Structure-Affinity relationships of novel σ2R/TMEM97 ligands.

The sigma 2 receptor (σ2R), which was recently identified as the transmembrane protein 97 (TMEM97), is increasingly attracting interest as a possible therapeutic target for indications in neuroscience. Toward identifying novel modulators of σ2R/TMEM97, we prepared a collection of benzoxazocine, benzomorphan, and methanobenzazepine ligands related to the known bioactive norbenzomorphans DKR-1677, FEM-1689, and EES-1686 and determined their Ki values for σ2R/TMEM97 and the sigma 1 receptor (σ1R). The σ2R/TMEM97 binding affinities and selectivities relative to σ1R of these new benzoxazocine, benzomorphan, and methanobenzazepine analogs are lower, often significantly lower, than their respective norbenzomorphan counterparts, suggesting the spatial orientation of pharmacophoric substituents is critical for binding to the two proteins. The benzoxazocine, benzomorphan, and methanobenzazepine congeners of DKR-1677 and FEM-1689 tend to be weakly selective for σ2R/TMEM97 versus σ1R, whereas EES-1686 derivatives exhibit the greatest selectivity, suggesting the size and/or nature of the substituent on the nitrogen atom of the scaffold may be important for selectivity. Computational docking studies were performed for the 1S,5R-and 1R,5S-enantiomers of DKR-1677, FEM-1689, and EES-1686 and their benzoxazocine, benzomorphan, and methanobenzazepine counterparts. These computations predict that the protonated amino group of each ligand forms a highly conserved salt bridge and a H-bonding interaction with Asp29 as well as a cation-π interaction with Tyr150 of σ2R/TMEM97. These electrostatic interactions are major driving forces for binding to σ2R/TMEM97 and are similar, though not identical, for each ligand. Other interactions within the well-defined binding pocket also tend to be comparable, but there are some major differences in how the hydrophobic aryl groups of various ligands interact with the protein surface external to the binding pocket. Overall, these studies show that the orientations of aryl and N-substituents on the norbenzomorphan and related scaffolds are important determinants of binding affinity of σ2R/TMEM97 ligands, and small changes can have significant effects upon binding profiles.

Benzomorphan and non-benzomorphan agonists differentially alter sigma-1 receptor quaternary structure, as does types of cellular stress.

Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.

Potent MOR Agonists from 2'-Hydroxy-5,9-dimethyl-N-phenethyl Substituted-6,7-benzomorphans and from C8-Hydroxy, Methylene and Methyl Derivatives of N-Phenethylnormetazocine.

(-)-5,9-Dimethyl-6,7-benzomorphan (normetazocine) derivatives with a para-OH or ortho-F substituent in the aromatic ring of the N-phenethyl moiety were synthesized and found to have subnanomolar potency at MOR, and both were fully efficacious in vitro. These new compounds, (1R,5R,9R)-6,11-dimethyl-3-(2-fluorophenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol and (1R,5R,9R)-6,11-dimethyl-3-(4-hydroxyphenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol, were more potent than the unsubstituted compound N-phenethylnormetazocine and about 30 or 40 times more potent than morphine, respectively. A variety of substituents in the ortho, meta, or para position in the aromatic ring of the N-phenethyl moiety were synthesized, 25 of these compounds, and found to have varying effects on potency and efficacy as determined by the forskolin-induced cAMP accumulation assay. The N-phenethyl moiety was also modified by increasing chain length to form a N-phenylpropyl side chain with and without a para-nitro moiety, and by an N-cinnamyl side chain. Also, an indole ethylamine normetazocine was synthesized to replace the N-phenethylamine side chain in normetazocine. The phenylpropylamine, propenylamine (cinnamyl) and the para-nitropropylamine had little or no MOR potency. The indole-ethylamine on the normetazocine nucleus, however, had moderate potency (MOR EC50 = 12 nM), and was fully efficacious (%Emax = 102%) in the cAMP assay. Retention of the N-phenethyl moiety and the addition of alkyl and alkenyl moieties on C8 in (-)-N-phenethylnormetazocine gave a C8-methylene derivative that had subnanomolar potency at MOR and a C8-methyl analog that had nanomolar potency. Five C8-substituted compounds were synthesized.

Design, synthesis, in vitro evaluation, and molecular modeling studies of N-substituted benzomorphans, analogs of LP2, as novel MOR ligands.

6,7-Benzomorphans have been investigated in medicinal chemistry for developing new drugs. This nucleus could be considered a versatile scaffold. The physicochemical properties of benzomorphan N-substituent are crucial in achieving a definite pharmacological profile at opioid receptors. Thus, the dual-target MOR/DOR ligands LP1 and LP2 were obtained through N-substituent modifications. Specifically, LP2, bearing as N-substituent the (2R/S)-2-methoxy-2- phenylethyl group, is a dual-target MOR/DOR agonist and is successful in animal models of inflammatory and neuropathic pain. To obtain new opioid ligands, we focused on the design and synthesis of LP2 analogs. First, the 2-methoxyl group of LP2 was replaced by an ester or acid functional group. Then, spacers of different lengths were introduced at N-substituent. In-vitro, their affinity profile versus opioid receptors has been performed through competition binding assays. Molecular modeling studies were conducted to deeply analyze the binding mode and the interactions between the new ligands and all opioid receptors.

LP1 and LP2: Dual-Target MOPr/DOPr Ligands as Drug Candidates for Persistent Pain Relief.

Although persistent pain is estimated to affect about 20% of the adult population, current treatments have poor results. Polypharmacology, which is the administration of more than one drug targeting on two or more different sites of action, represents a prominent therapeutic approach for the clinical management of persistent pain. Thus, in the drug discovery process the "one-molecule-multiple targets" strategy nowadays is highly recognized. Indeed, multitarget ligands displaying a better antinociceptive activity with fewer side effects, combined with favorable pharmacokinetic and pharmacodynamic characteristics, have already been shown. Multitarget ligands possessing non-opioid/opioid and opioid/opioid mechanisms of action are considered as potential drug candidates for the management of various pain conditions. In particular, dual-target MOPr (mu opioid peptide receptor)/DOPr (delta opioid peptide receptor) ligands exhibit an improved antinociceptive profile associated with a reduced tolerance-inducing capability. The benzomorphan-based compounds LP1 and LP2 belong to this class of dual-target MOPr/DOPr ligands. In the present manuscript, the structure-activity relationships and the pharmacological fingerprint of LP1 and LP2 compounds as suitable drug candidates for persistent pain relief is described.

Exploiting the Power of Stereochemistry in Drug Action: 3-[(2S,6S,11S)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide as Potent Sigma-1 Receptor Antagonist.

(+)-(2S,6S,11S)- and (-)-(2R,6R,11R)-Benzomorphan derivatives have a different binding affinity for sigma-1 (σ1R) and opioid receptors, respectively. In this study, we describe the synthesis of the (+)-enantiomer [(+)-LP1] of the benzomorphan MOR agonist/DOR antagonist LP1 [(-)-LP1]. The binding affinity of both (+)-LP1 and (-)-LP1 for σ1R and sigma-2 receptor (σ2R) was tested. Moreover, (+)-LP1 opioid receptor binding affinity was also investigated. Finally, (+)-LP1 was tested in a mouse model of inflammatory pain. Our results showed a nanomolar σ1R and binding affinity for (+)-LP1. Both (+)-LP1 and (-)-LP1 elicited a significant analgesic effect in a formalin test. Differently from (-)-LP1, the analgesic effect of (+)-LP1 was not reversed by naloxone, suggesting a σ1R antagonist profile. Furthermore, σ1R agonist PRE-084 was able to unmask the σ1R antagonistic component of the benzomorphan compound. (+)-LP1 could constitute an useful lead compound to develop new analgesics based on mechanisms of action alternative to opioid receptor activation.

Molecular modeling and biological studies show that some µ-opioid receptor agonists might elicit analgesia acting as MMP-9 inhibitors.

Aim: Despite the serious side effects, analgesics acting on opioid receptors are still considered the best way to get antinociception. Matrix metalloproteinases, a large family of zinc-dependent proteases implicated in many pathological conditions, such as diabetes and osteoarthritis, are also involved in inflammation and pain. Methodology & results: Looking for evidence of possible interactions of opioid pathways and inflammation mediators, molecular modeling studies of a series of recently developed µ-opioid receptor benzomorphanic agonists together with biological data on pain and inflammation molecular targets, allowed us to hypothesize a possible correlation between µ-opioid receptor system and MMP-9. Conclusion: A new compound, (-)-MML1017, emerged as a possible dual-acting agent able to interact selectively and potently with the two molecular targets.

(2S)-N-2-methoxy-2-phenylethyl-6,7-benzomorphan compound (2S-LP2): Discovery of a biased mu/delta opioid receptor agonist.

The pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan scaffold was investigated combining synthetic and pharmacological approaches. 2R- and 2S-diastereoisomers of the multitarget MOR/DOR antinociceptive ligand LP2 (1) were synthesized and their pharmacological profile was evaluated in in vitro and vivo assays. From our results, 2S-LP2 (5) showed an improved pharmacological profile in comparison to LP2 (1) and 2R-LP2 (4). 2S-LP2 (5) elicited an antinociceptive effect with a 1.5- and 3-times higher potency than LP2 (1) and R-antipode (4), respectively. In vivo effect of 2S-LP2 (5) was consistent with the improved MOR/DOR efficacy profile assessed by radioligand binding assay, to evaluate the opioid receptor affinity, and BRET assay, to evaluate the capability to promote receptor/G-protein and receptor/ß-arrestin 2 interaction. 2S-LP2 (5) was able to activate, with different efficacy, G-protein pathway over ß-arrestin 2, behaving as biased agonist at MOR and mainly at DOR. Considering the therapeutic potential of both multitarget MOR/DOR agonism and functional selectivity over G-protein, the 2S-LP2 (5) biased multitarget MOR/DOR agonist could provide a safer treatment opportunity.

Benzomorphan skeleton, a versatile scaffold for different targets: A comprehensive review.

Despite the fact that the benzomorphan skeleton has mainly been employed in medicinal chemistry for the development of opioid analgesics, it is a versatile structure. Its stereochemistry, as well as opportune modifications at the phenolic hydroxyl group and at the basic nitrogen, play a pivotal role addressing the benzomorphan-based compounds to a specific target. In this review, we describe the structure activity-relationships (SARs) of benzomorphan-based compounds acting at sigma 1 receptor (σ1R), sigma 2 receptor (σ2R), voltage-dependent sodium channel, N-Methyl-d-Aspartate (NMDA) receptor-channel complex and other targets. Collectively, the SARs data have highlighted that the benzomorphan nucleus could be regarded as a useful template for the synthesis of drug candidates for different targets.

Synthesis and Structure-Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino Analogues as Novel MOR Agonists.

The opioid pharmacological profile of cis-(-)-N-normetazocine derivatives is deeply affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a-d and 6a-d were characterized by flexible groups at the N-substituent that allow them to reposition themselves relative to cis-(-)-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays a KiMOR of 6.1 nM in a competitive binding assay, and an IC50 value of 11.5 nM and an Imax of 72% in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c, intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED50 of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural variations in the opioid receptor profile of cis-(-)-N-normetazocine derivatives and identify a new MOR agonist useful for the development of novel opioid analgesics for pain treatment.

Benzomorphan scaffold for opioid analgesics and pharmacological tools development: A comprehensive review.

Benzomorphan, derived by morphine skeleton simplification, has been the subject of exploration in medicinal chemistry for the development of new drugs and pharmacological tools to explore opioid pharmacology in vitro and in vivo. Building upon these evidences, the design and synthesis of benzomorphan-based compounds, appropriately modified at the basic nitrogen and/or the phenolic hydroxyl (8-OH) group, represent a valid and versatile strategy to obtain analgesics. In this review, to improve the body of information in this field, we report structure activity-relationships (SARs) of benzomorphan-based compounds analysing data literature of last 25 years. Collectively, SARs data highlighted that the benzomorphan nucleus represents a template in the achievement of a specific functional profile, by modifying N-substituent or 8-OH group.

Development of novel LP1-based analogues with enhanced delta opioid receptor profile.

Pain relief achieved by co-administration of drugs acting at different targets is more effective than that obtained with conventional MOR selective agonists usually associated to relevant side effects. It has been demonstrated that simultaneously targeting different opioid receptors is a more effective therapeutic strategy. Giving the promising role for DOR in pain management, novel LP1-based analogues with different N-substituents were designed and synthesized with the aim to improve DOR profile. For this purpose, we maintained the phenyl ring in the N-substituent of 6,7-benzomorphan scaffold linked to an ethyl spacer bearing a hydroxyl/methyl or methoxyl group at carbon 2 or including it in a 1,4-benzodioxane ring. LP1 analogues were tested by competition binding assays. Compounds 6 (KiMOR=2.47nM, KiDOR=9.6nM), 7 (KiMOR=0.5nM and KiDOR=0.8nM) and 9 (KiMOR=1.08nM, KiDOR=6.6nM) retained MOR affinity but displayed an improved DOR binding capacity as compared to LP1 (KiMOR=0.83nM, KiDOR=29.1nM). Moreover, GPI and MVD functional assays indicated that compounds 6 (IC50=49.2 and IC50=10.8nM), 7 (IC50=9.9 and IC50=11.8nM) and 9 (IC50=21.5 and IC50=4.4nM) showed a MOR/DOR agonist profile, unlike LP1 that was a MOR agonist/DOR antagonist (IC50=1.9 and IC50=1240nM). Measurements of their antinociceptive effect was evaluated by mice radiant tail flick test displaying for compounds 6, 7 and 9 ED50 values of 1.3, 1.0 and 0.9mg/kg, i.p., respectively. Moreover, the antinociceptive effect of compound 9 was longer lasting with respect to LP1. In conclusion the N-substituent nature of compounds 6, 7 and 9 shifts the DOR profile of LP1 from antagonism to agonism.

Dnmt1 regulates the myogenic lineage specification of muscle stem cells.

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.

An LP1 analogue, selective MOR agonist with a peculiar pharmacological profile, used to scrutiny the ligand binding domain.

The hypothesis that central analgesia with reduced side effects is obtainable by occupying an 'allosteric' site in the MOR ligand binding domain requires the development of new ligands with peculiar pharmacological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5, bearing a diphenylic N-substituent on the benzomorphan nucleus, showed an affinity (Kiµ=0.5±0.2nM) comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1 and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several reference compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for their interactions with 'orthosteric' and 'allosteric' binding sites.

Evaluation of N-substituent structural variations in opioid receptor profile of LP1.

The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9-15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (Ki(MOR)=38±4nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA2) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD50=2.0mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N-substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function.

Norbenzomorphan Framework as a Novel Scaffold for Generating Sigma†2 Receptor/PGRMC1 Subtype-Selective Ligands.

A novel structural class with high affinity and subtype selectivity for the sigma 2 receptor has been discovered. Preliminary structure-affinity relationship data are presented showing that 8-substituted 1,3,4,5-tetrahydro-1,5-methanobenzazepine (norbenzomorphan) derivatives elicit modest to high selectivity for the sigma 2 over the sigma 1 receptor subtype. Indeed, piperazine analogue 8-(4-(3-ethoxy-3-oxopropyl)piperazin-1-yl)-1,3,4,5-tetrahydro-1,5-methanobenzazepine-2-carboxylate (SAS-1121) is 574-fold selective for the sigma 2 over the sigma 1 receptor, thereby establishing it as one of the more subtype-selective sigma 2 binding ligands reported to date. Emerging evidence has implicated the sigma 2 receptor in multiple health disorders, so the drug-like characteristics of many of the selective sigma 2 receptor ligands disclosed herein, coupled with their structural similarity to frameworks found in known drugs, suggest that norbenzomorphan analogues may be promising candidates for further development into drug leads.

Predicted structures for kappa opioid G-protein coupled receptor bound to selective agonists.

Human kappa opioid receptor (κ-OR), a G protein-coupled receptor (GPCR), has been identified as a drug target for treatment of such human disorders as pain perception, neuroendocrine physiology, affective behavior, and cognition. In order to find more selective and active agonists, one would like to do structure based drug design. Indeed, there is an X-ray structure for an antagonist bound to κ-OR, but structures for activated GPCRs are quite different from those for the inactive GPCRs. Here we predict the ensemble of 24 low-energy structures of human kappa opioid receptor (κ-OR), obtained by application of the GEnSeMBLE (GPCR Ensemble of Structures in Membrane Bilayer Environment) complete sampling method, which evaluates 13 trillion combinations of tilt and rotation angles for κ-OR to select the best 24. To validate these structures, we used the DarwinDock complete sampling method to predict the binding sites for five known agonists (ethylketocyclazocine, bremazocine, pentazocine, nalorphine, and morphine) bound to all 24 κ-OR conformations. We find that some agonists bind selectively to receptor conformations that lack the salt bridge between transmembrane domains 3 and 6 as expected for active conformations. These 3D structures for κ-OR provide a structural basis for understanding ligand binding and activation of κ-OR, which should be useful for guiding subtype specific drug design.

Differential scanning calorimetry approach to investigate the transfer of the multitarget opioid analgesic LP1 to biomembrane model.

An emerging approach in pain management is the use of multitarget opioid ligands, owing an improved analgesic effect coupled to a reduced incidence of side effects. With a mu opioid receptor agonist/delta opioid receptor antagonist profile, the benzomorphan-based compound LP1 belongs to multitarget ligands class. Previous in vivo investigations showed that LP1 - subcutaneously administered as oxalate salt - was an antinociceptive agent as potent as morphine with a low tolerance-inducing capability. Because the renal toxicity of oxalate is known, an alternative approach allowing the administration of LP1 freebase could be more biocompatible. In this study the interaction of LP1 freebase and LP1 oxalate salt with multilamellar vesicles, as membrane model, was evaluated using differential scanning calorimetry technique. Despite the good membrane interaction showed by LP1 freebase, it was not capable to diffuse in the aqueous medium and to be uptaken by multilamellar vesicles. On the other hand, LP1 freebase possessed a good transfer profile by a liposomal carrier to a biomembrane model. Considering our findings and the need of safe formulations, studies for the development of a suitable carrier for a systemic administration of LP1 freebase are in progress.

Knockout subtraction autoradiography: a novel ex vivo method to detect heteromers finds sparse KOP receptor/DOP receptor heterodimerization in the brain.

Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [(3)H]bremazocine or with [(3)H]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist.

The multitarget opioid ligand LP1's effects in persistent pain and in primary cell neuronal cultures.

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.