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The association of opioids and non-steroidal anti-inflammatory drugs, to enhance pain relief and reduce the development of side effects, has been demonstrated. Given many reports concerning the antinociceptive and anti-inflammatory effects of Harpagophytum procumbens extracts, the aim of our study was to investigate the advantage of a co-administration of a subanalgesic dose of morphine preceded by a low dose of H. procumbens to verify this therapeutically useful association in a neuropathic pain model. Time course, registered with the association of the natural extract, at a dose that does not induce an antinociceptive effect, followed by a subanalgesic dose of morphine showed a well-defined antiallodynic and antihyperalgesic effect, suggesting a synergism as a result of the two-drug association. H. procumbens cooperates synergistically with morphine in resolving hyperalgesia and allodynia, two typical symptoms of neuropathic pain. The results support the strategy of using an adjuvant drug to improve opioid analgesic efficacy.

Harpagophytum procumbens (H. procumbens), also known as Devil's Claw, has been used to treat a wide range of pathological conditions, including pain, arthritis and inflammation. Inflammatory mediators, released at the site of injury, can sensitize nociceptive terminals and are responsible for allodynia and hyperalgesia. Carbon monoxide (CO), produced in a reaction catalyzed by the enzyme heme oxygenase (HO), may play a role in nociceptive processing and has also been recognized to act as a neurotransmitter or neuromodulator in the nervous system. This study was designed to investigate whether the HO/CO pathway is involved in the analgesic response of H. procumbens in carrageenan-induced hyperalgesia in rats. Mechanical allodynia and thermal hyperalgesia were evaluated by using von Frey filaments and the plantar test, respectively. The results of our experiments showed that pretreatment with the HO inhibitor ZnPP IX significantly decreased the antihyperalgesic effect produced by H. procumbens (800 mg/kg, i.p.) in carrageenan-injected rats. Consistently, the pretreatment with hemin, a HO-1 substrate, or CORM-3, a CO releasing molecule, before a low dose of H. procumbens (300 mg/kg, i.p.) induced a clear antiallodynic response in carrageenan injected rats. These results suggest the involvement of HO-1/CO system in the antiallodynic and antihyperalgesic effect of H. procumbens in carrageenan-induced inflammatory pain.

Objective and Design: The sigma 1 (σ1) receptor, which is widely distributed in the CNS in areas that are known to be important for pain control, may play a role in persistent pain characterized by the hypersensitivity of nociceptive transmission. Here, we investigated the effect of σ1 blockade in an inflammatory pain model., Treatment and Methods: An intraplantar injection of carrageenan (2 %) was used to induce paw inflammation. The effects of the σ1 antagonist (+)-MR200, given subcutaneously at a dose of 0.1, 0.25, 0.5,1, 1.5, and 2 mg/kg prior to injection of carrageenan, on inflammatory pain and inflammation were assessed. Mechanical allodynia with von Frey filaments, thermal hyperalgesia with the plantar test and edema evaluation with a plethysmometer were measured. Intergroup comparisons were assessed by one- or two-way analysis of variance when appropriate, followed by post-hoc tests (Dunnett's test for one-way or Bonferroni for two-way ANOVA)., Results: (+)-MR200 dose-dependently prevented allodynia and hyperalgesia induced by carrageenan. Furthermore, it reduced paw edema with a significant inhibition percentage of 37.82 % at 3 h after carrageenan treatment., Conclusions: The blockade of the σ1 receptor with the selective antagonist (+)-MR200 may contribute to the suppression of the typical symptoms of inflammatory pain.

The compound (−)-MRV3 [(−)-Methyl (1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-methyl]-1-phenylcyclopropanecarboxylate] has an assessed antagonistsigma 1 (σ1) profile and showed improved σ1/σ2 selectivity with respect to the parent compound(+)-MR200. The σ1 receptor is reported to play arole in both central sensitization and pain hypersensitivity,which suggests a potential use of σ1 antagonists forthe treatment of persistent pain conditions. The present study was performed to assess the effects of theselective σ1 antagonist (−)-MRV3, in carrageenan-inducedinflammatory hyperalgesia, allodynia and edema.Mechanical allodynia with a series of calibratedvon Frey’s filaments, thermal hyperalgesia with plantartest and edema evaluation with a plethysmometerwere measured. Subcutaneous (s.c.) treatment with(−)-MRV3 (1, 2, 3, 4, 5 mg/kg) dose-dependentlyreduced allodynia and hyperalgesia induced byintraplantar carageenan. Furthermore, treatment with(−)-MRV3 (3 mg/kg s.c.) also inhibited paw edemawith a significant inhibition of 61.53 % 3 h aftercarrageenan treatment [corrected]. These results provide a strongbasis for the use of σ1 receptor antagonists in thetreatment of inflammatory pain.

Persistent pain states, such as those caused by nerve injury or inflammation, are associated with altered sensations, allodynia and hyperalgesia, that are resistant to traditional analgesics. A contribution to development and maintenance in altered pain perception comes from nociceptive processing and descending modulation from supraspinal sites. A multitarget ligand seems to be useful for pain relief with a decreased risk of adverse events and a considerable analgesic efficacy. The multitarget MOR agonist-DOR antagonist LP1, (3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benazocin-3(2H)-yl]-N-phenylpropanamide, is a central acting antinociceptive agent with low potential to induce tolerance. LP1 was tested in models of neuropathic pain - induced by chronic constriction injury (CCI) of the left sciatic nerve - and inflammatory pain - produced by intraplantar injection of carrageenan. In CCI rats, subcutaneous (s.c.) LP1 (3 mg/kg) showed a significant antiallodynic effect, measured with von Frey filaments, and antihyperalgesic effect, evoked in response to a radiant heat stimulus with plantar test. Analogously, LP1 significantly reduced allodynic and hyperalgesic thresholds in a model of inflammatory pain induced by carrageenan. To evaluate the contribution of opioid receptor subtypes in LP1 antinociceptive effects, the multitarget LP1 profile was assessed using selective opioid antagonists. Moreover, functional electrophysiological in vitro assays, using primary cortical and spinal cord networks, allowed to define the "pharmacological fingerprint" of LP1. The EC₅₀ values in this functional screening seem to confirm LP1 as a potent opioid ligand (EC₅₀ = 0.35 fM and EC₅₀ = 44 pM in spinal cord and frontal cortex, respectively). Using a NeuroProof data-base of well characterised reference compounds, a similarity profile of LP1 to opioid and non-opioid drugs involved in pain modulation was detected. Our studies seem to support that multitarget ligand approach should be useful for persistent pain conditions in which mechanical allodynia and thermal hyperalgesia are significant components of the nociceptive response., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Herein we report the synthesis of new bifunctional sigma-1 (σ1)-selective ligands with antioxidant activity. To achieve this goal, we combined the structure of lipoic acid, a universal antioxidant, with an appropriate sigma aminic moiety. Ligands 14 and 26 displayed high affinity and selectivity for σ1 receptors (Kiσ1 = 1.8 and 5.5 nM; Kiσ2/σ1 = 354 and 414, respectively). Compound 26 exhibited in vivo antiopioid effects on kappa opioid (KOP) receptor-mediated analgesia. In rat liver and brain mitochondria (RLM, RBM), this compound significantly reduced the swelling and the oxidation of thiol groups induced by calcium ions. Our results demonstrate that the tested compound has protective effects against oxidative stress.

Aims: Opioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects., Main Methods: We explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague-Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist., Key Findings: Data obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia., Significance: LP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The neuropeptide Substance P (SP), that has a high affinity for the neurokinin 1 (NK1) receptor, is involved in modulation of pain transmission. Although SP is thought to have excitatory actions and promote nociception in the spinal cord, the peptide induces analgesia at the supraspinal level. The aim of this study was to evaluate the role of supraspinal SP and the NK1 receptor in inflammatory pain induced by injection of carrageenan in the hind paw of the rat. There are two nociceptive behavioral responses associated with this pain state: mechanical allodynia and heat hyperalgesia. Because the NK1 receptor colocalizes with the MOP receptor in supraspinal sites involved in pain modulation, we also decided to study the possible involvement of the opioid system on SP-induced analgesia. We found that treatment with SP, at doses of 3.5, 5 and 7 μg/5 μl/rat i.c.v., clearly showed inhibition of allodynia and hyperalgesia. Pretreatment with the selective NK1 antagonist L-733,060 (10mg/kg i.p.) blocked the SP-induced analgesia, suggesting the involvement of the NK1 receptor. This SP-induced analgesia was significantly reduced by administration of the opioid antagonist naloxone (3mg/kg s.c.). This reduction occurred when SP was administered either before or after the carrageenan injection. These results suggest a significant antinociceptive role for SP and the NK1 receptor in inflammatory pain at the supraspinal level, possibly through the release of endogenous opioids., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Powerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR. The aim of this work was to evaluate and compare the induction of tolerance to antinociceptive effects from treatment with LP1 and morphine., Main Methods: Here, we evaluated the pharmacological effects of LP1 administered at a dose of 4 mg/kg subcutaneously (s.c.) twice per day for 9 days to male Sprague-Dawley rats. In addition, the LP1 mechanism of action was assessed by measurement of LP1-induced [(35)S]GTPγS binding to the MOR and DOR., Key Findings: Data obtained from the radiant heat tail flick test showed that LP1 maintained its antinociceptive profile until the ninth day, while tolerance to morphine (10mg/kg s.c. twice per day) was observed on day 3. Moreover, LP1 significantly enhanced [(35)S]GTPγS binding in the membranes of HEK293 cells expressing either the MOR or the DOR., Significance: LP1 is a novel analgesic agent for chronic pain treatment, and its low tolerance-inducing capability may be correlated with its ability to bind both the MOR and DOR., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Nocistatin (NST) and Nociceptin/Orphanin FQ (N/OFQ) are derived from the same precursor protein, pre-proN/OFQ, and exert opposite effects on the modulation of pain signals. However, the role of the peripheral N/OFQ and the NOP receptor, which is located at the endings of sensory nerves, in inflammatory pain was not ascertained. NST administered intrathecally (i.t.) prevented the nociceptive effects induced by i.t. N/OFQ and PGE₂. Moreover an up regulation of N/OFQ was shown in the rat in response to peripheral inflammation. Here, we investigated the effects of intraplantar (i.pl.) administration of functional N/OFQ and NOP receptor antagonists in a rat model of inflammatory pain. Our findings showed that i.pl. injection of (±)-J 113397, a selective antagonist of the NOP receptor, and NST, the functional N/OFQ antagonist, prior to carrageenan significantly reduced the paw allodynic and thermal hyperalgesic threshold induced by the inflammatory agent. The resulting antiallodynic and antihyperalgesic effects by co-administering NST and (±)-J 113397 prior to carrageenan were markedly enhanced, and the basal latencies were restored. Thus, it is likely that the peripheral N/OFQ/NOP receptor system contributes to the abnormal pain sensitivity in an inflammatory state., (Copyright © 2011. Published by Elsevier Inc.)

Novel enantiomers and diastereoisomers structurally related to σ ligand (+)-MR200 were synthesized to improve σ(1)/σ(2) subtype selectivity. The selective σ(1) ligand (-)-8 showed an antagonist profile determined by phenytoin differential modulation of binding affinity in vitro, confirmed in vivo by an increase of κ opioid analgesia. The σ(2) ligand (-)-9 displayed agonist properties in an in vitro isolated organ bath assay and antiproliferative effects on LNCaP and PC3 prostate cancer cell lines.

Complex mechanisms of prostate cancer progression prompt to novel therapeutic strategies concerning a combination of drugs or of single molecules able to interact with more crucial targets. Histone deacetylase inhibitors and sigma ligands with mixed σ(1) antagonist and σ(2) agonist properties were proposed as new potential tools for treatment of prostate cancer. (±)-MRJF4 was synthesized as phenylbutyrate ester of haloperidol metabolite II, which is a molecule consisting of a histone deacetilase inhibitor (4-phenylbutyric acid) and a sigma ligand (haloperidol metabolite II). Antiproliferatives activities of 4-phenylbutyric acid, haloperidol metabolite II, equimolar mixture of both compounds and (±)-MRJF4 were evaluated in vitro on LNCaP and PC3 prostate cancer cells. Preliminary binding studies of (±)-MRJF4 for σ(1), σ(2), D(2) and D(3) receptors and inhibition HDAC activity were reported. MTT cell viability assays highlighted a notable increase of antiproliferative activity of (±)-MRJF4 (IC(50) = 11 and 13 μM for LNCaP and PC3, respectively) compared to 4-phenylbutyric acid, haloperidol metabolite II and the respective equimolar pharmacological association. (±)-MRJF4 was also used in combination with σ(1) agonist (+)-pentazocine and σ(2) antagonist AC927 in order to evaluate the role of σ receptor subtypes in prostate cancer cell death., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

The enantiomers of cis-(+/-)-methyl (1R,2S/1S,2R)-2-[(4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl)cyclopropanecarboxylate [1, (+/-)-PPCC], a selective sigma ligand, were synthesized. The (+)- and (-)-enantiomers bind predominantly to sigma(1) receptors and have a reduced sigma(2) affinity. Both individually restore the astroglial oxidative status modified by glutamate, counteracting also transglutaminase-2 overexpression. They exhibited in vivo anti-opioid effects on kappa opioid (KOP) receptor-mediated analgesia. Our findings demonstrate that the enantiomers display mainly sigma(1) agonist activity and that they have neuroprotective effects.

6,7-benzomorphan derivatives, exhibiting different mu, delta, and kappa receptor selectivity profiles depending on the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic agents. In this work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide spacer for the synthesis of a new series of 5,9-dimethyl-2'-hydroxy-6,7-benzomorphan derivatives (12-22). Data obtained by competition binding assays showed that the mu opioid receptor seems to prefer an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer and less hindered amide. Highly stringent features are required for delta receptor interaction, while an N-acetamide spacer and/or bulkier amide could preferentially lead to kappa receptor selectivity. In the propanamide series, compound 12 (named LP1) displayed high mu affinity (Ki=0.83 nM), good delta affinity (Ki=29 nM) and low affinity for the kappa receptor (Ki=110 nM), with a selectivity ratio delta/mu and kappa/mu of 35.1 and 132.5, respectively. Further, in the adenylyl cyclase assay, LP1 displayed a mu/delta agonist profile, with IC50 values of 4.8 and 12 nM at the mu and delta receptors, respectively. The antinociceptive potency of LP1 in the tail-flick test after sc administration in rat was comparable with the potency of morphine (ED50=2.03 and 2.7 mg/kg, respectively), and was totally reversed by naloxone. LP1, possessing a mu/delta agonist profile, could represent a lead in further developing benzomorphan-based ligands with potent in vivo analgesic activity and a reduced tendency to induce side effects., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The ventrolateral periaqueductal gray (vlPAG) is a major site of opioid analgesic action and a key locus for the development of morphine tolerance. Previous experimental evidence supports the hypothesis that the brain synthesizes and secretes neuropeptides, which act as a part of the homeostatic system to attenuate the effects of morphine and endogenous opioid peptides. Among the known antiopioid peptides, nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit various opioid effects, especially analgesia. The present study investigated the effect of NOP receptor blockade on the tolerance to morphine antinociception in the vlPAG. Systemic morphine (10mg/kg s.c. twice per day) induced an antinociceptive effect that diminished significantly on the third day when tolerance developed, as quantified by the tail flick and the hot plate tests. Intra vlPAG (i.vlPAG) administration of the NOP receptor antagonist (+/-)-J 113397 restored the opioid's analgesic effect. When (+/-)-J 113397 was administered beginning the first day preceding each morphine administration, tolerance did not develop, but it appeared if the NOP antagonist had been suspended. These data suggest that the N/OFQ in the vlPAG may play a key role in opioid-induced antinociceptive tolerance., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Nociceptin/Orphanin FQ (N/OFQ) counteracts supraspinal opioid effects and plays a role in antinociceptive morphine tolerance. Therefore, in the present study, the selective mu-opioid agonist [D-Ala(2)-NMe-Phe(4)-Gly-ol(5)]-enkephalin (DAMGO) was used. Repeated injection of DAMGO (1 microg/ 1 microl) into the ventrolateral periaqueductal gray (vlPAG), a key site for the development of opioid tolerance, induced analgesia that lasted up to 3 days. In DAMGO-tolerant rats, injection of the N/OFQ antagonist (+/-)-J 113397 (4 microg/1 microl), into the same site, restored the antinociceptive effect of DAMGO. If (+/-)-J 113397 treatment preceded each DAMGO injection, tolerance did not develop. Inhibition of N/OFQ signaling can reverse and prevent the development of DAMGO tolerance in the vlPAG. The results confirm that N/OFQ acts as a functional opioid antagonist., (2010 S. Karger AG, Basel.)

Aims: It has been well documented that ventrolateral periaqueductal gray (vlPAG) matter is a crucial component of the descending pain modulatory system in the chronic pain condition. The aim of the present study was to identify the role of the vlPAG Nociceptin/Orphanin FQ/NOP receptor system in allodynia, a nociceptive behavioral response associated with chronic pain., Main Methods: We used two animal models of persistent pain: chronic constriction injury (CCI) and inflammation induced by carrageenan. In each, Nociceptin/Orphanin FQ transmission was abolished using UFP-101, a selective NOP receptor antagonist, which was injected into the vlPAG at a dose of 18 microg/1 microl/rat., Key Findings: We found that treatment with the NOP antagonist reversed the decrease in allodynic threshold in CCI rats fourteen days after the ligature, which was the timepoint of the greatest reduction in threshold. Moreover, UFP-101 administered immediately prior to or 2 h after intra plantar (i.pl.) carrageenan injection prevented or reversed, respectively, allodynic behavior in rats with inflammation., Significance: Our findings support the hypothesis that the endogenous Nociceptin/Orphanin FQ/NOP receptor system is tonically active at the vlPAG level during neuropathic states or carrageenan inflammation.

The compound (1R,2S/1S,2R)-2-[4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(+/-)-PPCC] is a ligand with high affinity for sigma (sigma) sites of which the selectivity towards several other receptor systems has been demonstrated. Given the existence of a relationship between the sigma system and the kappa opioid (KOP)-mediated analgesia, to characterize the pharmacological properties of (+/-)-PPCC we analyzed its influence on the analgesic effect of the systemic injected kappa agonist (-)-U-50,488H comparing the effects with those shown by (+)-pentazocine and BD1047. The results demonstrate that the systemic administration of (+/-)-PPCC (1 mg/kg s.c.) does not modify basal tail-flick latency. Pre-treatment with (+/-)-PPCC, at the same dose, significantly decreased the antinociceptive effect of (-)-U-50,488H, analogously to the sigma compounds used. This study confirms that (+/-)-PPCC plays the role of sigma agonist in this model and strengthens the hypothesis of the sigma receptor modulatory role on KOP-mediated analgesia.

There is considerable interest in developing KOP Opioid receptor ligands as clinically useful analgesics. Moreover, compounds with mixed KOP receptor and mu-opioid peptide (MOP) receptor agonist/antagonist properties could have a better therapeutic potential. The benzomorphan-based synthetic ligands MPCB and CCB have been shown to bind KOP receptors with high affinity and selectivity. We report here a series of compounds synthesized to perform structure-affinity relationship (SAR) studies on MPCB. The aim of this study was to optimise KOP receptor-ligand interaction and to modulate MOP receptor selectivity. In the benzylamide analogue of MPCB (compound 9) the presence of a third aromatic nucleus, at an appropriate distance and conformation with respect to aromatic pharmacophoric residues, increased KOP receptor affinity by about 6-fold compared to MPCB (Ki = 35 nM and Ki = 240 nM, respectively). Instead, compound 28 with a tertiary amino group in the nitrogen substituent displayed a comparable KOP receptor affinity (Ki = 179 nM) but also high MOP receptor affinity (Ki = 45 nM). Thus, the present study shows that in benzomorphan-based ligands the presence of different functional groups in the nitrogen substituent, ranging from a positive charged amine to an additional aromatic ring, is able to promote the correct aligment of aromatic pharmacophoric residues with MOP and KOP receptor types. Evaluation of docking simulations of compounds 9 and 28 into the KOP and MOP receptor displayed selective ligand interactions with the important amino acid residues Tyr320 (TMVII) and Trp318 (TMVII), respectively.

Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are involved in various biological functions including pain. High density of NOP receptor has been found in the ventrolateral periaqueductal gray (vlPAG), the main output pathway involved in descending pain-control system. The aim of our work was to evaluate the involvement of the N/OFQ/NOP system in the modulation of MOP analgesia in the rat vlPAG using UFP-101, a selective NOP antagonist. N/OFQ significantly blocked DAMGO (a selective MOP agonist) analgesia, while UFP-101 enhanced the effect of the opioid given at a subanalgesic dose. These results confirm our hypothesis of an antiopioid role for N/OFQ in the vlPAG.

The proapoptotic cytokine TRAIL has been shown to enhance amyloid-beta-dependent neurotoxicity. Here are reported interactions between TRAIL and nitric oxide (NO) in cultured rat astrocytes in vitro. Rat astrocytes expressed all TRAIL receptor mRNAs and proteins. However, TRAIL failed in inducing apoptosis of astrocytes, whereas these cells released substantial amounts of nitrites. A TRAIL-neutralizing antibody was able to prevent LPS-induced iNOS expression in astrocytes. Interestingly, TRAIL induced its own expression in astrocytes. These data suggest that redundancy between TRAIL and NO in astrocytes could be fueling neuronal damage/death processes, potentially uncovering novel molecular targets for the treatment of neurodegenerative disorders.

Tumour growth is tightly related to new blood vessel formation, tissue remodelling and invasiveness capacity. A number of tissular factors fuel the growth of glioblastoma multiforme, the most aggressive brain neoplasm. In fact, gene array analyses demonstrated that the proapoptotic cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) inhibited mRNA expression of VEGF, along with those of matrix metalloproteinase-2 (MMP-2), its inhibitor tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), as well as the tumour invasiveness-related gene secreted protein acid rich in cysteine (SPARC) in different human glioblastoma cell lines. Particularly, VEGF mRNA and protein expression and release from glioblastoma cells were also inhibited by TRAIL. The latter also exerted antimitogenic effects on human umbilical vein endothelial cells (HUVECs). With the same cells, TRAIL inhibited new vessel formation in the in vitro matrigel model, as well as it exerted powerful inhibition of blood vessel formation induced by an angiogenic cocktail administered in subcutaneous pellets in vivo in the C57 mouse. Moreover, the expression of MMP-2, its inhibitor TIMP-2 and the tumour invasiveness-related protein SPARC were effectively inhibited by TRAIL in glioblastoma cell lines. In conclusion, our data indicate that TRAIL inhibits the orchestra of factors contributing to glioblastoma biological aggressiveness. Thus, the TRAIL system could be regarded as a molecular target to exploit for innovative therapy of this type of tumour.

The compound (+)-MR200 [(+)-methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate] is a sigma ligand with increased affinity and selectivity compared to the structurally related ligand haloperidol. From the results of a previous study on the modulation of a systemically injected KOP opioid agonist analgesia by (+)-MR200, we analysed the influence of this sigma ligand on the antinociceptive effect of centrally injected MOP, DOP, and KOP selective agonists using the tail-flick test in rats. The results obtained confirmed that systemic administration of (+)-MR200 (1mg/Kg s.c.) did not modify basal tail-flick latency. Pre-treatment with 1mg/Kg s.c. of (+)-MR200 provided a significant increase in the antinociceptive effect of DAMGO (100ng/rat i.c.v.) and DPDPE (20 microg/rat i.c.v.). Conversely to previous reports, pre-treatment with (+)-MR200 reversed, in these experimental conditions, U-50488H (100 microg/rat i.c.v.) analgesia. The mechanism involved in these effects was not clear, but provided additional data on a diverging modulator role of selective sigma-1 antagonists on KOP analgesia.

Nocistatin and Nociceptin/Orphanin FQ are two neuropeptides derived from the same precursor protein, pre-pro-Nociceptin. Nocistatin does not bind to Nociceptin/Orphanin FQ peptide (NOP) receptor but it antagonizes the allodynic and hyperalgesic effect of intrathecal (i.t.) Nociceptin. In this study, we examined the effect of Nocistatin on nociception and opioid analgesia by itself and the nociceptive effect of Nociceptin and antagonistic effect of nociceptin on opioid receptors in tail flick test when given the i.c.v. route. More precisely, supraspinal Nocistatin by itself had no significative effect on nociception and opioid analgesia in the tail flick test but, at the dose of 0.5ng/rat, it reversed the nociceptive effect of Nociceptin and also the antagonistic effect of Nociceptin against analgesia caused by the selective opioid agonists: DAMGO, DPDPE, Deltorphin II and U50 488H. These data suggest that Nocistatin antagonizes the effect of Nociceptin on opioid analgesia and could play an important role in the regulation of nociceptive transmission.

Glucocorticoids (GCs) exert via glucocorticoid receptors (GRs) potent anti-inflammatory and immunosuppressive effects. Emerging evidence indicates that an inflammatory process is involved in dopaminergic nigro-striatal neuronal loss in Parkinson's disease. We here report that the GR deficiency of transgenic (Tg) mice expressing GR antisense RNA from early embryonic life has a dramatic impact in "programming" the vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The GR deficiency of Tg mice exacerbates MPTP-induced toxicity to dopaminergic neurons, as revealed by both severe loss of tyrosine hydroxylase positive nigral neurons and sharp decreases in striatal levels of dopamine and its metabolites. In addition, the late increase in dopamine oxidative metabolism and ascorbic acid oxidative status in GR-deficient mice was far greater than in wild-type (Wt) mice. Inducible nitric oxide synthase (iNOS) was sharply increased in activated astrocytes, macrophages/microglia of GR-deficient as compared with Wt mice. Moreover, GR-deficient microglia produced three- to fourfold higher nitrite levels than Wt mice; these increases preceded the loss of dopaminergic function and were resistant to GR the inhibitory effect of GC, pointing to peroxynitrites as candidate neurotoxic effectors. The iNOS inhibitor N6-(1-iminoethyl)-L-lysine normalized vulnerability of Tg mice, thus establishing a novel link between genetic impairment of GR function and vulnerability to MPTP.

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1beta (IL-1beta), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly (S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1beta induced cartilage destruction.

Bidirectional communication between the neuroendocrine and immune systems plays a pivotal role in health and disease. Signals generated by the hypothalamic-pituitary-gonadal (HPG) axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids) are major players coordinating the development immune system function. Conversely, products generated by immune system activation exert powerful and longlasting effects on HPG axis activity. In the central nervous system (CNS), one chief neuroendocrine-immune (NEI) compartment is represented by the astroglial cell population and its mediators. Of special interest, the major supporting cells of the brain and the thymus, astrocytes and thymic epithelial cells, share a similar origin and a similar set of peptides, transmitters, hormones and cytokines functioning as paracrine/autocrine regulators. This may explain some fundamental analogies in LHRH regulation of both cell types during ontogeny and in adult life. Hence, the neuropeptide LHRH significantly modulates astrocyte and thymic cell development and function. Here we focus this work on LHRH neuron-glial signaling cascades which dictate major changes during LHRH neuronal differentiation and growth as well as in response to hormonal manipulations and pro-inflammatory challenges. The interplay between LHRH, growth factors, estrogens and pro-inflammatory mediators will be discussed, and the potential physiopathological implications of these findings summarized. The overall study highlights the plasticity of this intersystem cross-talk and emphasize neuron-glial interactions as a key regulatory level of neuroendocrine axes activity.

1. Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r-hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro. 2. r-hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl-2. 3. r-hGH restored mitogen-stimulated release of IL-2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r-hGH-dependent IL-2 release. 4. These results demonstrate that r-hGH protects irradiated PBL from death in a specific, receptor-mediated manner. Such effect of r-hGH on PBL involves activation of the antiapoptotic gene bcl-2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer.

The effects of intraperitoneal and intracerebroventricular administration of the inhibitor of endopeptidase EC 24.11 (enkephalinase), thiorphan, and the synthetic enkephalin analogue [D-Ala2-Met5]enkephalinamide (DALA) were investigated in cold-restraint-stressed rats. Drugs were administered alone or after pretreatment with naloxone or naloxone methiodide given 20 min prior to the drugs. Thiorphan and DALA, administered centrally (4 micrograms i.c.v./rat) or peripherally (400 micrograms/kg), induced a significant gastric protection. Prior treatment with naloxone s.c. (1 mg/kg) inhibited the effect induced by i.c.v. or i.p. injections of thiorphan or DALA. In contrast, s.c. administration of naloxone methiodide (1 mg/kg) did not affect the response induced by central administration of thiorphan or DALA, but was able to prevent that of thiorphan or DALA when they were administered i.p. These data strongly support the hypothesis of a central and peripheral involvement of endogenous opioid peptides in gastric protection in stressed rats.

The effects of intraperitoneal and intracerebroventricular administration of mu- and delta- selective opioid receptor agonists (DAGO and DPDPE, respectively) on gastric lesions, were investigated in cold-restraint-stressed rats. DAGO and DPDPE, peripherally and centrally administered, induced a significant gastric protection. Naloxone prevented the effects of both opioids whereas naltrindole prevented the gastric protection induced by DPDPE but not that by DAGO. The results suggest that mu- and delta-opioid agonists prevent gastric damage induced by stress through an involvement of both central and peripheral mu- and delta-opioid receptor subtypes.

Morphine and the synthetic opioid met-enkephalin analog [D-Ala2, MePhe4, Met(0)5ol] enkephalin (FK 33-824) injected intraperitoneally to rats at doses of 5-20 and 0.5-2 mg/kg respectively showed a protective effect on gastric lesion induced by cold-restraint stress. This protective effect was abolished by pretreatment with indomethacin. This suggests a role for prostaglandins in the protection, induced by opioids of the gastric mucosa against the development of stress-induced ulcers.

The endogenous opioid met-enkephalin intraventricularly adminstered to the rat at the dose of 100 microgram raised rectal temperature, whereas 400 microgram of the pentapeptide caused a diphasic effect, i.e., hypothermia followed by hyperthermia. Met-enkephalin was ineffective when administered i.p. The effects on temperature were substantially similar to those elicited, for both routes of administration, by morphine, which may either raise or lower rat temperature depending on the dose. More naloxone was required to antagonize thermic effects of met-enkephalin than morphine. Finally, there was a lack of effects on temperature for met-enkephalin centrally administered to morphine-tolerant animals, thus providing further evidence, in vivo, of cross tolerance between opiates and naturally occurring ligands of opiate receptors.

An enhanced prostaglandinlike activity is shown in homogenates of brain from rats treated intracerebroventricularly with 100 microgram of metenkephalin. The increase is significantly reduced by naloxone pretreatment. A relationship is proposed between generation of prostaglandins in the brain following met-enkephalin administration and hyperthermic effect of the opiatelike factor in the rat. Normalization of prostaglandinlike activity following chronic administration of met-enkephalin in the rat may also account for the development of tolerance to its thermic effect.

The objective of the present study was to obtain information on prostaglandin (PG) biosynthesis in placenta following narcotic administration. PG biosynthetic capacity was determined in whole rat placenta homogenates in the presence of Na arachidonate, by evaluating net production of PGs assayed against PGE1 on rat stomach strips. An enhanced prostaglandin-like activity is shown in homogenates of placenta from rats treated subcutaneously with 10 mg/kg of morphine. This increase was prevented by naloxone pre-treatment. Continual morphine administration during gestation, results in a normalization of placental biosynthetic capacity thus suggesting the development of a tolerance to the narcotic effect.

The effects of morphine HCl and a synthetic met-enkephalin analogue [D-Ala2,MePhe4,Met(O)5ol]enkephalin (FK 33-824) on gastric damage produced by the intraperitoneal administration of indomethacin (10 mg/kg i.p.) have been investigated. Rats intraperitoneally pretreated with morphine HCl (10 mg/kg i.p.) and FK 33-824 (1 mg/kg i.p.) showed a statistically significant reduction both of the number and intensity of lesions induced by indomethacin. This protection was reversed by naloxone HCl (2 mg/kg i.p.). The protective effect was not related to a reduction of gastric secretion since the antisecretory drug cimetidine (25 mg/kg i.p.) and methscopolamine bromide (10 mg/kg i.p.) did not significantly prevent mucosal damage under the same experimental conditions.

An increase was found in the opiate activity of pituitary extracts obtained rom rats injected with the neuroleptic drugs trifluoperazine and sulpiride. The increase of opiate activity, measured by bioassay, was particularly evident 2 h after the administration of sulpiride. Dexamethasone completely prevented the neuroleptic-induced effect. Trifluoperazine and sulpiride may have affected dopaminergic mechanisms regulating endorphin storage in the pituitary.