Quantitative Systems Pharmacology and Biased Agonism at Opioid Receptors: A Potential Avenue for Improved Analgesics

Chronic pain is debilitating and represents a significant burden in terms of personal and socio-economic costs. Although opioid analgesics are widely used in chronic pain treatment, many patients report inadequate pain relief or relevant adverse effects, highlighting the need to develop analgesics with improved efficacy/safety. Multiple evidence suggests that G protein-dependent signaling triggers opioid-induced antinociception, whereas arrestin-mediated pathways are credited with modulating different opioid adverse effects, thus spurring extensive research for G protein-biased opioid agonists as analgesic candidates with improved pharmacology. Despite the increasing expectations of functional selectivity, translating G protein-biased opioid agonists into improved therapeutics is far from being fully achieved, due to the complex, multidimensional pharmacology of opioid receptors. The multifaceted network of signaling events and molecular processes underlying therapeutic and adverse effects induced by opioids is more complex than the mere dichotomy between G protein and arrestin and requires more comprehensive, integrated, network-centric approaches to be fully dissected. Quantitative Systems Pharmacology (QSP) models employing multidimensional assays associated with computational tools able to analyze large datasets may provide an intriguing approach to go beyond the greater complexity of opioid receptor pharmacology and the current limitations entailing the development of biased opioid agonists as improved analgesics

Side chain effect in the modulation of αvβ3/α5β1 integrin activity via clickable isoxazoline-RGDmimetics: development of molecular delivery systems

View references (57) Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose \u3b1v\u3b23 and \u3b15\u3b21 integrins\u2019 potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as \u201cshuttles\u201d for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards \u3b15\u3b21 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated \u3b15\u3b21 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application

Functional Selectivity and Antinociceptive Effects of a Novel KOPr Agonist

Kappa opioid receptor (KOPr) agonists represent alternative analgesics for their low abuse potential, although relevant adverse effects have limited their clinical use. Functionally selective KOPr agonists may activate, in a pathway-specific manner, G protein-mediated signaling, that produces antinociception, over \u3b2-arrestin 2-dependent induction of p38MAPK, which preferentially contributes to adverse effects. Thus, functionally selective KOPr agonists biased toward G protein-coupled intracellular signaling over \u3b2-arrestin-2-mediated pathways may be considered candidate therapeutics possibly devoid of many of the typical adverse effects elicited by classic KOPr agonists. Nonetheless, the potential utility of functionally selective agonists at opioid receptors is still highly debated; therefore, further studies are necessary to fully understand whether it will be possible to develop more effective and safer analgesics by exploiting functional selectivity at KOPr. In the present study we investigated in vitro functional selectivity and in vivo antinociceptive effects of LOR17, a novel KOPr selective peptidic agonist that we synthesized. LOR17-mediated effects on adenylyl cyclase inhibition, ERK1/2, p38MAPK phosphorylation, and astrocyte cell proliferation were studied in HEK-293 cells expressing hKOPr, U87-MG glioblastoma cells, and primary human astrocytes; biased agonism was investigated via cAMP ELISA and \u3b2-arrestin 2 recruitment assays. Antinociception and antihypersensitivity were assessed in mice via warm-water tail-withdrawal test, intraperitoneal acid-induced writhing, and a model of oxaliplatin-induced neuropathic cold hypersensitivity. Effects of LOR17 on locomotor activity, exploratory activity, and forced-swim behavior were also assayed. We found that LOR17 is a selective, G protein biased KOPr agonist that inhibits adenylyl cyclase and activates early-phase ERK1/2 phosphorylation. Conversely to classic KOPr agonists as U50,488, LOR17 neither induces p38MAPK phosphorylation nor increases KOPr-dependent, p38MAPK-mediated cell proliferation in astrocytes. Moreover, LOR17 counteracts, in a concentration-dependent manner, U50,488-induced p38MAPK phosphorylation and astrocyte cell proliferation. Both U50,488 and LOR17 display potent antinociception in models of acute nociception, whereas LOR17 counteracts oxaliplatin-induced thermal hypersensitivity better than U50,488, and it is effective after single or repeated s.c. administration. LOR17 administered at a dose that fully alleviated oxaliplatin-induced thermal hypersensitivity did not alter motor coordination, locomotor and exploratory activities nor induced pro-depressant-like behavior. LOR17, therefore, may emerge as a novel KOPr agonist displaying functional selectivity toward G protein signaling and eliciting antinociceptive/antihypersensitivity effects in different animal models, including oxaliplatin-induced neuropathy

Experimental pharmacotherapy for dry eye disease: A review

none2noDry eye disease (DED) is a complex multifactorial disease showing heterogenous symptoms, including dryness, photophobia, ocular discomfort, irritation and burning but also pain. These symptoms can affect visual function leading to restrictions in daily life activities and reduction in work productivity with a consequently high impact on quality of life. Several pathological mechanisms contribute to the disease: evaporative water loss leads to impairment and loss of tear homeostasis inducing either directly or indirectly to inflamma-tion, in a self-perpetuating vicious cycle. Dysregulated ocular immune responses result in ocular surface damage, which further contributes to DED pathogenesis. Currently, DED treatment is based on a flexible stepwise approach to identify the most beneficial interven-tion. Although most of the available treatments may control to a certain extent some signs and symptoms of DED, they show significant limitations and do not completely address the needs of patients suffering from DED. This review provides an overview of the emerging experimental therapies for DED. Several promising therapeutic strategies are under development with the aim of dampening inflammation and restoring the homeostasis of the ocular surface microenvironment. Results from early phase clinical trials, testing the effects of EnaC blockers, TRPM8 agonist or mesenchymal stem cells in DED patients, are especially awaited to demonstrate their therapeutic value for the treatment of DED. Moreover, the most advanced experimental strategies in the pipeline for DED, tivanisiran, IL-1R antagonist EBI-005 and SkQ1, are being tested in Phase III clinical trials, still ongoing. Nevertheless, although promising results, further studies are still needed to confirm efficacy and safety of the new emerging therapies for DED.openBaiula M.; Spampinato S.Baiula M.; Spampinato S

Agonist-regulated endocytosis and desensitization of the human nociceptin receptor

A series of nociceptin receptor ligands has been investigated in relationship to their capability to promote receptor endocytosis, desensitization (evaluated as inhibition of forskolin-stimulated cAMP production) and compensatory upregulation of adenylyl cyclase activity in CHO-K1 cells expressing the cloned human nociceptin receptor. Nociceptin (NC), [Arg14, Lys15]NC-NH2 and NNC 63-0532 (0.01 nM-10 microM) induce a concentration-dependent endocytosis and recycling of the nociceptin receptor. This mechanism contributes to maintain receptor signaling as it counteracts desensitization development and enhances a compensatory upregulation of adenyl cyclase activity. In contrast, the partial agonists [Phe1,Psi(CH2NH)Gly2]NC(1-13)-NH2, Ac-RYYRIK-NH2 and Ac-RYYRWK-NH2 (up to 100 microM) fail to induce receptor endocytosis and cause a pronounced receptor desensitization that is not influenced by monensin, a blocker of recycling of the internalized receptors

Phase II drugs under investigation for allergic conjunctivitis

Ocular allergies comprise a spectrum of conditions that are underreported and underdiagnosed, and are frequently associated with rhinoconjunctivitis. Although allergic conjunctivitis is often not a sight-threatening condition, it could have a significant impact on a person's quality of life, morbidity and productivity. A variety of agents are available for the treatment of allergic conjunctivitis, including antihistamines, mast-cell stabilizers, dual action agents, glucocorticoids, calcineurin inhibitors and immunotherapy

Protein Kinase C-dependent up-regulation of human mu-opioid receptor gene (OPRM1) transcription is negatively influenced by the transcription factor REST

Mu-opioid receptor (MOPr) mediates several functions including pain transmission, respiration, cardiac and gastrointestinal function, and immune responses. Human mu-opioid receptor gene (OPRM1) expression is tightly modulated by several cytokines and neurotrophic factors in both neuronal and immune cells; particularly, OPRM1 expression is increased during neurogenesis, regulates the survival of maturing neurons and is implicated in ischemia-induced neuronal death. The Repressor Element 1 Silencing Transcription factor (REST), a regulator of a subset of genes in differentiating and post mitotic neurons, is implicated in OPRM1 transcriptional repression as well and it has been shown to counteract IGF-I-mediated up-regulation of its expression. Albeit several studies have been performed to characterize OPRM1 transcriptional regulation, extracellular signaling molecules and mechanisms involved in such processes are still not fully understood. In this study, both REST-expressing and REST-non expressing neuronal cells were employed to investigate the role of protein kinase C (PKC) on OPRM1 transcription, in the context of the potential influence of REST. OPRM1 endogenous mRNA levels were evaluated by Real Time PCR whereas OPRM1 promoter transcriptional activity was assessed by employing specific Luciferase/OPRM1 promoter reporter vectors. We observed that in native SH-SY5Y neuroblastoma cells, which endogenously express REST, PKC activation with phorbol 12-myristate 13-acetate (PMA, 16 nM; 24 h) significantly down regulates OPRM1 transcription and concomitantly elevates the REST binding activity to its repressor element 1 on the OPRM1 promoter. On the contrary, when REST expression is knocked-down by an antisense strategy or by retinoic acid-induced cell differentiation, PMA significantly up-regulates OPRM1 gene transcription. REST therefore seems to be crucial in determining the differential effect of PMA on OPRM1 transcription, as its presence in native SH-SY5Y cells is enough to favor OPRM1 down-regulation whereas its absence in antisense-treated or retinoic acid differentiated SH-SY5Y cells allows PKC-dependent up-regulation of OPRM1 transcription. PMA acts through a PKC-dependent pathway requiring downstream extracellular-signal-regulated kinase 1/2 and the transcription factor AP-1, as it was demonstrated by co-administration of PMA and specific PKC, JNK and MAPKK inhibitors. PKC inhibitor GF109203 prevented both PMA-induced down-regulation of OPRM1 transcription in native SH-SY5Y cells and PMA-mediated OPRM1 up-regulation in retinoic acid- differentiated SH-SY5Y cells. Furthermore, JNK and MAPKK inhibitors, SP600125 and PD98059 respectively, but not p38 and PI3K inhibitors, SB203580 and LY294002 respectively, prevented OPRM1 up-regulation in differentiated SH-SY5Y cells, thus suggesting that PKC modulates OPRM1 transcription through the activation of the transcription factor AP-1. This observation was also confirmed by employing decoys oligonucleotide specific for AP-1 inhibition. Experiments were also carried out in SH-SY5Y cells and PC-12 cells transfected with promoter/Luciferase reporter vectors containing different OPRM1 promoter fragments. In these assays we observed that OPRM1 promoter transcriptional activity is up-regulated by PMA-dependent PKC activation in PC12 cells, which lack of REST expression, as well as in SH-SY5Y cells either transfected with OPRM1 promoter constructs deficient in RE1 (the REST DNA binding element) or when REST is down-regulated in retinoic acid-differentiated cells, thus confirming the role played by REST in the differential modulation of OPRM1 transcription by PKC. The finding that PMA may up-regulate OPRM1 expression in differentiating neuroblastoma cells and that this effect depends on REST expression is new and is relevant in elucidating MOPr role in neurogenesis and in post-mitotic neurons. Several studies have demonstrated that OPRM1 gene expression is seen very early on in the embryonic rat brain with an increase observed during the critical period of neurogenesis, neuronal migration, and synaptogenesis, suggesting a role of this opioid receptor in brain developmental processes. Recently, it has been shown that MOPr regulates the survival of maturing neurons in adult hippocampal neurogenesis, therefore, MOPr could be included among the target genes regulated by PKC in a time-dependent manner during neurogenesis. Our results provide new insights into how OPRM1 transcription is regulated and may help to better understand its contribution to epigenetic modifications and reprogramming of neuronal cells exposed to PKC-activating agents in differentiated cells. These findings are relevant considering the potential involvement of such processes in chronic pain treatment

Agonist-Regulated Internalization and desensitization of the Human Nociceptin Receptor Expressed in CHO Cells

In this study we examined agonist-induced internalization of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by receptor binding assay on viable cells and confocal microscopy. The agonists nociceptin/orphanin FQ (NC), NC-NH2, NC(1-13)-NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 promote a rapid, concentration-dependent internalization of the hNOP receptor. Under the same conditions, [Phe1,ψ(CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, ψ(CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 failed to induce significant, concentration-dependent NOP receptor endocytosis; even when present at high concentrations (up to 1 mM) they promoted only an approximately 25-30% internalization of hNOP receptors. We also investigated hNOP receptor desensitization upon agonist challenge: ligand efficacy to inhibit forskolin-stimulated cAMP production. After 1 h exposure to NC, NC-NH2, NC(1-13)-NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 (5 μM) ≈20 to 30% of receptor desensitization was observed. Moreover, we found that the blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. The non-internalizing agonists [Phe1,ψ(CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, ψ(CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 (100 μM) resulted in a strong (67 and 74 %, respectively) receptor desensitization which was not influenced by monensin. Finally, CHO-hNOP cells exposed to the receptor-internalizing agonists for 24 h resulted in a significantly higher cAMP accumulation (defined supersensitization) compared with the non-internalizing agonists. In addition, blocking of receptor recycling by monensin led to a decrease of the cAMP accumulation only in cells exposed to internalizing agonists. These data show that prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways