Rosalia Bertorelli

CB2 receptor-mediated antihyperalgesia: possible direct involvement of neural mechanisms

In mouse the cannabinoid receptor 2 (CB2) agonists L768242 and (+)‐AM1241, at doses of 30 mg/kg i.p. and 1 and 3 mg/kg i.v., respectively, reduced the second phase of nocifensive behaviors elicited by formalin intraplantar injection. This effect was counteracted by the selective CB2 antagonist SR144528 (1 mg/kg i.p.). In rat (+)‐AM1241 (3 and 6 mg/kg i.v.) and L768242 (30 mg/kg i.p.) reduced allodynia elicited by L5–L6 spinal nerve ligation. SR144528 reverted these effects, supporting a CB2‐mediated action. To clarify the mechanisms underlying these effects we investigated CB2 gene expression and function in the nervous system. CB2 mRNA was expressed in spinal cord and dorsal root ganglia (DRG) of both sham and neuropathic rats and was up‐regulated in the ipsilateral spinal cord of neuropathic rats. Expression studies demonstrated the presence of CB2 mRNA in culture of spinal cord microglia. A biomarker, CGRP, was used to investigate modulation of DRG primary afferents by CB2 agonists. Both L768242 and (+)‐AM1241 dose dependently (EC50 of 3.6 and 4.5 nm, respectively) reduced capsaicin‐induced calcitonin gene‐related peptide (CGRP) release. Coadministration of SR144528 resulted in a rightforward shift (pKB 8.1 and 8.2 for (+)‐AM1241 and L768242, respectively) of the dose–response curve. Experiments on capsaicin‐induced CGRP release in tissue from CB1–/– mice ruled out a CB1‐mediated effect. These results confirm that CB2 is present in the central nervous system and suggest that CB2 agonists may elicit their analgesic effect by acting not only at non‐neuronal peripheral sites but also at neural level, making CB2 an attractive target for chronic pain treatment.

Adenosine A2A receptor antagonists are potential antidepressants : evidence based on pharmacology and A2A receptor knockout mice

Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce ‘depressant’ effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine‐mediated ‘depressant’ effect. We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity. Adenosine A2A receptor knockout mice were found to be less sensitive to ‘depressant’ challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 – 10 mg kg−1, i.p.) and KW 6002 (0.1 – 10 mg kg−1, p.o.) reduced the total immobility time in the tail suspension test. The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 – 10 mg kg−1) and ZM 241385 (15 – 60 mg kg−1) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg−1 reduced immobility of mice that were selectively bred for their spontaneous ‘helplessness’ in this assay. Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg−1 reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg−1 by 75 and 79%, respectively. Administration of the dopamine D2 receptor antagonist haloperidol (50 – 200 μg kg−1 i.p.) prevented the antidepressant‐like effects elicited by SCH 58261 (10 mg kg−1 i.p.) in forced swim test whereas it left unaltered its stimulant motor effects. In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape‐directed behaviour in two screening tests for antidepressants. Altogether the results support the hypothesis that blockade of the adenosine A2A receptor might be an interesting target for the development of effective antidepressant agents.

A catalytically silent FAAH-1 variant drives anandamide transport in neurons.

The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, that remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), that lacked amidase activity but bound anandamide with low micromolar affinity and facilitated its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, blocked these effects. We also identified a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, that prevented anandamide internalization in vitro, interrupted anandamide deactivation in vivo and exerted profound analgesic effects in rodent models of nociceptive and inflammatory pain, which were mediated by CB1 cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.

Characterization of the Potent and Highly Selective A2A Receptor Antagonists Preladenant and SCH 412348 [7-[2-[4-2,4-Difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in Rodent Models of Movement Disorders and Depression

The adenosine A2A receptor has been implicated in the underlying biology of various neurological and psychiatric disorders, including Parkinsons disease (PD) and depression. Preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] are potent competitive antagonists of the human A2A receptor (Ki = 1.1 and 0.6 nM, respectively) and have >1000-fold selectivity over all other adenosine receptors, making these compounds the most selective A2A receptor antagonists reported to date. Both compounds attenuate hypolocomotion induced by the A2A receptor agonist CGS-21680 [2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine], suggesting that they inhibit A2A receptor activity in vivo. Their high degree of selectivity and robust in vivo activity make preladenant and SCH 412348 useful tools to investigate the role of the A2A receptor system in animal models of PD and depression. Oral administration of preladenant and SCH 412348 (0.1–1 mg/kg) to rats potentiated 3,4-dihydroxy-l-phenylalanine (l-Dopa)-induced contralateral rotations after 6-hydroxydopamine lesions in the medial forebrain bundle and potently attenuated the cataleptic effects of haloperidol. Preladenant (1 mg/kg) inhibited l-Dopa-induced behavioral sensitization after repeated daily administration, which suggests a reduced risk of the development of dyskinesias. Finally, preladenant and SCH 412348 exhibited antidepressant-like profiles in models of behavioral despair, namely the mouse tail suspension test and the mouse and rat forced swim test. These studies demonstrate that preladenant and SCH 412348 are potent and selective A2A receptor antagonists and provide further evidence of the potential therapeutic benefits of A2A receptor inhibition in PD (with reduced risk of dyskinesias) and depression (one of the primary nonmotor symptoms of PD).

The selective A2A receptor antagonist SCH 58261 reduces striatal transmitter outflow, turning behavior and ischemic brain damage induced by permanent focal ischemia in the rat.

Adenosine A(2A) receptor antagonists have been proved protective in different ischemia models. In this study we verified if the protective effect of the selective A(2A) antagonist, SCH 58261, could be attributed to the reduction of the excitatory amino acid outflow induced by cerebral focal ischemia. A vertical microdialysis probe was inserted into the striatum of male Wistar rats and, after 24 h, permanent right intraluminal middle cerebral artery occlusion (MCAo) was induced. Soon after waking, rats showed a definite contralateral turning behavior, which persisted up to 7 h after MCAo. During 4 h after MCAo, glutamate, aspartate, GABA, adenosine and taurine outflow increased. SCH 58261 (0.01 mg/kg, i.p.), administered 5 min after MCAo, suppressed turning behavior and significantly reduced the outflow of glutamate, aspartate, GABA and adenosine. At 24 h after MCAo, the rats showed severe sensorimotor deficit and damage in both the striatum and cortex. SCH 58261 significantly reduced cortical damage but did not protect against the sensorimotor deficit. The protective effect of SCH 58261 against turning behavior and increased outflow of excitatory amino acids in the first hours after MCAo suggests the potential utility of selective adenosine A(2A) antagonists when administered in the first hours after ischemia. Furthermore, this study, for the first time, proposes that turning behavior after permanent intraluminal MCAo, be used as a precocious index of neurological deficit and neuronal damage.

Adenosine A2A Receptors and Neuroprotection

The adenosine A2A receptor subtype is one of the four adenosine receptors that have been identified in the mammalian organism. In addition to being found in blood vessels, platelets and polymorphonuclear leukocytes, the A2A receptors are abundant in the central nervous system, especially in the striatum. The recent development of selective A2A receptor ligands, in particular of receptor antagonists, makes it possible to elucidate the function of A2A receptors in normal and altered conditions. Pharmacological studies have shown that A2A receptor antagonists are potentially effective for treatment of neurodegenerative processes such as Parkinsons disease. Their activity is attributed to the close anatomical and functional links between A2A receptors and dopaminergic pathways in the basal ganglia. More recently, A2A receptor antagonists have proved to be active in models of cerebral ischemia. While the mechanisms underlying the role of A2A receptors in the hypoxia/ ischemia processes remains to be clarified, it is recognized that A2A receptor antagonists counteract the effects of excitatory aminoacids, which are massively released after cerebral ischemia. Another function of A2A receptors is related to protection from seizures, but further studies are needed to elucidate their specific interaction, if any, with neuronal excitability. Altogether, the great advance recently made with the discovery of selective A2A receptor ligands provides increasing information on the function of A2A receptors and opens new perspectives for treatment of neurological disorders.

Serotonergic Facilitation of Acetylcholine Release In Vivo from Rat Dorsal Hippocampus via Serotonin 5-HT3 Receptors

Abstract: The serotonin (5‐HT) releaser d‐fenfluramine and its active metabolite d‐norfenfluramine, or the 5‐HT‐uptake inhibitor citalopram, by increasing synaptic 5‐HT availability, facilitated in vivo release of acetylcholine (ACh) from dorsal hippocampi of freely moving rats as determined by the microdialysis technique. The effects of d‐norfenfluramine (7.5 mg/kg i.p.) and citalopram (10 μM, applied by reverse dialysis) were prevented by a 14‐day chemical lesion of the raphe nuclei, suggesting mediation by the 5‐HT system in the cholinergic action of the drugs. The increase in extracellular ACh content induced by d‐norfenfluramine (5 mg/kg i.p.) was antagonized by the 5‐HT3 receptor antagonists tropisetron (0.5 mg/kg i.p.) and DAU 6215 (60 μg/kg i.p.), but not by the mixed 5‐HT1 and 5‐HT2 receptor antagonist metergoline (2 mg/kg s.c.). In accordance with an involvement of the 5‐HT3 receptor in the ACh facilitation induced by d‐norfenfluramine is the finding that the selective 5‐HT3 receptor agonist 2‐methyl‐serotonin (250 μg i.c.v., or 10 μM applied by reverse dialysis) raised ACh release. The effect of the intracerebroventricular drug was prevented by the 5‐HT3 antagonists DAU 6215 (60 μg/kg i.p.) and ondansetron (60 μg/kg s.c.). These antagonists by themselves did not modify the basal ACh release, indicating that 5‐HT does not tonically activate the 5‐HT3 receptors involved. In conclusion, the overall regulatory control exerted by 5‐HT in vivo is to facilitate hippocampal ACh release. This is mediated by 5‐HT3 receptors probably located in the dorsal hippocampi.

Up-regulation of ORL-1 receptors in spinal tissue of allodynic rats after sciatic nerve injury

Nociceptin, acting through the opioid receptor-like 1 (ORL1) receptor, produces anti-nociception in several models of neuropathy. We examined the involvement of the ORL1 receptor system in the allodynia developed after sciatic nerve ligation. Allodynic rats were selected by the von Frey hair and treated intrathecally with nociceptin or morphine. The peptide induced dose-dependent anti-allodynic activities, while morphine was effective at the higher dose only. By the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay, the two described ORL1 receptor isoforms were up-regulated in the allodynic animals, but unmodified in non-allodynic rats. Both short and long ORL1 receptor mRNA isoforms increased in the ipsilateral lumbar enlargement (by 50% and 100%, respectively), while 50% and 60% increases were found in the ipsilateral L5-L6 dorsal root ganglia, respectively. No significant changes were observed for either the nociceptin precursor or mu-opioid receptor expression. Thus, the ORL1 receptor system seems to regulate the mechano-allodynia that developed after nerve damage, suggesting its potential role in the treatment of neuropathic pain.

Antinociceptive effects of the N-acylethanolamine acid amidase inhibitor ARN077 in rodent pain models.

TOC summary The N‐acylethanolamine acid amidase inhibitor ARN077 exerts profound antinociceptive effects in animal pain models by enhancing endogenous lipid signaling at peroxisome proliferator‐activated receptor‐α. N‐acylethanolamine acid amidase may be a new target for analgesic drugs. ABSTRACT Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator‐activated receptor‐α (PPAR‐α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N‐acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti‐inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA‐mediated FAE deactivation might regulate pain signaling. The present study examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose‐dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR‐α antagonist GW6471 and did not occur in PPAR‐α–deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR‐α.

Nociceptin and the ORL‐1 ligand [Phe1ψ (CH2‐NH)Gly2]nociceptin(1‐13)NH2 exert anti‐opioid effects in the Freund's adjuvant‐induced arthritic rat model of chronic pain

Stimulation of the opioid receptor‐like1 (ORL‐1) receptor by nociceptin (NC) produces hyperalgesia and reverses the antinociceptive effects induced by opioids. Most studies concerning the central effects of NC were conducted using acute pain models. The role NC may play in chronic inflammation remains unelucidated. The present study was undertaken to assess the action of NC in the Freunds adjuvant‐induced monoarthritic rat model. The effects of drugs known to act as analgesics in this model were evaluated. The effects of NC, NCNH2, and the ORL‐1 ligand, [Phe1ψ(CH2‐NH)Gly2]NC(1‐13)NH2 ([F/G]NC(1‐13)NH2), were also studied alone or in association with morphine. NC (1–30 nmol, i.c.v.) was inactive, whilst NCNH2 (10 nmol, i.c.v.) exerted hyperalgesic effects (−4.5±0.9 vs −0.7±0.8 s of vehicle‐treated animals). [F/G]NC(1‐13)NH2 (0.01–10 nmol, i.c.v.) induced hyperalgesia in the arthritic paw (−3.3±0.6 vs −0.3±0.5 s of vehicle‐treated animals; 10 nmol). Both NC (0.01–10 nmol, i.c.v.) and [F/G]NC(1‐13)NH2 (0.01–1 nmol, i.c.v), 30 min after morphine (3 mg kg−1, s.c.) induced an immediate and short‐lived reversal of morphine effects (2.6±0.3 vs 10.4±1.0 and 1.2±1.5 vs 9.3±1.1 s of morphine alone, respectively), therefore displaying anti‐opioid activity. In the Freunds adjuvant‐induced rat model of arthritis, both NC and [F/G]NC(1‐13)NH2 act as anti‐opioid peptides. Furthermore, NCNH2 and [F/G]NC(1‐13)NH2 induce hyperalgesia when given alone. Further investigations and the identification of a centrally acting ORL‐1 antagonist are necessary to better understand the role of NC in pain mechanisms.