice, weighing 20–30 g, were obtained from Center of the Laboratory Animals, Trakya University, and maintained under 12–12 h light–dark cycles at the temperature of 21 ± 2 °C; water and food were provided ad libitum. All experimental protocols of this study were approved by the local ‘‘Animal Care Ethics Committee’’.
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Effects of endocannabinoid system modulators on serotonin-induced scratching behavior As shown in Fig. 1, intradermal injection of 50 lg/50 lL of serotonin elicited intense scratching of the injected site
Attenuation of serotonin-induced itch responses
Fig. 1 Reduction of the number of serotonin-induced scratches by the FAAH inhibitor, URB597 (10 mg/kg, P \ 0.05), and the MAGL inhibitor, JZL184 (16 mg/kg, P \ 0.05), but not by the endocannabinoid transporter inhibitor, AM404 (10 mg/kg, n = 9 for each group)
Fig. 3 Effects of the CB1 receptor antagonist, AM251 (1 mg/kg), and the CB2 receptor antagonist, SR144528 (1 mg/kg), on attenuation of serotonin-induced scratches by the MAGL inhibitor, JZL184 (16 mg/kg, n = 9 for each group)
Table 1 Mechanism of action of the drugs studied Drugs
Mechanism of action
URB597
FAAH inhibitor
JZL184
MAGL inhibitor
AM404
endocannabinoid transporter inhibitor
AM251
CB1 receptor antagonist
SR144528
CB2 receptor antagonist
Discussion Fig. 2 Effects of the CB1 receptor antagonist, AM251 (1 mg/kg), and the CB2 receptor antagonist, SR144528 (1 mg/kg), on attenuation of serotonin-induced scratches by the FAAH inhibitor, URB597 (10 mg/kg, P \ 0.05, n = 9 for each group)
with the hind paws. The FAAH inhibitor, URB597 (10 mg/ kg, P \ 0.05, Fig. 1), and the MAGL inhibitor, JZL184 (16 mg/kg, P \ 0.05, Fig. 1) reduced the number of serotonin-induced scratches, whereas the endocannabinoid transporter inhibitor, AM404 (10 mg/kg, Fig. 1), did not produce any significant change in scratching behavior induced by serotonin. Effects of cannabinoid CB1 and CB2 receptor antagonists on the inhibitory action of FAAH and MAGL inhibitors on serotonin-induced scratches The inhibitory action of URB597, but not JZL184, was antagonized by the CB2 receptor antagonist, SR144528 (1 mg/kg, P \ 0.05, Fig. 2). On the other hand, the CB1 receptor antagonist, AM251 (1 mg/kg), had no effect on attenuation of serotonin-induced scratches neither by URB597 nor by JZL184 (Fig. 3).
Pruritus is a distressing symptom, reducing the quality of life, but generally controlled with difficulty by classic antipruritic agents, such as histamine H1 receptor antagonists. Taking into account the growing interest on the effect of modulation of endocannabinoids on pain sensation, we examined whether the same approach on endocannabinoids elicits anti-pruritic action. Elevating AEA and 2-AG levels through pharmacological inhibition of their catabolic enzymes or membrane transporters seems to be promising not only for reducing pain, but also itch sensation. Our findings demonstrate that, in line with previous reports, FAAH inhibition attenuate serotonin-induced scratching response in mice. In addition, we showed for the first time that, MAGL inhibition, but not antagonism of endocannabinoid transport, attenuate scratching behavior and this effect is mediated by cannabinoid CB2 receptors (Table 1). Blockade of FAAH by selective inhibitors leads to augmentation of endogenous AEA levels and produce analgesic effects in preclinical pain models, primarily via cannabinoid CB1, but also CB2 receptors (Kinsey et al. 2009; Russo et al. 2007; Jayamanne et al. 2006; Schlosburg et al. 2009b). Very little work has been performed in case of pruritus; FAAH suppression has been indicated to reduce scratching response through activation of cannabi-
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noid CB1 receptors (Schlosburg et al. 2009a). Furthermore, CB1 receptors are shown to mediate rimonabant-induced pruritic responses in mice (Schlosburg et al. 2011). Our results, on the other hand, indicate that FAAH inhibition by URB597 is reversed only by pharmacological antagonism of the CB2 receptor. URB597 reduced scratching in CB2 (-/-) mice to a lesser extend than CB (?/?) (Schlosburg et al. 2009a), and CB2 receptors are shown to mediate antinociception by endocannabinoids (Kinsey et al. 2009; Russo et al. 2007; Jayamanne et al. 2006; Schlosburg et al. 2009b). Thus, contribution of CB2 receptors to reduction of scratching behavior is expected. However, ineffectiveness of CB1 antagonists in our studies is surprising, since these receptors are suggested as the predominant receptor subtype in cannabinoid effect on both pain and inflammation, as well as itch (Kinsey et al. 2011; Schlosburg et al. 2011). Although there are similar observations indicating that inhibition or genetic deletion of FAAH have little effect on neuropathic pain behaviors (Lichtman et al. 2004; Jayamanne et al. 2006), differences in the sensation (itch vs. pain), itch model (serotonin vs. 48/80), animals (Balb/c vs. C57BL/6 J; female vs. male) and other methodologies may be the reason for this discrepancy. It is noteworthy that AEA also activates transient receptor potential vanilloid type-1 (TRPV-1) channels which may interfere with the analgesic effect of AEA, and simultaneous blockade of FAAH and TRPV-1 may be efficacious than using FAAH inhibitors alone (Maione et al. 2013; Starowicz and Di Marzo 2013). Prolonged elevation of 2-AG levels by MAGL inhibition is suggested to result in antinociception mediated by both CB1 and CB2 receptors (Guindon et al. 2007; Guindon and Hohmann 2009; Kinsey et al. 2009). To our knowledge, our findings indicating that selective MAGL inhibition attenuates serotonin-induced scratching behavior is the first data on the effect of MAGL inhibition on pruritus. Our results also suggest that this reduction in scratching response is changed neither with cannabinoid CB1 nor with CB2 antagonists. This is an unexpected finding, but it should be taken into consideration that other metabolic changes caused by MAGL inhibition, such as prevention of generation arachidonic acid, may mediate this anti-pruritic effect of MAGL inhibitors (Nomura et al. 2011). It will be relevant to mention here that dual FAAH/ MAGL inhibitors are developed, and can be useful against both pain and pruritus (Niphakis et al. 2012; Starowicz and Di Marzo 2013). Targeting the cellular uptake of AEA and 2-AG is another strategy to enhance extracellular endocannabinoid levels. While there are reports indicating that endocannabinoid transport inhibitors are eliciting analgesic effects in some pain models (Mitchell et al. 2007), it is generally accepted that antinociception produced by endocannabinoid
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transport inhibition is not as vigorous as that of FAAH or MAGL inhibition (Jhaveri et al. 2007). In our experiments, the endocannabinoid transport inhibitor AM404 had no effect on serotonin-induced scratches. In addition to the weak effect of transport inhibitors compared to degradative enzyme inhibitors, TRPV-1 activating effect of AM404 may also lie behind this result (Rawls et al. 2006). In earlier studies evidence was provided that selfgrooming, a behaviour that partially overlaps scratch responses to itch both in phenotype and central regulation, is connected to anxiety regulation and 5-HT2C receptors in rats (Graf et al. 2003). Furthermore, direct serotonin– endocannabinoid interactions have been described in the regulation of several behaviors and physiological functions including anxiety in humans and also rats. Among those 5-HT2C receptor–endocannabinoid receptor interactions were widely studied (Lazary et al. 2011). In addition, endocannabinoids and their receptors play a local role in the tone and permeability of blood vessels locally and also through central regulation. Similarly, serotonin also has local vascular effects, in addition to pain and inflammation. One should also pay attention to these, although they were not considered in the study design. Modulation of endocannabinoid system by inhibition of catabolic enzymes or cellular uptake of endocannabinoids is well known to be involved in pain processes, a very similar sensation to itch. FAAH inhibition was shown to attenuate scratching responses in a similar allergenic model. Our data extend these findings to include to antiscratching property of MAGL inhibition, but not antagonism of endocannabinoid transport. A limitation of this study is that TRPV-1 antagonists could be used, together either with FAAH or MAGL inhibitors, which will probably be tested in the near future. Another is that endocannabinoids levels could be measured directly which would help to strengthen our results and conclusions; taking into account these limitations, inhibition of catabolic enzymes, FAAH and MAGL, may represent a novel target for pruritus, with no apparent central side effects. Acknowledgments This work was supported by a grant from Trakya University Research Council (TUBAP-2012/187). The authors have no conflicts of interests to report.
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