Gabriella Giagnoni

The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and anti-hyperalgesic effects in a murine model of neuropathic pain: involvement of CB(1), TRPV1 and PPARgamma receptors and neurotrophic factors.

Abstract Palmitoylethanolamide (PEA) is an endogenous lipid that is thought to be involved in endogenous protective mechanisms activated as a result of stimulation of inflammatory response. In spite of the well demonstrated anti‐inflammatory properties of PEA, its involvement in controlling pain pathways still remains poorly characterized. On this basis, we tested the efficacy of PEA in vivo against a peculiar persistent pain, such as neuropathic one. PEA was administered i.p. to mice with chronic constriction injury of sciatic nerve (CCI) once a day for one week starting the day after the lesion. This therapeutic regimen evoked a relief of both thermal hyperalgesia and mechanical allodynia in neuropathic mice. Various selective receptor antagonists were used in order to clarify the relative contribution of cannabinoid, vanilloid and peroxisome proliferator‐activated receptor to PEA‐induced effects. The results indicated that CB1, PPARγ and TRPV1 receptors mediated the antinociception induced by PEA, suggesting that the most likely mechanism might be the so‐called “entourage effect” due to the PEA‐induced inhibition of the enzyme catalyzing the endocannabinoid anandamide (AEA) degradation that leads to an enhancement of its tissue levels thus increasing its analgesic action. In addition, the hypothesis that PEA might act through the modulation of local mast cells degranulation is sustained by our findings showing that PEA significantly reduced the production of many mediators such as TNFα and neurotrophic factors, like NGF. The findings presented here, in addition to prove the beneficial effects of PEA in chronic pain, identify new potential targets for analgesic medicine.

Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat

The antiinflammatory activity of synthetic cannabinoid nabilone in the rat model of carrageenan‐induced acute hindpaw inflammation was compared with that of the endocannabinoid palmitoylethanolamide and the nonsteroidal antiinflammatory drug indomethacin. Preliminary experiments in rats used a tetrad of behavioural tests, specific for tetrahydrocannabinol‐type activity in the CNS. These showed that the oral dose of nabilone 2.5 mg kg−1 had no cannabinoid psychoactivity. Intraplantar injection of carrageenan (1% w v−1) elicited a time‐dependent increase in paw volume and thermal hyperalgesia. Nabilone (0.75, 1.5, 2.5 mg kg−1, p.o.), given 1 h before carrageenan, reduced the development of oedema and the associated hyperalgesia in a dose‐related manner. Nabilone 2.5 mg kg−1, palmitoylethanolamide 10 mg kg−1 and indomethacin 5 mg kg−1, given p.o. 1 h before carrageenan, also reduced the inflammatory parameters in a time‐dependent manner. The selective CB2 cannabinoid receptor antagonist {N‐[(1S)‐endo‐1,3,3‐trimethyl bicyclo [2.2.1]heptan‐2‐yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide} (SR 144528), 3 mg kg−1 p.o. 1 h before nabilone and palmitoylethanolamide, prevented the anti‐oedema and antihyperalgesic effects of the two cannabinoid agonists 3 h after carrageenan. Our findings show the antiinflammatory effect of nabilone and confirm that of palmitoylethanolamide indicating that these actions are mediated by an uncharacterized CB2‐like cannabinoid receptor.

Glial TLR4 Receptor as New Target to Treat Neuropathic Pain: Efficacy of a New Receptor Antagonist in a Model of Peripheral Nerve Injury in Mice

Neuropathic pain remains a prevalent clinical problem because it is often poorly responsive to the currently used analgesics, thus it is crucial the identification of new potential targets and drugs. Recent evidence indicated that microglial cells in the spinal cord are critically involved in the development and maintenance of neuropathic pain, with a pivotal role of toll‐like receptor 4 (TLR4). Binding of an endogenous ligand to TLR4 might be considered an important step in the regulation of microglia activity in pain facilitation, suggesting that a mechanism aimed to inhibit such a binding could be effective against neuropathic pain. We have synthesized new ligands to TLR4 with antagonistic activity. In the present work we evaluated the efficacy of the most potent TLR4 antagonist synthesized by us (FP‐1), administered in mice with painful neuropathy. The repeated treatment of neuropathic mice with FP‐1 (5–10 mg/kg, i.p.) evoked a relief of both thermal hyperalgesia and mechanical allodynia, whereas the administration of the highest dose to TLR4 knockout neuropathic mice revealed that in the absence of TLR4 receptor, the compound lost its efficacy. As consequence of TLR4 binding, the repeated treatment with FP‐1 prevented the activation of the transcription factor NF‐kB and the TNFα overproduction in the spinal cord. Together, our findings support the previous evidence indicative for a contribution of glial TLR4 to the initiation of neuropathic pain, suggest it as potential innovative target to treat this debilitating disease, and propose FP‐1 as lead compound for the development of new effective drugs.

Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid, cannabidiol, in a rat model of acute inflammation

Cannabidiol (CBD), a nonpsychoactive marijuana constituent, was recently shown as an oral antihyperalgesic compound in a rat model of acute inflammation. We examined whether the CBD antihyperalgesic effect could be mediated by cannabinoid receptor type 1 (CB1) or cannabinoid receptor type 2 (CB2) and/or by transient receptor potential vanilloid type 1 (TRPV1). Rats received CBD (10 mg kg−1) and the selective antagonists: SR141716 (N‐(piperidin‐1‐yl)‐5‐(4‐chlorophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) for CB1, SR144528 (N‐[(1S)‐endo‐1,3,3‐trimethylbicyclo[2.2.1]heptan‐2‐yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide) for CB2 and capsazepine (CPZ) for TRPV1 receptors. The intraplantar injection of carrageenan in rats induced a time‐dependent thermal hyperalgesia, which peaked at 3 h and decreased at the following times. CBD, administered 2 h after carrageenan, abolished the hyperalgesia to the thermal stimulus evaluated by plantar test. Neither SR141716 (0.5 mg kg−1) nor SR144528 (3 and 10 mg kg−1) modified the CBD‐induced antihyperalgesia; CPZ partially at the lowest dose (2 mg kg−1) and fully at the highest dose (10 mg kg−1) reversed this effect. These results demonstrate that TRPV1 receptor could be a molecular target of the CBD antihyperalgesic action.

Therapeutic effect of the endogenous fatty acid amide, palmitoylethanolamide, in rat acute inflammation: inhibition of nitric oxide and cyclo‐oxygenase systems

The anti‐inflammatory activity of the endogenous fatty acid amide palmitoylethanolamide and its relationship to cyclo‐oxygenase (COX) activity, nitric oxide (NO) and oxygen free radical production were investigated in the rat model of carrageenan‐induced acute paw inflammation and compared with the nonsteroidal anti‐inflammatory drug (NSAID) indomethacin. Palmitoylethanolamide (1, 3, 5, 10 mg kg−1; p.o.) and indomethacin (5 mg kg−1; p.o.) were administered daily after the onset of inflammation for three days and the paw oedema was measured daily; 24 h after the last dose (fourth day) the rats were killed and the COX activity and the content of nitrite/nitrate (NO2−/NO3−), malondialdehyde (MDA), endothelial and inducible nitric oxide synthase (eNOS and iNOS) were evaluated in the paw tissues. Palmitoylethanolamide had a curative effect on inflammation, inhibiting the carrageenan‐induced oedema in a dose‐ and time‐dependent manner. This effect was not reversed by the selective CB2 receptor antagonist (N‐[(1S)‐endo‐1,3,3‐trimethylbicyclo[2.2.1]heptan‐2yl]‐5‐(4‐chloro‐3‐methylphenyl)‐1‐(4‐methylbenzyl)pyrazole‐3 carboxamide) (SR144528), 3 mg kg−1 p.o. On the fourth day after carrageenan injection, COX activity and the level of NO2−/NO3−, eNOS and MDA were increased in the inflamed paw, but iNOS was not present. Palmitoylethanolamide (10 mg kg−1) and indomethacin markedly reduced these increases. Our findings show, for the first time, that palmitoylethanolamide has a curative effect in a model of acute inflammation. The inhibition of COX activity and of NO and free radical production at the site of inflammation might account for this activity.

Chronic CP-55,940 alters cannabinoid receptor mRNA in the rat brain: an in situ hybridization study.

Using in situ hybridization we found that chronic treatment with CP-55,940 (0.4 mg kg-1, i.p. daily for 11 days), a synthetic cannabinoid receptor ligand, changed cannabinoid receptor mRNA levels in rat brain. CP-55,940 produced the expected tolerance: the decrease in locomotor activity (75%) caused by an acute dose was diminished to 25% after the 11 days of treatment. Thirty minutes after the last injection the animals were killed and in situ hybridization indicated that the levels of cannabinoid receptor mRNA in the caudate-putamen were reduced by 33%, with no alteration in the other brain areas. We suggest that the altered cannabinoid receptor expression is part of the adaptive changes underlying cannabinoid tolerance.

Effect of the cannabinoid CB1 receptor antagonist, SR141716, on nociceptive response and nerve demyelination in rodents with chronic constriction injury of the sciatic nerve.

&NA; Many reports have shown the efficacy of cannabinoid agonists in chronic pain, whereas no report exists concerning the potential effect of cannabinoid antagonists following prolonged treatment. We tested the effects of repeated administration of the selective cannabinoid receptor type 1 (CB1) antagonist, SR141716 (rimonabant), in rats with chronic constriction injury of the sciatic nerve (CCI), an animal model of neuropathic pain. The repeated oral administration of SR141716 (1, 3 and 10 mg/kg, once a day for 1 week, from day 7 after the injury) dose dependently attenuated both thermal and mechanical hyperalgesia. A similar effect was observed in CCI wild‐type mice, whereas SR141716 was unable to elicit pain relief in CB1 knockout mice, suggesting CB1 receptors involvement in the SR141716‐induced antihyperalgesia. The antihyperalgesic activity of SR141716 was associated with a significant reduction of several pro‐inflammatory and pro‐nociceptive mediators such as tumor necrosis factor alpha (TNFα), prostaglandin‐E2 (PGE2), lipoperoxide and nitric oxide (NO) levels. The histological analysis of sciatic nerve sections showed a marked degeneration of myelinated fibers in CCI rats, which was substantially reduced after repeated administration of SR141716. This suggests that the compound may favour myelin repair and consequently promote long‐lasting functional recovery. This was confirmed by the maintenance of recovery for at least four weeks after treatment discontinuation. In conclusion, the present findings suggest that SR141716 is effective not only in alleviating neuropathic pain but also in favouring the nerve myelin repair.

The inhibition of monoacylglycerol lipase by URB602 showed an anti-inflammatory and anti-nociceptive effect in a murine model of acute inflammation.

2‐arachidonoylglycerol (2‐AG) is an endocannabinoid whose hydrolysis is predominantly catalysed by the enzyme monoacylglycerol lipase (MAGL). The development of MAGL inhibitors could offer an opportunity to investigate the anti‐inflammatory and anti‐nociceptive role of 2‐AG, which have not yet been elucidated. On these bases, URB602, a MAGL inhibitor, was tested in a murine model of inflammation/inflammatory pain.

Repeated treatment with the synthetic cannabinoid WIN 55,212-2 reduces both hyperalgesia and production of pronociceptive mediators in a rat model of neuropathic pain

The antinociceptive properties of cannabinoids in persistent pain are not fully elucidated. We investigated the effect of repeated treatment with the synthetic cannabinoid receptor agonist WIN 55,212‐2 on the neuropathic pain induced in rats by chronic constriction of the sciatic nerve. WIN 55,212‐2 administered daily throughout the development of neuropathy reversed the hyperalgesia, at a dose (0.1 mg kg−1, s.c.) that had no effect on the nociceptive responses of either paw contralateral to the sciatic ligation or of animals subjected to sham surgery. At 14 days after injury, the levels of mediators known to be involved in neuropathic pain, such as prostaglandin E2, NO and the neuronal NOS, were increased. Repeated treatment with WIN 55,212‐2 abolished these increases. In the light of the current clinical need for neuropathic pain treatments, these findings indicate that cannabinoid agonists, at doses devoid of psychoactive effects, could constitute important compounds for the development of new analgesics.

AM404, an inhibitor of anandamide uptake, prevents pain behaviour and modulates cytokine and apoptotic pathways in a rat model of neuropathic pain

1 An attractive alternative to the use of direct agonists at the cannabinoid receptor type 1 (CB1) in the control of neuropathic pain may be to potentiate the actions of endogenous cannabinoids. Thus, the effects of AM404, an inhibitor of anandamide uptake, were assessed in an experimental model of neuropathic pain in rats. 2 Daily treatment with AM404 prevented, time‐ and dose‐dependently, the development of thermal hyperalgesia and mechanical allodynia in neuropathic rats. Antagonists at cannabinoid CB1 or CB2 receptors, or at the transient receptor potential vanilloid type 1 receptor, each partially reversed effects induced by AM404. A complete reversal was obtained when the three antagonists were given together, suggesting that all three receptors are involved. 3 AM404 treatment affected two pathways involved in the generation and maintenance of neuropathic pain, one mediated by nitric oxide (NO) and the other by cytokines. AM404 completely prevented the overproduction of NO and the overexpression of nNOS, inhibited the increase in tumour necrosis factor α (TNFα) and enhanced the production of interleukin‐10. Both NO and TNFα are known to contribute to the apoptotic process, which plays an important role in the establishment of chronic pain states. AM404 treatment prevented the increase in the ratio between pro‐ and anti‐apoptotic gene bax/bcl‐2 expression observed in the spinal cord of neuropathic rats. 4 Taken together, these findings suggest that inhibition of endocannabinoid uptake, by blocking the putative anandamide carrier, results in the relief of neuropathic pain and may represent a novel strategy for treating chronic pain.