318 results on '"Lambert DG"'
Search Results
52. Anaesthesiology in China. Response to Br J Anaesth 2019; 123: 559-64.
- Author
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Lambert DG
- Subjects
- China, Anesthesiology
- Published
- 2019
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53. The good, the bad, and the ugly: the many faces of opioids.
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Hemmings HC Jr and Lambert DG
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- Analgesics, Opioid pharmacology, Analgesics, Opioid therapeutic use, Humans, Immune Tolerance drug effects, Neoplasms immunology, Neoplasms surgery, Opioid-Related Disorders etiology, Opioid-Related Disorders prevention & control, Pain Management methods, Prescription Drug Overuse adverse effects, Analgesics, Opioid adverse effects
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- 2019
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54. Hot topics in opioid pharmacology: mixed and biased opioids.
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Azzam AAH, McDonald J, and Lambert DG
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- Analgesics, Opioid adverse effects, Drug Combinations, Drug Design, Drug Therapy, Combination, Humans, Ligands, Receptors, Opioid, delta antagonists & inhibitors, Receptors, Opioid, mu agonists, Analgesics, Opioid pharmacology
- Abstract
Analgesic design and evaluation have been driven by the desire to create high-affinity high-selectivity mu (μ)-opioid peptide (MOP) receptor agonists. Such ligands are the mainstay of current clinical practice, and include morphine and fentanyl. Advances in this sphere have come from designing pharmacokinetic advantage, as in rapid metabolism for remifentanil. These produce analgesia, but also the adverse-effect profile that currently defines this drug class: ventilatory depression, tolerance, and abuse liability. The MOP receptor is part of a family, and there are significant functional interactions between other members of the family (delta [δ]-opioid peptide [DOP], kappa [κ]-opioid peptide [KOP], and nociceptin/orphanin FQ receptor [NOP]). Experimentally, MOP agonism and DOP antagonism produce anti-nociception (animals) with no tolerance, and low doses of MOP and NOP ligands synergise to antinociceptive advantage. In this latter context, the lack of effect of NOP agonists on ventilation is an additional advantage. Recent development has been to move towards low-selectivity multifunctional 'mixed ligands', such as cebranopadol, or ligand mixtures, such as Targinact®. Moreover, the observation that β-arrestin coupling underlies the side-effect profile for MOP ligands (from knockout animal studies) led to the discovery of biased (to G-protein and away from β-arrestin intracellular signalling) MOP ligands, such as oliceridine. There is sufficient excitement in the opioid field to suggest that opioid analgesics without significant side-effects may be on the horizon, and the 'opioid Holy Grail' might be in reach., (Copyright © 2019 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2019
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55. Mixed mu-nociceptin/orphanin FQ opioid receptor agonists and the search for the analgesic holy grail.
- Author
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Lambert DG
- Subjects
- Analgesics, Opioid adverse effects, Animals, Drug Design, Humans, Ligands, Nociceptin Receptor, Analgesics, Opioid pharmacology, Receptors, Opioid agonists
- Published
- 2019
- Full Text
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56. A preclinical ultrasound method for the assessment of vascular disease progression in murine models.
- Author
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Janus J, Kanber B, Mahbuba W, Beynon C, Ramnarine KV, Lambert DG, Samani NJ, Stringer EJ, and Kelly ME
- Abstract
Introduction: The efficacy of preclinical ultrasound at providing a quantitative assessment of mouse models of vascular disease is relatively unknown. In this study, preclinical ultrasound was used in combination with a semi-automatic image processing method to track arterial distension alterations in mouse models of abdominal aortic aneurysm and atherosclerosis., Methods: Longitudinal B-mode ultrasound images of the abdominal aorta were acquired using a preclinical ultrasound scanner. Arterial distension was assessed using a semi-automatic image processing algorithm to track vessel wall motion over the cardiac cycle. A standard, manual analysis method was applied for comparison., Results: Mean arterial distension was significantly lower in abdominal aortic aneurysm mice between day 0 and day 7 post-onset of disease (p < 0.01) and between day 0 and day 14 (p < 0.001), while no difference was observed in sham control mice. Manual analysis detected a significant decrease (p < 0.05) between day 0 and day 14 only. Atherosclerotic mice showed alterations in arterial distension relating to genetic modification and diet. Arterial distension was significantly lower (p < 0.05) in Ldlr
-/- (++/--) mice fed high-fat western diet when compared with both wild type (++/++) mice and Ldlr-/- (++/--) mice fed chow diet. The manual method did not detect a significant difference between these groups., Conclusions: Arterial distension can be used as an early marker for the detection of arterial disease in murine models. The semi-automatic analysis method provided increased sensitivity to differences between experimental groups when compared to the manual analysis method.- Published
- 2019
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57. N/OFQ-NOP System in Peripheral and Central Immunomodulation.
- Author
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Kadhim S, Bird MF, and Lambert DG
- Subjects
- Animals, Immunomodulation, Ligands, Nociceptin, Opioid Peptides metabolism, Receptors, Opioid metabolism
- Abstract
Classical opioids (μ: mu, MOP; δ: delta, DOP and κ: kappa, KOP) variably affect immune function; they are immune depressants and there is good clinical evidence in the periphery. In addition, there is evidence for a central role in the control of a number of neuropathologies, e.g., neuropathic pain. Nociceptin/Orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor, NOP; peripheral and central activation can modulate immune function. In the periphery, NOP activation generally depresses immune function, but unlike classical opioids this is in part driven by NOP located on circulating immune cells. Peripheral activation has important implications in pathologies like asthma and sepsis. NOP is expressed on central neurones and glia where activation can modulate glial function. Microglia, as resident central 'macrophages', increase/infiltrate in pain and following trauma; these changes can be reduced by N/OFQ. Moreover, the interaction with other glial cell types such as the ubiquitous astrocytes and their known cross talk with microglia open a wealth of possibilities for central immunomodulation. At the whole animal level, clinical ligands with wide central and peripheral distribution have the potential to modulate immune function, and defining the precise nature of that interaction is important in mitigating or even harnessing the adverse effect profile of these important drugs.
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- 2019
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58. Perioperative medicine and UK plc.
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Ackland GL, Galley HF, Shelley B, and Lambert DG
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- Anesthesia methods, Drug Discovery methods, Drug Discovery trends, Humans, Multimorbidity trends, Perioperative Care methods, United Kingdom, Anesthesia trends, Perioperative Care trends, State Medicine trends
- Published
- 2019
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59. Ketamine and depression.
- Author
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Hirota K and Lambert DG
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- Anti-Inflammatory Agents pharmacology, Catecholamines metabolism, Depression drug therapy, Humans, Ketamine adverse effects, Ketamine therapeutic use, Receptors, N-Methyl-D-Aspartate antagonists & inhibitors, Antidepressive Agents pharmacology, Ketamine pharmacology
- Published
- 2018
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60. Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.
- Author
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Bird MF, Guerrini R, Willets JM, Thompson JP, Caló G, and Lambert DG
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- Animals, CHO Cells, Cells, Cultured, Cricetulus, HEK293 Cells, Humans, Neutrophils metabolism, Receptors, Opioid metabolism, Nociceptin, Fluorescent Dyes chemistry, Opioid Peptides chemistry, Receptors, Opioid analysis
- Abstract
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/OFQ
ATTO594 ) 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., (© 2018 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)- Published
- 2018
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61. Nociceptin/orphanin FQ receptor ligands and translational challenges: focus on cebranopadol as an innovative analgesic.
- Author
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Calo G and Lambert DG
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- Analgesics, Non-Narcotic adverse effects, Analgesics, Non-Narcotic therapeutic use, Clinical Trials as Topic, Humans, Indoles adverse effects, Indoles therapeutic use, Receptors, Opioid, mu agonists, Spiro Compounds adverse effects, Spiro Compounds therapeutic use, Nociceptin Receptor, Analgesics, Non-Narcotic pharmacology, Indoles pharmacology, Receptors, Opioid agonists, Spiro Compounds pharmacology
- Abstract
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., (Copyright © 2018 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2018
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62. Opioids, gliosis and central immunomodulation.
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Kadhim S, McDonald J, and Lambert DG
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- Animals, Astrocytes drug effects, Gliosis metabolism, Humans, Immunomodulation drug effects, Microglia drug effects, Neuralgia physiopathology, Neuroglia drug effects, Receptors, Opioid drug effects, Analgesics, Opioid pharmacology, Neuralgia drug therapy, Neuroglia metabolism
- Abstract
Neuropathic pain is a common health problem that affects millions of people worldwide. Despite being studied extensively, the cellular and molecular events underlying the central immunomodulation and the pathophysiology of neuropathic pain is still controversial. The idea that 'glial cells are merely housekeepers' is incorrect and with respect to initiation and maintenance of neuropathic pain, microglia and astrocytes have important roles to play. Glial cells differentially express opioid receptors and are thought to be functionally modulated by the activation of these receptors. In this review, we discuss evidence for glia-opioid modulation of pain by focusing on the pattern of astrocyte and microglial activation throughout the progress of nerve injury/neuropathic pain. Activation of astrocytes and microglia is a key step in central immunomodulation in terms of releasing pro-inflammatory markers and propagation of a 'central immune response'. Inhibition of astrocytes before and after induction of neuropathic pain has been found to prevent and reverse neuropathic pain, respectively. Moreover, microglial inhibitors have been found to prevent (but not to reverse) neuropathic pain. As they are expressed by glia, opioid receptors are expected to have a role to play in neuropathic pain.
- Published
- 2018
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63. In vitro and in vivo characterization of the bifunctional μ and δ opioid receptor ligand UFP-505.
- Author
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Dietis N, Niwa H, Tose R, McDonald J, Ruggieri V, Filaferro M, Vitale G, Micheli L, Ghelardini C, Salvadori S, Calo G, Guerrini R, Rowbotham DJ, and Lambert DG
- Subjects
- Animals, CHO Cells, Cricetulus, Injections, Spinal, Ligands, Male, Rats, Wistar, Receptors, Opioid, mu metabolism, Analgesics pharmacology, Analgesics therapeutic use, Oligopeptides pharmacology, Oligopeptides therapeutic use, Pain drug therapy, Receptors, Opioid, delta metabolism, Receptors, Opioid, mu agonists
- Abstract
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. GTPγ
35 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., (© 2018 The British Pharmacological Society.)- Published
- 2018
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64. 2017: A year of change for British Journal of Anaesthesia.
- Author
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Lambert DG
- Subjects
- Societies, Medical, United Kingdom, Anesthesiology trends, Periodicals as Topic trends
- Published
- 2018
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65. Mitochondrial pharmacology turns its sights on the Ca 2+ uniporter.
- Author
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Storey NM and Lambert DG
- Abstract
Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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66. In vitro pharmacological characterization of a novel unbiased NOP receptor-selective nonpeptide agonist AT-403.
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Ferrari F, Malfacini D, Journigan BV, Bird MF, Trapella C, Guerrini R, Lambert DG, Calo' G, and Zaveri NT
- Abstract
Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP), a member of the opioid receptor family. We recently identified a new high affinity and highly selective NOP agonist AT-403. In this study, we characterized the functional profile of AT-403 and compared it to other known nonpeptide NOP agonists Ro 65-6570, Ro 2q, SCH-221510, MCOPPB, AT-202 and SCH-486757, using the following assays: GTPγ[
35 S] stimulated binding, calcium mobilization assay in cells-expressing human NOP or classical opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, and the electrically stimulated mouse vas deferens bioassay. All compounds behaved as NOP full agonists consistently showing the following rank order of potency MCOPPB > AT-403 > Ro 65-6570 = Ro 2q > SCH-221510 > AT-202 > SCH-486757. AT-403 and MCOPPB displayed the highest NOP selectivity both at human and murine receptors. Interestingly, while all the other nonpeptide NOP agonists displayed bias toward G protein-mediated signaling in the BRET assay, AT-403, similar to the natural ligand N/OFQ, behaved as an unbiased agonist, activating G-protein-mediated function as well as arrestin recruitment. AT-403 may be a useful nonpeptide tool compound to study the pharmacology of NOP activation in disease states., (© 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)- Published
- 2017
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67. Urotensin-II peptidomimetic incorporating a non-reducible 1,5-triazole disulfide bond reveals a pseudo-irreversible covalent binding mechanism to the urotensin G-protein coupled receptor.
- Author
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Pacifico S, Kerckhoffs A, Fallow AJ, Foreman RE, Guerrini R, McDonald J, Lambert DG, and Jamieson AG
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- Humans, Models, Molecular, Protein Binding, Protein Conformation, Disulfides chemistry, Peptidomimetics chemistry, Peptidomimetics metabolism, Receptors, G-Protein-Coupled metabolism, Triazoles chemistry, Urotensins chemistry
- Abstract
The urotensin-II receptor (UTR) is a class A GPCR that predominantly binds to the pleiotropic cyclic peptide urotensin-II (U-II). U-II is constrained by a disulfide bridge that induces a β-turn structure and binds pseudo-irreversibly to UTR and is believed to result in a structural rearrangement of the receptor. However, it is not well understood how U-II binds pseudo-irreversibly and the nature of the reorganization of the receptor that results in G-protein activation. Here we describe a series of U-II peptidomimetics incorporating a non-reducible disulfide bond structural surrogate to investigate the feasibility that native U-II binds to the G protein-coupled receptor through disulfide bond shuffling as a mechanism of covalent interaction. Disubstituted 1,2,3-triazoles were designed with the aid of computational modeling as a non-reducible mimic of the disulfide bridge (Cys5-Cys10) in U-II. Solid phase synthesis using CuAAC or RuAAC as the key macrocyclisation step provided four analogues of U-II(4-11) incorporating either a 1,5-triazole bridge (5, 6) or 1,4-triazole bridge (9, 10). Biological evaluation of compounds 5, 6, 9 and 10 was achieved using in vitro [
125 I]UII binding and [Ca2+ ]i assays at recombinant human UTR. Compounds 5 and 6 demonstrated high affinity (KD ∼ 10 nM) for the UTR and were also shown to bind reversibly as predicted and activate the UTR to increase [Ca2+ ]i . Importantly, our results provide new insight into the mechanism of covalent binding of U-II with the UTR.- Published
- 2017
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68. Pharmacological studies on the NOP and opioid receptor agonist PWT2-[Dmt 1 ]N/OFQ(1-13).
- Author
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Cerlesi MC, Ding H, Bird MF, Kiguchi N, Ferrari F, Malfacini D, Rizzi A, Ruzza C, Lambert DG, Ko MC, Calo G, and Guerrini R
- Subjects
- Animals, CHO Cells, Chemistry Techniques, Synthetic, Cricetinae, Cricetulus, Female, Humans, Macaca mulatta, Male, Oligopeptides chemistry, Recombinant Proteins metabolism, Nociceptin Receptor, Oligopeptides chemical synthesis, Oligopeptides pharmacology, Receptors, Opioid agonists
- Abstract
An innovative chemical strategy named peptide welding technology (PWT) has been developed for the facile synthesis of tetrabranched peptides. [Dmt
1 ]N/OFQ(1-13)-NH2 acts as a universal agonist for nociceptin/orphanin FQ (N/OFQ) and classical opioid receptors. The present study investigated the pharmacological profile of the PWT derivative of [Dmt1 ]N/OFQ(1-13)NH2 (PWT2-[Dmt1 ]) in several assays in vitro and in vivo after spinal administration in monkeys subjected to the tail withdrawal assay. PWT2-[Dmt1 ] mimicked the effects of [Dmt1 ]N/OFQ(1-13)-NH2 displaying full agonist activity, similar affinity/potency and selectivity at human recombinant N/OFQ (NOP) and opioid receptors in receptor binding, stimulation of [35 S]GTPγS binding, calcium mobilization in cells expressing chimeric G proteins, and BRET studies for measuring receptor/G-protein and receptor/β-arrestin 2 interaction. In vivo in monkeys PWT2-[Dmt1 ] elicited dose-dependent and robust antinociceptive effects being more potent and longer lasting than [Dmt1 ]N/OFQ(1-13)-NH2 . The analgesic action of PWT2-[Dmt1 ] was sensitive to the NOP receptor antagonist J-113397, but not naltrexone. Thus, the present study demonstrated that the tetrabranched derivative of [Dmt1 ]N/OFQ(1-13)-NH2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide but displays higher potency and longer lasting action in vivo., (Copyright © 2016 Elsevier B.V. All rights reserved.)- Published
- 2017
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69. An Unusual Clinical Presentation of Solitary Fibrous Tumor in the Oral Cavity.
- Author
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de Morais EF, Moreira DG, Oliveira VA, Rodrigues RR, Germano AR, and Freitas RA
- Abstract
Solitary fibrous tumor is a rare neoplasm of mesenchymal origin that usually affects the pleura. This rarity becomes more relevant in the oral cavity since the clinical features are nonspecific. A 66-year-old female patient presented with a 3-month history of a swelling in the floor of the mouth, measuring 2 cm in greatest diameter, and pain symptomatology. Occlusal and panoramic radiographs showed no bone involvement. Ultrasonography of the submandibular and parotid salivary glands revealed normal morphology, dimensions, and echogenicity. During this exam, a nodular image of low echogenicity measuring about 2.7 × 1.8 cm was detected. An excisional biopsy was performed and histopathological analysis revealed a well-defined tumor-like lesion with alternation between hypercellular areas without a defined pattern and hypocellular areas. On immunohistochemistry, the tumor was positive for CD34 and CD99 and negative for α -SMA, S-100, and bcl-2. Combining the histopathological and immunohistochemical features, the diagnosis was solitary fibrous tumor. The patient is under periodical clinical follow-up and shows no signs of recurrence 7 months after surgical excision of the tumor. The combination of clinical-pathological and immunohistochemical features is necessary for the diagnosis., Competing Interests: All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria, educational grants, participation in speakers' bureaus, membership, employment, consultancies, stock ownership, or other equity interest, expert testimony, or patent-licensing arrangements) or nonfinancial interest (such as personal or professional relationships, affiliations, and knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
- Published
- 2017
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70. Effects of cannabinoid receptor activation by CP55,940 on normal bladder function and irritation-induced bladder overactivity in non-awake anaesthetised rats.
- Author
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Bakali E, Mbaki Y, Lambert DG, Elliott RA, Mason R, and Tincello DG
- Subjects
- Acetic Acid, Administration, Intravesical, Animals, Disease Models, Animal, Female, Indoles administration & dosage, Piperidines administration & dosage, Pyrazoles administration & dosage, Rats, Rats, Sprague-Dawley, Treatment Outcome, Urinary Bladder physiopathology, Urinary Bladder, Overactive chemically induced, Urinary Bladder, Overactive physiopathology, Urination drug effects, Urodynamics drug effects, Cannabinoid Receptor Agonists pharmacology, Cyclohexanols pharmacology, Urinary Bladder drug effects, Urinary Bladder, Overactive drug therapy
- Abstract
Introduction and Hypothesis: This study was designed to evaluate the effects of CP55,940 on normal bladder function in vivo and examine whether it suppresses urinary frequency induced by nociceptive stimuli in the bladder. Cannabinoid receptor (CBR) activity may be involved in the regulation of bladder function. However, the role of CBR subtypes in micturition has yet to be established. CP55,940 is a synthetic analogue of tetrahydrocannabidiol, which is a psychoactive ingredient of the Cannabis plant., Methods: Cystometry under urethane anaesthesia was performed to evaluate the effect of intravesical delivery of CP55,940 with or without administration of CB1 antagonist AM251 or CB2 antagonist AM630 on bladder function in female rats. The effects of CP55,940 were also examined in rats with urinary irritation induced by intravesical infusion of acetic acid., Results: Infusion of CP55,940 significantly (p < 0.05) increased micturition interval (MI) and bladder capacity (BC) by 52 % and decreased maximal voiding pressure (MP) by 25 %. Pretreatment with AM251 or AM630 before CP55,940 administration prevented CP55,940-induced increases in MI, BC and reduced MP. Acetic acid induced urinary frequency as evidenced by a reduction in MI and was suppressed by CP55,940., Conclusions: CP55,940 decreases bladder activity and urinary frequency induced by nociceptive stimuli, probably by suppression of bladder afferent activity. Effects of CP55,940 were abolished by both CBR antagonists. This data implicates a role for the endocannabinoid system in bladder mechanoafferent function in rats. In addition, our results show that CP55,940 reverses urinary frequency exemplified in an overactive bladder model, suggesting it could be an effective treatment for patients with lower urinary tract symptoms.
- Published
- 2016
- Full Text
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71. Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo).
- Author
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Bird MF, Cerlesi MC, Brown M, Malfacini D, Vezzi V, Molinari P, Micheli L, Di Cesare Mannelli L, Ghelardini C, Guerrini R, Calò G, and Lambert DG
- Subjects
- Animals, CHO Cells, Calcium metabolism, Cricetulus, Guinea Pigs, HEK293 Cells, Humans, Male, Peptides chemistry, Peptides pharmacology, Rats, Rats, Sprague-Dawley, Receptors, Opioid chemistry, Receptors, Opioid metabolism, Receptors, Opioid, mu chemistry, Receptors, Opioid, mu metabolism, Nociceptin Receptor, Opioid Peptides chemistry, Peptides chemical synthesis, Receptors, Opioid agonists, Receptors, Opioid, mu agonists
- Abstract
Introduction: Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ., Methods: We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats., Results: DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats., Conclusion: Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.
- Published
- 2016
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72. Nociceptin/orphanin FQ (N/OFQ) modulates immunopathology and airway hyperresponsiveness representing a novel target for the treatment of asthma.
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Singh SR, Sullo N, Matteis M, Spaziano G, McDonald J, Saunders R, Woodman L, Urbanek K, De Angelis A, De Palma R, Berair R, Pancholi M, Mistry V, Rossi F, Guerrini R, Calò G, D'Agostino B, Brightling CE, and Lambert DG
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- Animals, Asthma drug therapy, Asthma pathology, Cells, Cultured, Female, Humans, Inflammation immunology, Male, Mice, Mice, Inbred BALB C, Middle Aged, Opioid Peptides administration & dosage, Receptors, Opioid genetics, Receptors, Opioid immunology, Respiratory Hypersensitivity drug therapy, Respiratory Hypersensitivity pathology, Nociceptin Receptor, Nociceptin, Asthma immunology, Opioid Peptides immunology, Respiratory Hypersensitivity immunology
- Abstract
Background and Purpose: There is evidence supporting a role for the nociceptin/orphanin FQ (N/OFQ; NOP) receptor and its endogenous ligand N/OFQ in the modulation of neurogenic inflammation, airway tone and calibre. We hypothesized that NOP receptor activation has beneficial effects upon asthma immunopathology and airway hyperresponsiveness. Therefore, the expression and function of N/OFQ and the NOP receptor were examined in healthy and asthmatic human airway tissues. The concept was further addressed in an animal model of allergic asthma., Experimental Approach: NOP receptor expression was investigated by quantitative real-time PCR. Sputum N/OFQ was determined by RIA. N/OFQ function was tested using several assays including proliferation, migration, collagen gel contraction and wound healing. The effects of N/OFQ administration in vivo were studied in ovalbumin (OVA)-sensitized and challenged mice., Key Results: NOP receptors were expressed on a wide range of human and mouse immune and airway cells. Eosinophils expressed N/OFQ-precursor mRNA and their number correlated with N/OFQ concentration. N/OFQ was found in human sputum and increased in asthma. Additionally, in asthmatic human lungs N/OFQ immunoreactivity was elevated. NOP receptor activation inhibited migration of immunocytes and increased wound healing in airway structural cells. Furthermore, N/OFQ relaxed spasmogen-stimulated gel contraction. Remarkably, these findings were mirrored in OVA-mice where N/OFQ treatment before or during sensitization substantially reduced airway constriction and immunocyte trafficking to the lung, in particular eosinophils. N/OFQ also reduced inflammatory mediators and mucin production., Conclusions and Implications: We demonstrated a novel dual airway immunomodulator/bronchodilator role for N/OFQ and suggest targeting this system as an innovative treatment for asthma., (© 2016 The British Pharmacological Society.)
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- 2016
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73. Evidence for nociceptin/orphanin FQ (NOP) but not µ (MOP), δ (DOP) or κ (KOP) opioid receptor mRNA in whole human blood.
- Author
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Al-Hashimi M, McDonald J, Thompson JP, and Lambert DG
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- Adult, Female, Humans, Male, Middle Aged, Polymerase Chain Reaction, Reference Values, Nociceptin Receptor, RNA, Messenger metabolism, Receptors, Opioid metabolism, Receptors, Opioid, delta metabolism, Receptors, Opioid, kappa metabolism, Receptors, Opioid, mu metabolism
- Abstract
Background: While it is well known that opioids depress the immune system, the site(s) of action for this depression is highly controversial. Immune modulation could occur directly at the immune cell or centrally via the hypothalamic-pituitary-adrenal axis. In a number of studies using individual enriched immune cell populations we have failed to detect classical µ (MOP), δ (DOP) and κ (KOP) receptors. The non-classical nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is expressed on all cells examined thus far. Our hypothesis was that immune cells do not express classical opioid receptors and that using whole blood would definitively answer this question., Methods: Whole blood (containing all immune cell types) was incubated with opioids (morphine and fentanyl) commonly encountered in anaesthesia and with agents mimicking sepsis [lipopolysaccharide (LPS) and peptidoglycan G (PepG)]. Opioid receptor mRNA expression was assessed by endpoint polymerase chain reaction (PCR) with gel visualisation and quantitative PCR., Results: Classical MOP, DOP, and KOP receptors were not detected in any of the samples tested either at rest or when challenged with opioids, LPS or PepG. Commercial primers for DOP did not perform well in quantitative PCR, so the absence of expression was confirmed using a traditional gel-based approach. NOP receptors were detected in all samples; expression was unaffected by opioids and reduced by LPS/PepG combinations., Conclusions: Classical opioid receptors are not expressed on circulating immune cells., (© The Author 2016. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
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- 2016
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74. Cannabinoid receptor expression in the bladder is altered in detrusor overactivity.
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Bakali E, McDonald J, Elliott RA, Lambert DG, and Tincello DG
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- Aged, Female, Humans, Male, Middle Aged, Urinary Bladder, Overactive physiopathology, Receptors, Cannabinoid biosynthesis, Urinary Bladder metabolism, Urinary Bladder, Overactive metabolism
- Abstract
Introduction: Immunohistochemical (IHC) evidence shows that cannabinoid receptors (CB) are expressed in human bladders and cannabinoid agonists are known to inhibit detrusor contractility. However, the mechanism for this inhibition remains unknown. In addition, the role of CB in detrusor overactivity (DO) is under-investigated. The aim of this study was to compare CB expression in normal and DO human bladders and to further characterise these receptors., Methods: Polymer chain reaction (PCR) was used to detect differences in CB transcripts in bladder samples. Differences in CB protein expression was assessed by IHC. Immunofluorescence (IF) was used to evaluate co-localisation of CB with nerve fibres. Receptor density and binding affinity were measured using the cannabinoid radioligand [(3)H]-CP-55,940., Results: There were higher levels of CB1 transcripts in the urothelium of patients with DO and lower levels in the detrusor, compared with normal bladders. Radioligand binding revealed CB density of 421 ± 104 fmol/mg protein in normal human bladders. IHC confirmed these findings at the protein level. IF staining demonstrated co-localisation of CB1 with choline acetyltransferase-(ChAT)-positive nerves in the detrusor and co-localisation with PGP9.5 in both urothelium and detrusor. CB2 was co-localised with both ChAT and PGP9.5 in the urothelium and the detrusor., Conclusions: Cannabinoid receptor expression is reduced in the detrusor of patients with DO, which may play a role in the pathophysiology of the disease. Co-localisation of CB receptors with cholinergic nerves may suggest that CB1, being localised on pre- and postsynaptic terminals, could influence neurotransmitter release. Our findings suggest the potential role of cannabinoid agonists in overactive bladder pharmacotherapy.
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- 2016
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75. Opioids and neovascularization; pro or anti?
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Mahbuba W and Lambert DG
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- Humans, Neovascularization, Pathologic physiopathology, Receptors, Opioid physiology, Signal Transduction physiology, Vascular Endothelial Growth Factor A physiology, Analgesics, Opioid adverse effects, Neovascularization, Pathologic chemically induced
- Published
- 2015
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76. Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.
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Ayakannu T, Taylor AH, Willets JM, Brown L, Lambert DG, McDonald J, Davies Q, Moss EL, and Konje JC
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- Algorithms, Carcinoma, Endometrioid pathology, Case-Control Studies, Cyclophilin A genetics, Endometrial Neoplasms pathology, Female, Gene Expression Regulation, Neoplastic, Humans, Mitochondrial Proteins genetics, Neoplasm Grading, Predictive Value of Tests, Receptor, Cannabinoid, CB1 genetics, Reference Values, Reproducibility of Results, Ribosomal Proteins genetics, Software, beta Karyopherins genetics, Biomarkers, Tumor genetics, Carcinoma, Endometrioid genetics, Endometrial Neoplasms genetics, Gene Expression Profiling standards, Real-Time Polymerase Chain Reaction standards
- Abstract
Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2015
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77. Simultaneous targeting of multiple opioid receptor types.
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Bird MF and Lambert DG
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- Acute Pain drug therapy, Analgesics, Opioid administration & dosage, Analgesics, Opioid adverse effects, Analgesics, Opioid therapeutic use, Chronic Pain drug therapy, Drug Synergism, Humans, Molecular Targeted Therapy adverse effects, Morphine adverse effects, Morphine therapeutic use, Receptors, Opioid, delta drug effects, Drug Tolerance physiology, Morphine administration & dosage, Receptors, Opioid, delta antagonists & inhibitors, Receptors, Opioid, mu drug effects
- Abstract
Purpose of Review: This article aims to discuss the multitarget concept for opioid receptor ligands framed on early observations that activating MOP (mu:μ) receptor whilst simultaneously blocking DOP (delta:δ) receptors reduces the onset of morphine tolerance. The review period is ostensibly calendar year 2014 but the new work in 2013 is also covered., Recent Findings: Two molecules of interest with MOP agonist/DOP agonist and MOP agonist/DOP antagonist profiles were described: Rv-Jim-C3 and 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), respectively. Both were effective in neuropathic pain (wherein classical single target opioids have low efficacy) with the latter having a predicted reduced tolerance profile. BU0807 is a buprenorphine derivative with mixed MOP/NOP agonist activity and this was shown to be effective in abdominal pain. SR16435 and GRT6005 (cebranopadol) are mixed MOP/MOP agonists with varying degrees of partial agonism. Both displayed significant antinociceptive activity and reduced tolerance potential in preclinical models., Summary: There is growing evidence for and interest in the design and evaluation of mixed opioids that extend beyond the MOP/DOP pairing to now include NOP. Indeed, a mixed MOP/NOP ligand is close to the clinic; this will reinvigorate the search for other mixed molecules with reduced side-effect profiles.
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- 2015
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78. Development and characterisation of novel fentanyl-delta opioid receptor antagonist based bivalent ligands.
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Bird MF, Vardanyan RS, Hruby VJ, Calò G, Guerrini R, Salvadori S, Trapella C, McDonald J, Rowbotham DJ, and Lambert DG
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- Animals, Arrestins metabolism, CHO Cells, Cricetinae, Cricetulus, Drug Discovery, Guanosine 5'-O-(3-Thiotriphosphate) metabolism, Ligands, Receptors, Opioid, delta physiology, Receptors, Opioid, mu physiology, beta-Arrestins, Dipeptides pharmacology, Fentanyl pharmacology, Receptors, Opioid, delta antagonists & inhibitors, Tetrahydroisoquinolines pharmacology
- Abstract
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 β-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 β-arrestin assays, compared with fentanyl (with response at maximum concentration (RMC):13.62), all compounds showed reduced ability to activate β-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., (© The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
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- 2015
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79. Structure activity studies of nociceptin/orphanin FQ(1-13)-NH2 derivatives modified in position 5.
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Guerrini R, Marzola E, Trapella C, Pacifico S, Cerlesi MC, Malfacini D, Ferrari F, Bird MF, Lambert DG, Salvadori S, and Calo G
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- Animals, CHO Cells, Cricetinae, Cricetulus, Humans, Protein Binding physiology, Structure-Activity Relationship, Nociceptin Receptor, Nociceptin, Opioid Peptides chemistry, Opioid Peptides metabolism, Receptors, Opioid metabolism
- Abstract
Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1-13)-NH2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1-13)-NH2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X(5)]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [(35)S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X(5)]N/OFQ(1-13)-NH2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo/hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
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- 2015
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80. Cebranopadol: a first in-class example of a nociceptin/orphanin FQ receptor and opioid receptor agonist.
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Lambert DG, Bird MF, and Rowbotham DJ
- Subjects
- Animals, Humans, Nociceptin Receptor, Indoles pharmacology, Receptors, Opioid agonists, Spiro Compounds pharmacology
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- 2015
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81. Properdin levels in human sepsis.
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Stover CM, McDonald J, Byrne S, Lambert DG, and Thompson JP
- Abstract
Properdin is a normal serum protein that increases the production of complement activation products by binding C3b integral to convertase complexes and amplifying their activity at the site of activation. Thereby, it not only can aid in the resolution of infection but also contribute to tissue damage. In human sepsis, circulating complement C3 concentrations are decreased, though C3 is described as a positive acute phase reactant. However, properdin levels in human sepsis have not been reported. In this study, serum from 81 critically ill patients (predominately abdominal and respiratory sepsis) were analyzed for properdin levels at defined points of their stay in the intensive care unit (ICU) and compared with 61 age and sex-matched healthy volunteers. Properdin concentrations were significantly decreased in patients with sepsis on admission to ICU, but increased after clinical recovery to exceed levels observed in healthy volunteers. Properdin concentrations at ICU admission were decreased in non-survivors of sepsis compared to survivors, but this did not correlate with APACHE II score. However, pathologically low properdin levels (<7 μg/ml) were related to increased duration of treatment.
- Published
- 2015
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82. International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, urotensin II-related peptide, and their receptor: from structure to function.
- Author
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Vaudry H, Leprince J, Chatenet D, Fournier A, Lambert DG, Le Mével JC, Ohlstein EH, Schwertani A, Tostivint H, and Vaudry D
- Subjects
- Amino Acid Sequence, Animals, Hormone Antagonists pharmacology, Humans, Intracellular Signaling Peptides and Proteins, Ligands, Molecular Sequence Data, Peptide Hormones antagonists & inhibitors, Peptide Hormones chemistry, Peptide Hormones genetics, Protein Conformation, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled genetics, Signal Transduction, Structure-Activity Relationship, Urotensins antagonists & inhibitors, Urotensins chemistry, Urotensins genetics, Peptide Hormones metabolism, Receptors, G-Protein-Coupled metabolism, Urotensins metabolism
- Abstract
Urotensin II (UII) is a cyclic neuropeptide that was first isolated from the urophysis of teleost fish on the basis of its ability to contract the hindgut. Subsequently, UII was characterized in tetrapods including humans. Phylogenetic studies and synteny analysis indicate that UII and its paralogous peptide urotensin II-related peptide (URP) belong to the somatostatin/cortistatin superfamily. In mammals, the UII and URP genes are primarily expressed in cholinergic neurons of the brainstem and spinal cord. UII and URP mRNAs are also present in various organs notably in the cardiovascular, renal, and endocrine systems. UII and URP activate a common G protein-coupled receptor, called UT, that exhibits relatively high sequence identity with somatostatin, opioid, and galanin receptors. The UT gene is widely expressed in the central nervous system (CNS) and in peripheral tissues including the retina, heart, vascular bed, lung, kidney, adrenal medulla, and skeletal muscle. Structure-activity relationship studies and NMR conformational analysis have led to the rational design of a number of peptidic and nonpeptidic UT agonists and antagonists. Consistent with the wide distribution of UT, UII has now been shown to exert a large array of biologic activities, in particular in the CNS, the cardiovascular system, and the kidney. Here, we review the current knowledge concerning the pleiotropic actions of UII and discusses the possible use of antagonists for future therapeutic applications., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)
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- 2015
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83. Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system.
- Author
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Thomas RC, Bath MF, Stover CM, Lambert DG, and Thompson JP
- Subjects
- Animals, Cycloheptanes pharmacology, Disease Models, Animal, Dose-Response Relationship, Drug, Interleukin-1beta metabolism, Mice, Mice, Inbred BALB C, Opioid Peptides pharmacology, Piperidines pharmacology, Rats, Rats, Wistar, Sepsis chemically induced, Sepsis pathology, Tumor Necrosis Factor-alpha metabolism, Nociceptin Receptor, Nociceptin, Lipopolysaccharides toxicity, Opioid Peptides metabolism, Receptors, Opioid metabolism, Sepsis metabolism
- Abstract
The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2mg/kg produced a profound response within 5h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25mg/kg resulted in marked lethargy before 24h. Splenic interleukin-1β mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1β and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose-response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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84. Nociceptin system as a target in sepsis?
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Thomas R, Stover C, Lambert DG, and Thompson JP
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- Animals, Humans, Pain physiopathology, Sepsis therapy, Vasodilation physiology, Nociceptin Receptor, Nociceptin, Opioid Peptides metabolism, Receptors, Opioid metabolism, Sepsis physiopathology
- Abstract
The nociceptin system comprises the nociceptin receptor (NOP) and the ligand nociceptin/orphanin FQ (N/OFQ) that binds to the receptor. The archetypal role of the system is in pain processing but the NOP receptor is also expressed on immune cells. Activation of the NOP receptor is known to modulate inflammatory responses, such as mast-cell degranulation, neutrophil rolling, vasodilation, increased vascular permeability, adhesion molecule regulation and leucocyte recruitment. As there is a loss of regulation of inflammatory responses during sepsis, the nociceptin system could be a target for therapies aimed at modulating sepsis. This review details the known effects of NOP activation on leucocytes and the vascular endothelium and discusses the most recent human and animal data on the role of the nociceptin system in sepsis.
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- 2014
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85. In vitro and in vivo pharmacological characterization of nociceptin/orphanin FQ tetrabranched derivatives.
- Author
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Rizzi A, Malfacini D, Cerlesi MC, Ruzza C, Marzola E, Bird MF, Rowbotham DJ, Salvadori S, Guerrini R, Lambert DG, and Calo G
- Subjects
- Animals, CHO Cells, Cells, Cultured, Cricetulus, Dose-Response Relationship, Drug, Humans, Locomotion drug effects, Male, Mice, Mice, Knockout, Molecular Conformation, Opioid Peptides chemical synthesis, Receptors, Opioid deficiency, Structure-Activity Relationship, Opioid Peptides chemistry, Opioid Peptides pharmacology, Receptors, Opioid agonists
- Abstract
Background and Purpose: An innovative chemical approach, named peptide welding technology (PWT), allows the synthesis of multibranched peptides with extraordinary high yield, purity and reproducibility. With this approach, three different tetrabranched derivatives of nociceptin/orphanin FQ (N/OFQ) have been synthesized and named PWT1-N/OFQ, PWT2-N/OFQ and PWT3-N/OFQ. In the present study we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide., Experimental Approach: The following in vitro assays were used: receptor and [(35)S]-GTPγS binding, calcium mobilization in cells expressing the human N/OFQ peptide (NOP) receptor, or classical opioid receptors and chimeric G proteins, electrically stimulated mouse vas deferens bioassay. In vivo experiments were performed; locomotor activity was measured in normal mice and in animals with the NOP receptor gene knocked out [NOP(-/-)]., Key Results: In vitro PWT derivatives of N/OFQ behaved as high affinity potent and rather selective full agonists at human recombinant and animal native NOP receptors. In vivo PWT derivatives mimicked the inhibitory effects exerted by the natural peptide on locomotor activity showing 40-fold higher potency and extremely longer lasting action. The effects of PWT2-N/OFQ were no longer evident in NOP(-/-) mice., Conclusions and Implications: The results showed that the PWT can be successfully applied to the peptide sequence of N/OFQ to generate tetrabranched derivatives characterized by a pharmacological profile similar to the native peptide and associated with a higher potency and marked prolongation of action in vivo., (© 2014 The British Pharmacological Society.)
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- 2014
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86. Functional plasticity of the N/OFQ-NOP receptor system determines analgesic properties of NOP receptor agonists.
- Author
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Schröder W, Lambert DG, Ko MC, and Koch T
- Subjects
- Analgesics pharmacology, Analgesics therapeutic use, Animals, Calcium Channels, N-Type metabolism, Humans, Pain drug therapy, Protein Isoforms metabolism, Receptors, Opioid chemistry, Nociceptin Receptor, Pain metabolism, Receptors, Opioid agonists, Receptors, Opioid metabolism
- Abstract
Despite high sequence similarity between NOP (nociceptin/orphanin FQ opioid peptide) and opioid receptors, marked differences in endogenous ligand selectivity, signal transduction, phosphorylation, desensitization, internalization and trafficking have been identified; underscoring the evolutionary difference between NOP and opioid receptors. Activation of NOP receptors affects nociceptive transmission in a site-specific manner, with antinociceptive effects prevailing after peripheral and spinal activation, and pronociceptive effects after supraspinal activation in rodents. The net effect of systemically administered NOP receptor agonists on nociception is proposed to depend on the relative contribution of peripheral, spinal and supraspinal activation, and this may depend on experimental conditions. Functional expression and regulation of NOP receptors at peripheral and central sites of the nociceptive pathway exhibits a high degree of plasticity under conditions of neuropathic and inflammatory pain. In rodents, systemically administered NOP receptor agonists exerted antihypersensitive effects in models of neuropathic and inflammatory pain. However, they were largely ineffective in acute pain while concomitantly evoking severe motor side effects. In contrast, systemic administration of NOP receptor agonists to non-human primates (NHPs) exerted potent and efficacious antinociception in the absence of motor and sedative side effects. The reason for this species difference with respect to antinociceptive efficacy and tolerability is not clear. Moreover, co-activation of NOP and μ-opioid peptide (MOP) receptors synergistically produced antinociception in NHPs. Hence, both selective NOP receptor as well as NOP/MOP receptor agonists may hold potential for clinical use as analgesics effective in conditions of acute and chronic pain., (© 2014 The British Pharmacological Society.)
- Published
- 2014
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87. Long-term activation of polymorph cannabinoid receptors does not affect receptor gene transcription.
- Author
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Beishon L, McDonald J, Thompson JP, and Lambert DG
- Subjects
- Cells, Cultured, HL-60 Cells, Humans, Neutrophils metabolism, Receptors, Cannabinoid biosynthesis, Transcription, Genetic
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- 2014
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88. Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder.
- Author
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Bakali E, Elliott RA, Taylor AH, Lambert DG, Willets JM, and Tincello DG
- Subjects
- Cell Line, Humans, Protein Binding physiology, Receptors, Cannabinoid, Urinary Bladder cytology, Urothelium cytology, Cannabinoids metabolism, Receptor, Cannabinoid, CB1 metabolism, Receptors, G-Protein-Coupled metabolism, Urinary Bladder metabolism, Urothelium metabolism
- Abstract
To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.
- Published
- 2014
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89. Radioimmunoassay, enzyme and non-enzyme-based immunoassays.
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Grange RD, Thompson JP, and Lambert DG
- Subjects
- Enzyme-Linked Immunosorbent Assay methods, Humans, Immunoassay methods, Immunoenzyme Techniques methods, Radioimmunoassay methods
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- 2014
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90. The Nociceptin/Orphanin FQ system is modulated in patients admitted to ICU with sepsis and after cardiopulmonary bypass.
- Author
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Thompson JP, Serrano-Gomez A, McDonald J, Ladak N, Bowrey S, and Lambert DG
- Subjects
- Aged, Case-Control Studies, Critical Care, Cytokines blood, Cytokines metabolism, Female, Gene Expression Regulation, Humans, Intensive Care Units, Male, Middle Aged, Opioid Peptides genetics, RNA, Messenger genetics, Receptors, Opioid genetics, Receptors, Opioid metabolism, Sepsis therapy, Time Factors, Nociceptin, Cardiopulmonary Bypass adverse effects, Opioid Peptides metabolism, Postoperative Complications, Sepsis etiology, Sepsis metabolism
- Abstract
Background and Objectives: Nociceptin/Orphanin FQ (N/OFQ) is a non-classical endogenous opioid peptide that modulates immune function in vitro. Its importance in inflammation and human sepsis is unknown. The objectives of this study were to determine the relationship between N/OFQ, transcripts for its precursor (pre-pro-N/OFQ [ppNOC]) and receptor (NOP), inflammatory markers and clinical outcomes in patients undergoing cardiopulmonary bypass and with sepsis., Methods: A prospective observational cohort study of 82 patients admitted to Intensive Care (ICU) with sepsis and 40 patients undergoing cardiac surgery under cardiopulmonary bypass (as a model of systemic inflammation). Sixty three healthy volunteers, matched by age and sex to the patients with sepsis were also studied. Clinical and laboratory details were recorded. Polymorph ppNOC and NOP receptor mRNA were determined using quantitative PCR. Plasma N/OFQ was determined using ELISA and cytokines (TNF- α, IL-8, IL-10) measured using radioimmunoassay. Data from patients undergoing cardiac surgery were recorded before, 3 and 24 hours after cardiopulmonary bypass. ICU patients with sepsis were assessed on Days 1 and 2 of ICU admission, and after clinical recovery., Main Results: Plasma N/OFQ concentrations increased (p<0.0001) on Days 1 and 2 of ICU admission with sepsis compared to matched recovery samples. Polymorph ppNOC (p= 0.019) and NOP mRNA (p<0.0001) decreased compared to healthy volunteers. TNF-α, IL-8 and IL-10 concentrations increased on Day 1 compared to matched recovery samples and volunteers (p<0.0001). Similar changes (increased plasma N/OFQ, [p=0.0058], decreased ppNOC [p<0.0001], increased IL-8 and IL-10 concentrations [both p<0.0001]) occurred after cardiac surgery but these were comparatively lower and of shorter duration., Conclusions: The N/OFQ system is modulated in ICU patients with sepsis with similar but reduced changes after cardiac surgery under cardiopulmonary bypass. Further studies are required to clarify the role of the N/OFQ system in inflammation and sepsis, and the mechanisms involved.
- Published
- 2013
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91. Can anesthetic techniques or drugs affect cancer recurrence in patients undergoing cancer surgery?
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Niwa H, Rowbotham DJ, Lambert DG, and Buggy DJ
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- Clinical Trials as Topic, Humans, Multicenter Studies as Topic, Prospective Studies, Randomized Controlled Trials as Topic, Retrospective Studies, Anesthesia adverse effects, Anesthetics adverse effects, Neoplasm Recurrence, Local etiology, Neoplasms surgery
- Abstract
Despite the development of effective chemotherapy and radiotherapy, surgery remains the mainstay treatment of many cancers, requiring anesthesia. Almost all cancer deaths after primary surgery are attributable to recurrence or metastases. Recently it has been hypothesized that the perioperative anesthetic management of cancer patients could potentially affect the risk of recurrence and metastases, which implies a key role for anesthesiologists in choosing anesthetic agents and techniques that optimize the balance between the metastatic potential of the tumor versus its elimination by antimetastatic immune defenses. This review summarizes available experimental information on the potential effects of common anesthetic agents and techniques on cancer metastases and the conflicting retrospective clinical data on regional anesthesia in various types of cancer. A number of prospective, randomized, multicenter, clinical trials are in progress, and their results are eagerly awaited.
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- 2013
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92. The nociceptin/orphanin FQ receptor antagonist UFP-101 reduces microvascular inflammation to lipopolysaccharide in vivo.
- Author
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Brookes ZL, Stedman EN, Brown NJ, Hebbes CP, Guerrini R, Calo G, Reilly CS, and Lambert DG
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- Animals, CHO Cells, Cardiovascular System drug effects, Cardiovascular System pathology, Cardiovascular System physiopathology, Cricetinae, Cricetulus, Fluorescein-5-isothiocyanate metabolism, Inflammation metabolism, Leukocyte Rolling drug effects, Male, Microcirculation drug effects, Microvessels drug effects, Microvessels pathology, Opioid Peptides metabolism, Rats, Rats, Wistar, Receptors, Opioid metabolism, Recombinant Proteins metabolism, Nociceptin Receptor, Nociceptin, Inflammation pathology, Narcotic Antagonists, Opioid Peptides pharmacology
- Abstract
Microvascular inflammation occurs during sepsis and the endogenous opioid-like peptide nociceptin/orphanin FQ (N/OFQ) is known to regulate inflammation. This study aimed to determine the inflammatory role of N/OFQ and its receptor NOP (ORL1) within the microcirculation, along with anti-inflammatory effects of the NOP antagonist UFP-101 (University of Ferrara Peptide-101) in an animal model of sepsis (endotoxemia). Male Wistar rats (220 to 300 g) were administered lipopolysaccharide (LPS) for 24 h (-24 h, 1 mg kg(-1); -2 h, 1 mg kg(-1) i.v., tail vein). They were then either anesthetised for observation of the mesenteric microcirculation using fluorescent in vivo microscopy, or isolated arterioles (~200 µm) were studied in vitro with pressure myography. 200 nM kg(-1) fluorescently labelled N/OFQ (FITC-N/OFQ, i.a., mesenteric artery) bound to specific sites on the microvascular endothelium in vivo, indicating sparse distribution of NOP receptors. In vitro, arterioles (~200 µm) dilated to intraluminal N/OFQ (10(-5)M) (32.6 + 8.4%) and this response was exaggerated with LPS (62.0 +7.9%, p=0.031). In vivo, LPS induced macromolecular leak of FITC-BSA (0.02 g kg(-1) i.v.) (LPS: 95.3 (86.7 to 97.9)%, p=0.043) from post-capillary venules (<40 µm) and increased leukocyte rolling as endotoxemia progressed (p=0.027), both being reduced by 150 nmol kg(-1) UFP-101 (i.v., jugular vein). Firstly, the rat mesenteric microcirculation expresses NOP receptors and secondly, NOP function (ability to induce dilation) is enhanced with LPS. UFP-101 also reduced microvascular inflammation to endotoxemia in vivo. Hence inhibition of the microvascular N/OFQ-NOP pathway may have therapeutic potential during sepsis and warrants further investigation.
- Published
- 2013
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93. Opioids and immune modulation: more questions than answers.
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Al-Hashimi M, Scott SW, Thompson JP, and Lambert DG
- Subjects
- Animals, Humans, Hypothalamo-Hypophyseal System drug effects, Hypothalamo-Hypophyseal System immunology, Models, Animal, Pituitary-Adrenal System drug effects, Pituitary-Adrenal System immunology, Analgesics, Opioid immunology, Analgesics, Opioid pharmacology, Immunomodulation drug effects, Immunomodulation immunology, Receptors, Opioid drug effects, Receptors, Opioid immunology
- Abstract
Opioid addicts are more likely to present with infections suggesting opioids are immune modulators. The potential sites/mechanism(s) for this modulation are controversial and on close inspection not well supported by the current literature. It has long been assumed that opioid-induced immune modulation occurs via a combination of direct actions on the immune cell itself, via the hypothalamic-pituitary-adrenal (HPA) axis, or both. Opioid receptors are classified as MOP (μ, mu), DOP (δ, delta), and KOP (κ, kappa)--classical naloxone sensitive receptors--or NOP (the receptor for nociceptin/orphanin FQ), which is naloxone insensitive. Opioids currently used in clinical practice predominantly target the MOP receptor. There do not appear to be classical opioid receptors present on immune cells. The evidence for HPA activation is also poor and shows some species dependence. Most opioids used clinically or as drugs of abuse do not target the NOP receptor. Other possible target sites for immune modulation include the sympathetic nervous system and central sites. We are currently unable to accurately define the cellular target for immune modulation and suggest further investigation is required. Based on the differences observed when comparing studies in laboratory animals and those performed in humans we suggest that further studies in the clinical setting are needed.
- Published
- 2013
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94. Measurement of [Ca²⁺]i in whole cell suspensions using Fura-2.
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Patel A, Hirst RA, Harrison C, Hirota K, and Lambert DG
- Subjects
- Animals, Cell Line, Humans, Tissue Culture Techniques, Calcium metabolism, Fura-2 metabolism
- Abstract
Use of Fura-2 in whole cell suspensions to measure changes in intracellular Ca(2+) is probably one of the simplest, yet most widely used protocols described in this volume. Whole cell suspensions are loaded with Fura-2 and then placed into a cuvette-based fluorimetric system (measuring 510 nm emission at alternating 340/340 nm excitation). Cells can be stimulated with agonists and antagonists to enable temporal response profiling and concentration-response curves to be constructed. The protocol can be used for a wide range of cells including those transfected with Ca(2+)-signaling proteins, e.g., receptors and channels. Loading characteristics and the need for agents to retain loaded dye (e.g., probenecid) need to be determined empirically. Calibration of whole cell suspensions to convert the fluorescent signal into Ca(2+) is simply performed using Triton-X lysis (to determine R (max)) and EGTA chelation (to determine R (min)).
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- 2013
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95. Ratiometric [Ca²⁺]i measurements in adherent cell-lines using the NOVOstar microplate reader.
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Hunt BD and Lambert DG
- Subjects
- Animals, CHO Cells, Cell Line, Cricetinae, Fura-2 metabolism, Humans, Calcium metabolism
- Abstract
The control of free ionized intracellular calcium concentration ([Ca(2+)](i)) is an established mechanism of cellular activation, regulating a diverse range of cellular events. Consequentially, experimental measurement of [Ca(2+)](i) is a potent technique for the medical science laboratory. The NOVOstar microplate reader is a versatile system, which may be easily configured to measure [Ca(2+)](i). Moreover, the relatively low cost of this system makes it an attractive one for researchers adhering to a modest budget, whilst allowing medium throughput to be achieved.These methods serve as a starting point for researchers wishing to measure intracellular calcium concentration in adherent cell-lines using the NOVOstar plate reader. Briefly, adherent cells are seeded into well plates 1 day prior to calcium determinations being made. On the day of the experiment, autofluorescence values of individual wells of cells are determined prior to the cells being loaded with the fluorophore, fura-2. [Ca(2+)](i) determinations are acquired by activating a predefined program within the NOVOstar software; full parameters are provided within this chapter for this purpose. Fluorescence ratio values may be easily calibrated to give absolute intracellular calcium concentrations (nM). Calibration involves determining experimental fluorescence at calcium-saturating and calcium-free conditions; ionomycin and EGTA are used to produce these two conditions respectively. Finally, mathematical calculation of absolute intracellular calcium concentration is described by use of the Grynkiewicz equation.
- Published
- 2013
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96. The effects of nociceptin peptide (N/OFQ)-receptor (NOP) system activation in the airways.
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Singh SR, Sullo N, D'Agostino B, Brightling CE, and Lambert DG
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- Animals, Anti-Asthmatic Agents pharmacology, Anti-Asthmatic Agents therapeutic use, Asthma drug therapy, Asthma immunology, Cough immunology, Cough metabolism, Humans, Immunomodulation, Respiratory System drug effects, Respiratory System immunology, Respiratory System metabolism, Nociceptin Receptor, Nociceptin, Asthma metabolism, Opioid Peptides physiology, Receptors, Opioid metabolism, Signal Transduction
- Abstract
The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide (NOP) receptor. It is cleaved from a larger precursor identified as prepronociceptin (ppN/OFQ). NOP is a member of the seven transmembrane-spanning G-protein coupled receptor (GPCR) family. ppN/OFQ and NOP receptors are widely distributed in different human tissues. Asthma is a complex heterogeneous disease characterized by variable airflow obstruction, bronchial hyper-responsiveness and chronic airway inflammation. Limited therapeutic effectiveness of currently available asthma therapies warrants identification of new drug compounds. Evidence from animal studies suggests that N/OFQ modulates airway contraction and inflammation. Interestingly up regulation of the N/OFQ-NOP system reduces airway hyper-responsiveness. In contrast, inflammatory cells central to the inflammatory response in asthma may be both sources of N/OFQ and respond to NOP activation. Hence paradoxical dysregulation of the N/OFQ-NOP system may potentially play an important role in regulating airway inflammation and airway tone. To date there is no data on N/OFQ-NOP expression in the human airways. Therefore, the potential role of N/OFQ-NOP system in asthma is unknown. This review focuses on its physiological effects within airways and potential value as a novel asthma therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2013
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97. Immunity and early pregnancy events: are endocannabinoids the missing link?
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Bambang KN, Lambert DG, Lam PM, Quenby S, Maccarrone M, and Konje JC
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- Animals, Cell Movement, Cytokines, Embryo Implantation, Female, Humans, Immune Tolerance, Placenta immunology, Pregnancy, Signal Transduction immunology, Endocannabinoids immunology, Fertility immunology, Killer Cells, Natural immunology, Receptor, Cannabinoid, CB1 immunology, T-Lymphocytes immunology
- Abstract
The success of pregnancy is dependent on a number of different cell types and signalling pathways, including immune cells which play a vital role in implantation. Immune cells express transcripts for all of the components of the endocannabinoid system, but the role of this system in the function of reproductive tract immune cells is still unclear. In this review, we present the hypothesis that the endocannabinoid signalling system is central to an endocannabinoid-immune-reproductive axis, and that it acts as the link via which immune cells exert their vital influence on implantation and foetal tolerance. Pubmed and Web of Science databases were searched for studies published since 1975 which explore the interaction between the endocannabinoid system and the immune system, the endocannabinoid system in pregnancy as well as the role of immune cells in pregnancy. There is evidence that the endocannabinoid system has established effects in several immune cell lineages including NK cells and T lymphocytes known to be crucial in the development of normal pregnancy. These effects include regulation of cytokine production, chemotaxis and proliferation. The immune system plays a critical role in placental development and foetal tolerance, achieving this through a large number of cytokines and chemokines. We conclude that there are intricate molecular interactions involved in the success of early pregnancy and that the endocannabinoid system, potentially interacting with the immune system, is a key contributor to these events., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2012
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98. In vitro siRNA-mediated knockdown of the UT receptor: implications of density on the efficacy of a range of UT ligands.
- Author
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Hunt BD, Ng LL, and Lambert DG
- Subjects
- Animals, CHO Cells, Calcium metabolism, Cricetinae, Cricetulus, Gene Knockdown Techniques, Humans, Receptors, G-Protein-Coupled metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Peptide Fragments pharmacology, RNA, Small Interfering genetics, Receptors, G-Protein-Coupled genetics, Urotensins pharmacology
- Abstract
Urotensin-II (U-II) is the peptide agonist for the U-II receptor (UT). Putative UT antagonists, urantide and UFP-803, have been found to have variable efficacy in a range of assays. We have used siRNA-mediated RNA interference to probe the efficacy of these ligands compared to U-II. Knockdown of human UT occurs in the same cellular background with the same coupling machinery allowing relative efficacy to be probed. CHO cells stably expressing 1,110 fmol/mg protein of human UT (CHOhUT) were transfected with s194454, s194455 (UT-targeting), or a negative control siRNA using siPORT amine transfection reagent. After 48 h,silencing was assessed using quantitative PCR in a duplex assay format. Functional consequences of silencing were assessed by measuring [Ca2+]i in Fura-2 loaded cells using the NOVOstar plate reader. Silencing with s194455 was greater than that with s194454 (93.5±2.8% and 73.0±2.5%knockdown of UT mRNA respectively at 10−7 M, p00.006).Both s194455 and s194454 knocked down UT mRNA expression with equal potency (EC50 1.38 and 0.45 nM). The negative control did not affect UT mRNA expression. U-II(10−6M) increased [Ca2+]i 630±69, 402±49 and 190±14nM,urantide (10−6 M) increased [Ca2+]i 408±55, 191±40, and 131±10 nM and UFP-803 (10−6 M) increased [Ca2+]i 134±23, 83±11 and 53±3nM for negative control siRNA, s194454 and s194455, respectively.We have demonstrated silencing of UT mRNA and a reduction of absolute efficacy of three UT ligands. However, we were unable to resolve any changes in relative efficacy for urantide and UFP-803. This is likely to result from a high starting expression and retention of a receptor/coupling reserve.
- Published
- 2012
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99. Evaluation of primary opioid receptor antibodies for use in western blotting.
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Niwa H, Rowbotham DJ, and Lambert DG
- Subjects
- Animals, Antibody Specificity, Blotting, Western methods, Brain metabolism, CHO Cells, Cricetinae, Cricetulus, Rats, Autoantibodies immunology, Receptors, Opioid immunology
- Published
- 2012
- Full Text
- View/download PDF
100. Direct effect of morphine on breast cancer cell function in vitro: role of the NET1 gene.
- Author
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Ecimovic P, Murray D, Doran P, McDonald J, Lambert DG, and Buggy DJ
- Subjects
- Animals, Breast Neoplasms metabolism, CHO Cells, Cell Line, Tumor, Cell Movement drug effects, Cell Proliferation drug effects, Cricetinae, Cricetulus, Female, Humans, Oncogene Proteins physiology, Receptors, Opioid analysis, Analgesics, Opioid pharmacology, Breast Neoplasms pathology, Morphine pharmacology, Oncogene Proteins genetics
- Abstract
Background: Experimental data suggest that postoperative analgesia in general and opioids in particular may affect the risk of metastases after primary cancer surgery. Perioperative single-gene activation may also spark metastatic disease. The NET1 gene promotes migration in adenocarcinoma cells. We investigated opioid receptor expression in both breast cancer cell lines and the direct effect of morphine and NET-1 on breast cancer cell migration in vitro., Methods: Proliferation and migration of oestrogen receptor-negative MDA-MB-231 and oestrogen receptor-positive MCF7 breast cancer cells were studied after incubation with morphine 10-100 ng ml(-1) and control. NET1 gene expression was determined by polymerase chain reaction. The effect of NET1 on cell migration was determined using gene silencing with siRNA and stimulation with lysophosphatidic acid (LPA). The effect of morphine on NET1 expression and migration of cells with silenced NET1 was investigated., Results: The NET1 gene was expressed in both cell lines and stimulated by LPA (2.9-fold in MCF7 and 78-fold in MDA-MB-231). NET1 expression was decreased by 96% after gene silencing in both cell lines with corresponding changes in migration. Despite the lack of opioid receptor expression, morphine increased the expression of NET1 (by 94% in MCF7 and by 263% in MDA-MB-231 cells). Morphine also increased migration by 17-27% and 7-53% in MCF7 and MDA-MB-231, respectively. Silencing the NET1 gene reversed the effect of morphine on migration., Conclusions: The NET1 gene, but not opioid receptors, is expressed in breast adenocarcinoma cells and may facilitate their migration. Morphine increased both expression of NET1 and cell migration but not when NET1 was silenced, implying that NET1 contributes to mediating the direct effect of morphine on breast cancer cell migration.
- Published
- 2011
- Full Text
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