In vitro pharmacological characterization of standard and new lysophosphatidic acid receptor antagonists using dynamic mass redistribution assay.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that acts as an agonist of six G protein-coupled receptors named LPA receptors (LPA1-6). LPA elicits diverse intracellular events and modulates several biological functions, including cell proliferation, migration, and invasion. Overactivation of the LPA-LPA receptor system is reported to be involved in several pathologies, including cancer, neuropathic pain, fibrotic diseases, atherosclerosis, and type 2 diabetes. Thus, LPA receptor modulators may be clinically relevant in numerous diseases, making the identification and pharmacodynamic characterization of new LPA receptor ligands of strong interest. In the present work, label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of some LPA1 and LPA2 standard antagonists at the recombinant human LPA1 and LPA2 receptors. These results are compared to those obtained in parallel experiments with the calcium mobilization assay. Additionally, the same experimental protocol has been used for the pharmacological characterization of the new compound CHI. KI 16425, RO 6842262, and BMS-986020 behaved as LPA1 inverse agonists in DMR experiments and as LPA1 antagonists in calcium mobilization assays. Amgen compound 35 behaved as an LPA2 antagonist, while Merck compound 20 from WO2012028243 was detected as an LPA2 inverse agonist using the DMR test. Of note, for all the compounds, similar potency values were estimated by DMR and calcium assay. The new compound CHI was found to be an LPA1 inverse agonist, but with potency lower than that of the standard compounds. In conclusion, we have demonstrated that DMR assay can be successfully used to characterize LPA1 and LPA2 ligands. Compared to the classical calcium mobilization assay, DMR offers some advantages, in particular allowing the identification of inverse agonists. Finally, in the frame of this study, a new LPA1 inverse agonist has been identified.

MOP and NOP receptor interaction: Studies with a dual expression system and bivalent peptide ligands.

Opioids targeting mu;µ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEKMOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEKMOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[35S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, DermorphinATTO488 and the NOP fluorescent ligand N/OFQATTO594. In HEKMOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEKMOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[35S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands.

In vitro and in vivo pharmacological characterization of the synthetic opioid MT-45.

MT-45 is a synthetic opioid that was developed in the 1970s as an analgesic compound. However, in recent years MT-45 has been associated with multiple deaths in Europe and has been included in the class of novel psychoactive substances known as novel synthetic opioids (NSOs). Little is known about the pharmaco-toxicological effects of MT-45. Therefore, we used a dynamic mass redistribution (DMR) assay to investigate the pharmacodynamic profile of this NSO in vitro compared with morphine. We then used in vivo studies to investigate the effect of the acute systemic administration of MT-45 (0.01-15 mg/kg i.p.) on motor and sensorimotor (visual, acoustic and tactile) responses, mechanical and thermal analgesia, muscle strength and body temperature in CD-1 male mice. Higher doses of MT-45 (6-30 mg/kg i.p.) were used to investigate cardiorespiratory changes (heart rate, respiratory rate, SpO2 saturation and pulse distention). All effects of MT-45 were compared with those of morphine. In vitro DMR assay results demonstrated that at human recombinant opioid receptors MT-45 behaves as a potent selective mu agonist with a slightly higher efficacy than morphine. In vivo results showed that MT-45 progressively induces tail elevation at the lowest dose tested (0.01 mg/kg), increased mechanical and thermal antinociception (starting from 1 to 6 mg/kg), decreased visual sensorimotor responses (starting from 3 to 6 mg/kg) and reduced tactile responses, modulated motor performance and induced muscle rigidity at higher doses (15 mg/kg). In addition, at higher doses (15-30 mg/kg) MT-45 impaired the cardiorespiratory functions. All effects were prevented by the administration of the opioid receptor antagonist naloxone. These findings reveal the risks associated with the ingestion of opioids and the importance of studying these drugs and undertaking more clinical studies of the current molecules to better understand possible therapeutic interventions in the case of toxicity.

Nociceptin/orphanin FQ receptor ligands and translational challenges: focus on cebranopadol as an innovative analgesic.

Opioids are characterised as classical (mu, delta, and kappa) along with the non-classical nociceptin/orphanin FQ (N/OFQ) receptor or NOP. Targeting NOP has therapeutic indications in control of the cardiovascular and respiratory systems and micturition, and a profile as an antidepressant. For all of these indications, there are translational human data. Opioids such as morphine and fentanyl (activating the mu receptor) are the mainstay of pain treatment in the perioperative period, despite a challenging side-effect profile. Opioids in general have poor efficacy in neuropathic pain. Moreover, longer term use is associated with tolerance. There is good evidence interactions between opioid receptors, and receptor co-activation can reduce side-effects without compromising analgesia; this is particularly true for mu and NOP co-activation. Recent pharmaceutical development has produced a mixed opioid/NOP agonist, cebranopadol. This new chemical entity is effective in animal models of nociceptive and neuropathic pain with greater efficacy in the latter. In animal models, there is little evidence for respiratory depression, and tolerance (compared with morphine) only develops after long treatment periods. There is now early phase clinical development in diabetic neuropathy, cancer pain, and low back pain where cebranopadol displays significant efficacy. In 1996, N/OFQ was formally identified with an innovative analgesic profile. Approximately 20 yr later, cebranopadol as a clinical ligand is advancing through the human trials process.

Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.

BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594 ) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. EXPERIMENTAL APPROACH: We assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). KEY RESULTS: N/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594 , movement of ligand and receptor from the cell surface to the cytosol can be measured. CONCLUSIONS AND IMPLICATIONS: In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ-driven cellular responses.

In vitro and in vivo characterization of the bifunctional µ and δ opioid receptor ligand UFP-505.

BACKGROUND AND PURPOSE: Targeting more than one opioid receptor type simultaneously may have analgesic advantages in reducing side-effects. We have evaluated the mixed µ opioid receptor agonist/ δ opioid receptor antagonist UFP-505 in vitro and in vivo. EXPERIMENTAL APPROACH: We measured receptor density and function in single µ, δ and µ /δ receptor double expression systems. GTPγ35 S binding, cAMP formation and arrestin recruitment were measured. Antinociceptive activity was measured in vivo using tail withdrawal and paw pressure tests following acute and chronic treatment. In some experiments, we collected tissues to measure receptor densities. KEY RESULTS: UFP-505 bound to µ receptors with full agonist activity and to δ receptors as a low efficacy partial agonist At µ, but not δ receptors, UFP-505 binding recruited arrestin. Unlike morphine, UFP-505 treatment internalized µ receptors and there was some evidence for internalization of δ receptors. Similar data were obtained in a µ /δ receptor double expression system. In rats, acute UFP-505 or morphine, injected intrathecally, was antinociceptive. In tissues harvested from these experiments, µ and δ receptor density was decreased after UFP-505 but not morphine treatment, in agreement with in vitro data. Both morphine and UFP-505 induced significant tolerance. CONCLUSIONS AND IMPLICATIONS: In this study, UFP-505 behaved as a full agonist at µ receptors with variable activity at δ receptors. This bifunctional compound was antinociceptive in rats after intrathecal administration. In this model, dual targeting provided no advantages in terms of tolerance liability. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Medico legal aspects on neuromonitoring in thyroid surgery: informed consent on malpractice claims.

AIM: The purpose of this paper is to provide a forensic profile framework of neuromonitoring in thyroid surgery, regarding the information given to the patient and its classification as part of professional liability in the event of recurrent injury. METHOD: Evaluation and reflections on the required behaviour of the surgeon on providing details on the operation before the informed consent is given and to outline the possible legal implications regarding professional liability as a result of recurrent injury. In particular, if it is an obligation to inform the patient about using this method and if it is possible for the surgeon to freely choose whether to employ this method, which is still burdened by a certain percentage of error and for that reason it cannot be defined a "standard of care". RESULTS: To recognize neuromonitoring the role of standard of care in surgery of the thyroid means attribute a role of method able to avoid the surgeon to cause iatrogenic damage to the laryngeal nerve. For the foregoing reasons that is not true, determining false positives and false negatives, and this can be a double edged sword for the surgeon. CONCLUSIONS: Although the progress in the field of thyroid surgery made in the last decade, currently there is no scientific reassuring evidence to completely avoid the possibility of producing an iatrogenic lesion of the laryngeal nerve. Information given to the patient prior to surgery should respect the requirements of completeness, freedom and honesty in order to allow the patient to self-determination.

Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test.

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. The NOP selective agonist Ro 65-6570 (0.1-1mgkg-1) and morphine (0.1-10mgkg-1) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01-0.1mgkg-1) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory.

A staged screening of registered drugs highlights remyelinating drug candidates for clinical trials.

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds' activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.

Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic-versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands.

BACKGROUND AND PURPOSE: Nociceptin/orphanin FQ (N/OFQ) receptor (NOP) agonists produce anxiolytic-like effects in rodents while antagonists promote antidepressant-like effects. The aim of this study was to investigate the effect on anxiety and depression of NOP receptor partial agonists such as the peptides [F/G]N/OFQ(1-13)NH2 and UFP-113 and the non-peptide AT-090. EXPERIMENTAL APPROACH: In vitro AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 were tested for their ability to promote NOP/G-protein and NOP/ß-arrestin 2 interaction, using a bioluminescence resonance energy transfer assay. In vivo, they were tested in mice in the elevated plus maze (EPM) and in the forced swim (FST) tests. NOP partial agonists effects were systematically compared to those of full agonists (N/OFQ and Ro 65-6570) and antagonists (UFP-101 and SB-612111). KEY RESULTS: In vitro, AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 promoted NOP/G protein interaction, with maximal effects lower than those evoked by N/OFQ and Ro 65-6570. AT-090 behaved as a NOP partial agonist also in inducing ß-arrestin 2 recruitment, while UFP-113 and [F/G]N/OFQ(1-13)NH2 were inactive in this assay. In vivo, AT-090 induced anxiolytic-like effects in the EPM but was inactive in the FST. Opposite results were obtained with UFP-113 and [F/G]N/OFQ(1-13)NH2. CONCLUSIONS AND IMPLICATIONS: NOP ligands producing similar effects on NOP/G protein interaction (partial agonism) but showing different effects on ß-arrestin 2 recruitment (partial agonism vs antagonism) elicited different actions on anxiety and mood. These results suggest that the action of a NOP ligand on emotional states is better predicted based on its ß-arrestin 2 rather than G-protein efficacy.

Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and ß-Arrestins 2.

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or ß-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/ß-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gß1 or NOP/ß-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiae yeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coli F4ac.

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic F4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of CNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 10(10) cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 10(10) cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 10(11) cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 10(8) cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P = 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P = 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P = 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P = 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P = 0.05). Blood concentrations of dodecenoyl-L-carnitine (P < 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P = 0.01 and P< 0.01, respectively), and α-amino adipic acid (P < 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P = 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P < 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

Spinal antinociceptive effects of the novel NOP receptor agonist PWT2-nociceptin/orphanin FQ in mice and monkeys.

BACKGROUND AND PURPOSE: Using an innovative chemical approach, peptide welding technology (PWT), a tetrabranched derivative of nociceptin/orphanin FQ (N/OFQ) has been generated and pharmacologically characterized. Both in vitro and in vivo PWT2-N/OFQ displayed the same pharmacological profile to the natural ligand. It was more potent and produced longer-lasting effects. The aim of the present study was to investigate the spinal effects of PWT2-N/OFQ in nociceptive and neuropathic pain models in mice and non-human primates. EXPERIMENTAL APPROACH: Tail withdrawal assay in mice and monkeys was used as a nociceptive pain model and mechanical threshold in mice subjected to chronic constriction injury was used as a neuropathic pain model. The antinociceptive effects of spinally administered N/OFQ and PWT2-N/OFQ were assessed in these models. KEY RESULTS: PWT2-N/OFQ mimicked the spinal antinociceptive effects of N/OFQ both in nociceptive and neuropathic pain models in mice as well as in non-human primates displaying 40-fold higher potency and a markedly prolonged duration of action. The effects of N/OFQ and PWT2-N/OFQ were sensitive to the N/OFQ receptor (NOP) antagonist SB-612111, but not to opioid receptor antagonists. CONCLUSIONS AND IMPLICATIONS: The present study has demonstrated that PWT2-N/OFQ mimicked the antinociceptive effects of the natural peptide in rodents and non-human primates acting as a potent and longer-lasting NOP-selective agonist. More generally, PWT derivatives of biologically active peptides can be viewed as innovative pharmacological tools for investigating those conditions and states in which selective and prolonged receptor stimulation promotes beneficial effects.

Acetylcholine contributes to control the physiological inflammatory response during the peri-implantation period.

BACKGROUND: Maternal antigen-presenting cells attracted to the pregnant uterus interact with trophoblast cells and modulate their functional profile to favour immunosuppressant responses. Non-neuronal cholinergic system is expressed in human cytotrophoblast cells and in immune cells with homeostatic regulatory functions. AIM: The aim of this work was to evaluate whether non-neuronal acetylcholine conditions maternal monocyte and DC migration and activation profiles. METHODS: We used an in vitro model resembling maternal-placental interface represented by the co-culture of human trophoblast cells (Swan-71 cell line) and monocytes or DC. RESULTS: When cytotrophoblast cells were treated with neostigmine (Neo) to concentrate endogenous acetylcholine levels, monocyte migration was increased. In parallel, high levels of IL-10 and decreased levels of TNF-α were observed upon interaction of maternal monocytes with trophoblast cells. This effect was synergized by Neo and was prevented by atropine, a muscarinic acetylcholine receptor antagonist. Similarly, trophoblast cells increased the migration of DC independently of Neo treatment; however, enhanced IL-10 and MCP-1 synthesis in trophoblast-DC co-cultures with no changes in TNF-α and IL-6 was observed. In fact, there were no changes in HLA-DR, CD86 or CD83 expression. Finally, trophoblast cells treated with Neo increased the expression of two antigen-presenting cells attracting chemokines, MCP-1, MIP-1α and RANTES through muscarinic receptors, and it was prevented by atropine. CONCLUSIONS: Our present results support a novel role of acetylcholine synthesized by trophoblast cells to modulate antigen-presenting cell migration and activation favouring an immunosuppressant profile that contributes to immune homeostasis maintenance at the maternal-foetal interface.

Development and characterisation of novel fentanyl-delta opioid receptor antagonist based bivalent ligands.

BACKGROUND: Opioid tolerance is a limiting factor in chronic pain. Delta opioid peptide (DOP)(δ) receptor antagonism has been shown to reduce tolerance. Here, the common clinical mu opioid peptide (MOP)(µ) receptor agonist fentanyl has been linked to the DOP antagonist Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydrisoquinoline-3-carboxylic acid) to create new bivalent compounds. METHODS: Binding affinities of bivalents(#9, #10, #11, #12 and #13) were measured in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, Kappa opioid peptide (KOP)(κ) and nociceptin/orphanin FQ opioid peptide (NOP) receptors. Functional studies, measuring GTPγ[(35)S] or ß-arrestin recruitment, were performed in membranes or whole cells respectively expressing MOP and DOP. RESULTS: The new bivalents bound to MOP (pKi : #9:7.31; #10:7.58; #11:7.91; #12:7.94; #13:8.03) and DOP (#9:8.03; #10:8.16; #11:8.17; #12:9.67; #13:9.71). In GTPγ[(35)S] functional assays, compounds #9(pEC50:6.74; intrinsic activity:0.05) #10(7.13;0.34) and #11(7.52;0.27) showed weak partial agonist activity at MOP. Compounds #12 and #13, with longer linkers, showed no functional activity at MOP. In antagonist assays at MOP, compounds #9 (pKb:6.87), #10(7.55) #11(7.81) #12(6.91) and #13(7.05) all reversed the effects of fentanyl. At DOP, all compounds showed antagonist affinity (#9:6.85; #10:8.06; #11:8.11; #12:9.42; #13:9.00), reversing the effects of DPDPE ([D-Pen(2,5)]enkephalin). In ß-arrestin assays, compared with fentanyl (with response at maximum concentration (RMC):13.62), all compounds showed reduced ability to activate ß-arrestin (#9 RMC:1.58; #10:2.72; #11:2.40; #12:1.29; #13:1.58). Compared with fentanyl, the intrinsic activity was: #9:0.12; #10:0.20; #11:0.18; #12:0.09 and #13:0.12. CONCLUSIONS: The addition of a linker between fentanyl and Dmt-Tic did not alter the ability to bind to MOP and DOP, however a substantial loss in MOP functional activity was apparent. This highlights the difficulty in multifunctional opioid development.

Differentiated thyroid cancer: feasibility of loboisthmectomy in an endemic region.

AIM: The aim of the present retrospective study was to assess the feasibility of loboisthmectomy for the treatment of differentiated thyroid cancer in a endemic area, evaluating the histopathological features and the results of a case series of 1154 patients. PATIENTS AND METHODS: The clinical records of 1154 patients submitted to total thyroidectomy in our Department were retrospectively reviewed to analyze the histopathological characters and the results. RESULTS: In 1044 cases (90.5%) a papillary cancer was observed, in 110 (9.5%) a follicular carcinoma; microcarcinomas were 399 (34.5%). Multifocality was present in 323 cases (28%), in 142 unilateral (12.3%) and in 181 bilateral (15.7%). Thyroiditis coexisted in 472 patients (40.9%), multinodular goiter in 404 (35%), Graves' disease in 48 (4.1%), and multinodular toxic goiter in 38 (3.3%). Complications were: postoperative bleeding in 20 patients (1.7%), transient unilateral vocal cord paralysis in 20 (1.7%) definitive in 10 (0.86%), a transient bilateral paralysis in 1 (0.08%), a transient hypoparathyroidism in 351 (30.4%), and a definitive in 24 (2.07%). Nodal recurrence occurred in 25 patients (2.16%). CONCLUSIONS: Total thyroidectomy remains the safest treatment in differentiated thyroid cancer, especially if performed in high volume centers in which complications can be minimized. Loboisthmectomy can be a viable and safe alternative in small (< 1 cm) unifocal tumors in patients at low risk. Loboisthmectomy is limited in endemic areas by the association with other thyroid diseases. A correct and detailed information of the patient is essential before planning surgery.

Stable planar mesoscopic photonic crystal cavities.

Mesoscopic self-collimation (MSC) in mesoscopic photonic crystals with high reflectivity is exploited to realize a novel high Q-factor cavity by means of mesoscopic PhC planar mirrors. These mirrors efficiently confine a mode inside a planar Fabry-Perot-like cavity, that results from a beam focusing effect that stabilizes the cavity even for small beam sizes, resembling the focusing behavior of curved mirrors. Moreover, they show an improved reflectivity with respect to their standard distributed Bragg reflector counterparts that allows higher compactness. A Q-factor higher than 104 has been achieved for an optimized 5-period-long mirror cavity. The optimization of the Q-factor and the performances in terms of energy storage, field enhancement, and confinement are detailed.

Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid.

The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the lpv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist.