The development of intracellular fatty acid amide hydrolase inhibitors (FAAH), that sustain the time of action of anandamide (AEA), the main endocannabinoid, represents a therapeutic option for the treatment of various clinical disorders, including migraine pain. AEA is released on demand during an inflammatory process and it is rapidly degraded by FAAH activity, that generates two inactive metabolites: arachidonic acid and ethanolamine, both of which are devoid of affinity for cannabinoid receptors. It is well known that the systemic administration of nitroglycerin (NTG), a nitric oxide donor, evokes delayed migraine-like headaches in migraineurs. In rodents, NTG administration induces neuronal activation in specific brain areas and a state of hyperalgesia, associated with a reduced pain threshold at the tail flick test, or an increased sensitivity to nociceptive tests (plantar and orofacial formalin test, tail flick test). The present study was aimed at investigating the therapeutic potential of two new FAAH inhibitors, ARN-14633 and ARN-14280, synthesized from URB597, to be a relatively selective global FAAH inhibitor. We tested the effects of the two inhibitors on the NTG-induced hyperalgesia at the orofacial formalin test, in the animal model of migraine through the quantification of nocifensive behavior and gene expression of some mediators, involved in pain transmission and inflammation. Sprague Dawley rats received ARN-14633 (1 mg/g, i. p.) or ARN-14280 (3 mg/kg, i. p. ) or vehicle 3 hours after the administration of NTG (10mg/kg, i.p.) or NTG vehicle, in order to identify a possible abortive effect. All rats were sacrificed 4 h after NTG or vehicle to evaluate: (i) response in the orofacial formalin test (ii) and mRNA expression levels (c-fos, neuropeptides, nNOS and cytokines) in specific central and peripheral areas (cervical cord and trigeminal ganglion ipsilateral to formalin injection, medulla-pons in toto) by rtPCR analysis. The latency of 4 h for NTG injection was selected in agreement with previous observations showing that it corresponds to the timing of maximal neuronal activation in brain areas relevant for migraine. The findings obtained showed that both FAAH inhibitors significantly reduced the NTG-induced hyperalgesia in the second phase of the orofacial formalin test. This behavioral response was associated with a significant decrease in gene expression of c-Fos, a neuronal activation marker, induced by NTG administration in all the evaluated areas. In addition, this reduction was accompanied by a decrease in CGRP and SP mRNA levels in all the areas, although with different entities. Finally, the gene expression levels of pro-inflammatory cytokines (IL-1beta, Il-6, TNF-alpha), significantly increased by NTG administration in the same areas, were reduced by the FAAH inhibitors, with a stronger effect of ARN-14633. By contrast, no significant effects were found in baseline conditions (in the absence of NTG). The results obtained in this study promote the research of FAAH inhibitors in migraine pain, offering a valid therapeutic approach. However, there is still a need for further studies on the use of these molecules, either for chronic treatment and to understand the role of other biologically active metabolites that may influence analgesic effects.
Lo sviluppo degli inibitori dell’idrolasi intracellulare degli acidi grassi amidici (FAAH), il cui effetto rende maggiore e più prolungata l’azione dell’anandamide (AEA), principale endocannabinoide, rappresenta un’opzione terapeutica per il trattamento di diversi disturbi clinici, compreso il dolore emicranico. L’AEA viene prodotta su richiesta durante un processo infiammatorio e la sua attività viene rapidamente interrotta dall’attività della FAAH, generando due metaboliti inattivi: acido arachidonico ed etanolamina, privi di affinità per i recettori cannabinoidi. È noto che la somministrazione sistemica di nitroglicerina (NTG), donatore di ossido nitrico, evoca nei soggetti emicranici una crisi di cefalea con caratteri simil spontanei, che si manifesta abitualmente con un intervallo di alcune ore. Nell’animale tale procedura causa l’attivazione neuronale di specifiche aree cerebrali, associata a uno stato di iperalgesia, percepibile come ridotta soglia algica al Tail Flick test, o aumentato comportamento nocifensivo al formalin test plantare e oro-facciale. Il presente studio è stato finalizzato ad investigare le potenzialità terapeutiche di due nuovi inibitori della FAAH, ARN-14633 e ARN-14280, sintetizzati a partire dall’URB597, noto inibitore periferico e centrale dell’enzima. Gli effetti dei due inibitori sono stati testati nel modello animale di emicrania basato sulla somministrazione di NTG, associata al formalin test oro-facciale, attraverso l’analisi del comportamento nocifensivo e dell’espressione genica di alcuni mediatori implicati nella trasmissione del dolore e nell’infiammazione. Ratti Sprague Dawley sono stati trattati con ARN-14633 (1 mg/g, i.p.) e ARN-14280 (3 mg/kg, i.p) o veicolo 3 ore dopo la somministrazione di NTG (10mg/kg, i.p.) o veicolo NTG, al fine di identificarne un possibile effetto abortivo. Gli animali sono stati sottoposti al formalin test oro-facciale e al termine del quale alcune aree centrali e periferiche del sistema nervoso (midollo cervicale, ganglio trigeminale ipsilaterale all’iniezione di formalina, area bulbo pontina in toto) sono state prelevate per l’analisi in rtPCR. La latenza di 4 ore per l’iniezione di NTG è stata scelta in accordo con precedenti osservazioni che hanno mostrato che essa corrisponde al tempo di massima attivazione nelle aree cerebrali rilevanti per l’emicrania. I risultati ottenuti hanno mostrato che ARN-14633 e ARN-14280 hanno ridotto significativamente l’iperalgesia indotta dalla NTG nella seconda fase del formalin test oro-facciale. L’effetto sull’espressione genica di c-Fos, marcatore di attivazione neuronale, indotto dalla somministrazione di NTG in tutte le aree è stato ridotto in maniera significativa da entrambi gli inibitori. A tale riduzione è risultata associata la diminuzione dei livelli di mRNA di CGRP e SP nelle varie aree, anche se con entità diversa. Infine, l’espressione genica delle citochine pro-infiammatorie (Il-1beta, Il-6, TNF-alpha), significativamente incrementate dalla NTG, è stata ridotta maggiormente dall’ARN-14633 a livello del ganglio trigeminale e aree centrali. Al contrario, nessun effetto significativo è emerso in condizioni basali (in assenza di NTG). I risultati ottenuti promuovono la ricerca sugli inibitori idrolasici della FAAH nell’ambito del dolore emicranico, offrendo un valido approccio terapeutico. Tuttavia, si evidenzia la necessità di effettuare ulteriori studi sull’utilizzo di tali molecole, sia in vista di un trattamento cronico, sia per comprendere il ruolo di altri metaboliti biologicamente attivi che possono influenzare gli effetti analgesici.
Modulazione del dolore emicranico attraverso due nuovi inibitori del catabolismo dell’anandamide: valutazione degli effetti in un modello animale di emicrania
CORVINO, MARIASOLE
2018/2019
Abstract
The development of intracellular fatty acid amide hydrolase inhibitors (FAAH), that sustain the time of action of anandamide (AEA), the main endocannabinoid, represents a therapeutic option for the treatment of various clinical disorders, including migraine pain. AEA is released on demand during an inflammatory process and it is rapidly degraded by FAAH activity, that generates two inactive metabolites: arachidonic acid and ethanolamine, both of which are devoid of affinity for cannabinoid receptors. It is well known that the systemic administration of nitroglycerin (NTG), a nitric oxide donor, evokes delayed migraine-like headaches in migraineurs. In rodents, NTG administration induces neuronal activation in specific brain areas and a state of hyperalgesia, associated with a reduced pain threshold at the tail flick test, or an increased sensitivity to nociceptive tests (plantar and orofacial formalin test, tail flick test). The present study was aimed at investigating the therapeutic potential of two new FAAH inhibitors, ARN-14633 and ARN-14280, synthesized from URB597, to be a relatively selective global FAAH inhibitor. We tested the effects of the two inhibitors on the NTG-induced hyperalgesia at the orofacial formalin test, in the animal model of migraine through the quantification of nocifensive behavior and gene expression of some mediators, involved in pain transmission and inflammation. Sprague Dawley rats received ARN-14633 (1 mg/g, i. p.) or ARN-14280 (3 mg/kg, i. p. ) or vehicle 3 hours after the administration of NTG (10mg/kg, i.p.) or NTG vehicle, in order to identify a possible abortive effect. All rats were sacrificed 4 h after NTG or vehicle to evaluate: (i) response in the orofacial formalin test (ii) and mRNA expression levels (c-fos, neuropeptides, nNOS and cytokines) in specific central and peripheral areas (cervical cord and trigeminal ganglion ipsilateral to formalin injection, medulla-pons in toto) by rtPCR analysis. The latency of 4 h for NTG injection was selected in agreement with previous observations showing that it corresponds to the timing of maximal neuronal activation in brain areas relevant for migraine. The findings obtained showed that both FAAH inhibitors significantly reduced the NTG-induced hyperalgesia in the second phase of the orofacial formalin test. This behavioral response was associated with a significant decrease in gene expression of c-Fos, a neuronal activation marker, induced by NTG administration in all the evaluated areas. In addition, this reduction was accompanied by a decrease in CGRP and SP mRNA levels in all the areas, although with different entities. Finally, the gene expression levels of pro-inflammatory cytokines (IL-1beta, Il-6, TNF-alpha), significantly increased by NTG administration in the same areas, were reduced by the FAAH inhibitors, with a stronger effect of ARN-14633. By contrast, no significant effects were found in baseline conditions (in the absence of NTG). The results obtained in this study promote the research of FAAH inhibitors in migraine pain, offering a valid therapeutic approach. However, there is still a need for further studies on the use of these molecules, either for chronic treatment and to understand the role of other biologically active metabolites that may influence analgesic effects.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/19795