Background and PurposeIn animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ-NOP system in freshly isolated volunteer human B- and T-cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis. Experimental ApproachB- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQ(ATTO594), N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPG alpha iq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25-plex assay format. Cells were challenged with LPS/PepG. Key ResultsCD19-positive B-cells bound N/OFQ(ATTO594); they also contain N/OFQ. Stimulation with CXCL13/IL-4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL-4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM-CSF release in an N/OFQ sensitive manner. CD3-positive T-cells did not bind N/OFQ(ATTO594); they did contain N/OFQ. Stimulation with CXCL12/IL-6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQ(ATTO594) binding. In LPS/PepG-treated cells, N/OFQ reduced migration to CXCL12/IL-6. LPS/PepG increased GM-CSF release in an N/OFQ sensitive manner. Conclusions and ImplicationsWe suggest both a constitutive and sepsis-inducible N/OFQ-NOP receptor autocrine regulation of B- and T-cell function, respectively. These NOP receptors variably inhibit migration and reduce GM-CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

Excessively high or low body weight, with cellular imbalance between nutrient and energy supply and demand, causes physiological changes that may affect drug dosing, handling, and pharmacokinetic properties.1 Furthermore, the pathological changes resulting from extremes of weight and their associated co-morbidities affect the metabolism and elimination of many drugs. This paper summarises the measures used to characterise body weight, the physiological effects of extreme body weights, and their implications for pharmacokinetics and drug dosing in anaesthesia and critical care.

Arrhythmias are abnormalities of cardiac rate or rhythm occurring for a variety of reasons. They are common in the perioperative period and in intensive care. Causes may reflect an underlying heritable predisposition, the presence of new pathology either of the heart or conducting system, or as a result of systemic illness. Targets for antiarrhythmics include myocardial ion channels, muscarinic or nicotinic acetylcholine receptors, adrenergic or adenosine receptors. Arrhythmias may cause cardiac arrest and haemodynamic compromise, requiring rapid identification and corrective treatment either of rate or rhythm. Even where stable, arrhythmias present an increased risk of thromboembolic events requiring the use of anticoagulation. Treatment may be directed at controlling heart rate or rhythm to restore the circulation and tissue perfusion. Strategies may include prevention or correction of precipitating factors (such as electrolyte abnormalities or sepsis) and sometimes non-pharmacological treatments (cardioversion, surgical ablation or pacing). Antiarrhythmic drugs are often required. The targets, mechanisms and clinical guidelines are reviewed for common antiarrhythmic agents.

Inadequate end organ perfusion and tissue hypoxia is an end point of many disease processes in critical illness. Maintenance of blood flow and hence tissue oxygenation is critical to the management of intensive care patients. End organ blood flow is determined by a balance of myocardial factors (stroke volume and heart rate) and vascular factors (vasodilation and constriction). Global blood flow is determined by a balance of neurohormonal factors, with local autoregulation ultimately determining regional flow. Pharmacological manipulation of both the myocardium and vasculature at the level of the autonomic nervous system (via α or β adrenoceptors), myocardium (e.g. calcium sensitization via levosimendan), or locally (e.g. via sympathectomy) is commonly used in anaesthesia to mitigate the effects of critical illness and to maintain organ perfusion, either through increasing vascular tone or cardiac output. This article considers the global control of the system through to local and regional regulation of blood flow, and how the system may be manipulated at every stage.

Arrhythmias can occur in medical or surgical practice and are common in the perioperative period and in intensive care. Their occurrence may reflect a pre-existing condition or predisposition, or arise de novo. Arrhythmias must be identified promptly and managed appropriately as they may become unstable, compromise cardiac output and risk cardiac arrest. In many cases, this involves prevention or correction of precipitating factors and sometimes non-pharmacological treatments (cardioversion or surgical ablation). However, anti-arrhythmic drugs are often required. A sound understanding of drug mechanisms, guidelines and evidence will aid choice of therapy. This article describes the mechanisms of action of the common anti-arrhythmic agents, their use in clinical practice and a review of recent guidelines for the management of common arrhythmias.

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 (10-5M) (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.

When it was first discovered in the 1500s, opium was heralded as the most potent analgesic. The Latin name for morphine tincture, laudanum (to praise), illustrates the high regard in which it was held. The many adverse effects associated with its use (now attributed to non-specific opioid receptor cross-activation between subtypes) necessitate caution in its use. Even today, the adverse effects still remain problematic and are clinically limiting, both in the acute setting and in chronic use, despite ample research into opioid receptor subtype specific agonists, antagonists and bifunctional molecules.The adverse effects, such as constipation, nausea, dysphoria, respiratory depression and itching, have necessitated the drive to develop nonopioid- related analgesics, both as sole agents and as part of multimodal, opioid-sparing regimens. For example, paracetamol has been shown to reduce patient-controlled analgesia (PCA) morphine doses by 20% when used as an adjunct for acute post-surgical pain. Additionally, whilst effective for acute pain, opiate analgesia is of limited effectiveness for chronic and neuropathic pain states, such as phantom limb pain. This is partly due to problems with chronic administration and pharmacologic tolerance although also relates to different mechanisms of acute (e.g. surgical) and chronic pain and involvement of additional neurotransmitters such as substance P, gamma-aminobutyric acid and glutamate.This article will give an overview of the pain pathway relative to ther-apeutic targets, revisit some common non-opioid analgesic agents and highlight some novel targets and drugs in development.

Pain is a major cause of distress, both physical and psychological and is also associated with increased inpatient hospital stays, poor wound healing and prolonged rehabilitation. Opioids are a mainstay of treatment for acute pain, although they are associated with significant morbidity through adverse effects such as respiratory depression, psychosis, and nausea and vomiting. The non-opiate analgesics avoid some of these effects, although are not panaceas, non-steroidal anti-inflammatory drugs (NSAIDS), for example, are associated with renal and gastric damage, which precludes their use in elderly patients and those with renal impairment. This article reviews the underlying pain pathways and the ways in which these pathways may be modulated in the pursuit of side-effect-free analgesia. Existing medications target these pathways, although there are evolving targets for analgesics. The advantages and disadvantages of the current non-opioid mainstays of treatment are -reviewed. Other centrally acting medications, both research (nociceptin, neuropeptides, cannabinoids) and clinical (gabapentin, carbamazepine) are also considered. Combinations of existing analgesics underlie the principles of multimodal analgesia; non-opioid adjuncts are known to reduce postsurgical opioid requirements and improve rehabilitation and discharge. The answer may lie in synergistic dual-acting medications, which are discussed with reference to the COX-inhibiting nitric oxide donors and dual lipoxygenase/cyclooxygenase inhibitors.

Department of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center (A.R., E.C.G., G.M., B.S., S.Z., D.R., G.C.) and Department of Pharmaceutical Science and Biotechnology Center (C.T.) University of Ferrara, 44100 Ferrara, Italy. Department of Experimental Medicine and Public Health (R.C.) University of Camerino, 62032 Camerino, Italy. JPET Fast Forward. Published on February 28, 2007 as DOI:10.1124/jpet.106.116780

Knockout and pharmacological studies demonstrated that the activation of delta opioid peptide (DOP) receptors produces antidepressant-like effects in rodents. Here we report the results obtained with the novel DOP ligand H-Dmt-Tic-NH-CH(2)-Bid (UFP-502). UFP-502 bound with high affinity (pK(i) 9.43) to recombinant DOP receptors displaying moderate selectivity over MOP and KOP. In CHO(hDOP) [(35)S]GTPgammaS binding and mouse vas deferens experiments, UFP-502 behaved as a potent (pEC(50) 10.09 and 10.70, respectively) full agonist. In these preparations, naloxone, naltrindole and N,N(CH(3))(2)Dmt-Tic-OH showed similar pA(2) values against UFP-502 and DPDPE and the same rank order of potency. In vivo in mice, UFP-502 mimicked DPDPE actions, producing a significant reduction of immobility time after intracerebroventricular administration in the forced swimming test and a clear antinociceptive effect after intrathecal injection in the tail withdrawal assay. However, while the effects of DPDPE were fully prevented by naltrindole those evoked by UFP-502 were unaffected (tail withdrawal assay) or only partially reversed (forced swimming test). In conclusion, UFP-502 represents a novel and useful chemical template for the design of selective agonists for the DOP receptor.

A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), has been generated by combining in the N/OFQ-NH2 sequence two chemical modifications, [Arg14,Lys15] and [(pF)Phe4], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHOhNOP cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(±)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA2 = 7.75–8.12) and UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ-NH2)(pA2 = 6.91–7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP–/–). In vivo, UFP-102 (0.01–0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1–10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP–/– mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.

To date, J-113397 represents the most potent and selective non peptide NOP receptor antagonist widely used in pharmacological studies. However, the synthesis, purification, and enantiomer separation of this molecule, which contains two chiral centers, is rather difficult and low-yielding. Here, we synthesized and tested a series of simplified J-113397 analogues to investigate the importance of the stereochemistry and the influence of the substituents at position 3 of the piperidine nucleus and on the nitrogen atom of the benzimidazolidinone nucleus. The compound coded as Trap-101, an achiral analogue of J-113397, combines a pharmacological profile similar to that of the parent compound with a practical, high-yielding preparation.

You could use these materials in one or two day workshops according to space in the curriculum to develop a reflective approach to practice which prioritises safety.