Studies on cannabinoid effects in intestinal tissue and in neuroblastoma

The growing numbers of putative receptors and allosteric sites in which cannabinoids have been observed to act upon has called in to question the established action of cannabinoids through solely the CB1 receptor in the guinea-pig ileum myenteric plexus. Previous work within our laboratory demonstrated cannabinoid inhibition of nicotine induced currents in cultured myenteric neurones through a mechanism expressing different pharmacological properties to those acknowledged as being ('. possessed by the CBI· receptor. To examine-this the effects of cannabinoid receptor ligands were examined on nicotine evoked contractions of the guinea-pig ileum myenteric plexus-longitudinal muscle preparation and on nicotine evoked currents in primary cultures ofmyenteric neurones. Contraction evoked by 100 JlM nicotine was inhibited by the cannabinoid agonist CP 55,940 (ICso 215 nM) but inhibition was not blocked by the CB1 receptor selective antagonist SR141716. Nicotinic log concentration-response curves showed a combination of some rightward displacement and decrease in maximal contraction ,. upon exposure to CP 55,940 suggesting non-competitive inhibition. In the presence of the voltage-gated sodium channel antagonist tetrodotoxin (TTX), both CP 55,940 and SR141716 inhibited contraction and acted additively when applied in conjunction to produce greater inhibition of nicotine evoked contraction. Some degree of stereoselectivity was observed for the cannabinoid agonist WIN 55,212-2 over the stereoisomer WIN 55,212-3, but in the presence ofTTX both stereoisomers displayed equal potency in inhibiting nicotine evoked contraction. Contractions evoked in the presence of the nicotinic receptor antagonist tubocurarine and those evoked by 10 JlM nicotine showed greater CP 55,940 inhibition (ICso 0.74 and 0.55 nM respectively) than that observed with 100 JlM nicotine. CP 55,940 inhibition of contraction evokedby 10 IlM nicotine displayed SR141716 sensitivity with log concentration-response. curves showing a parallel rightward shift (lCso 19.5 nM). Whole cell patch clamp of cultured myenteric neurones confirmed cannabinoid inhibition of nicotine evoked currents and displayed similar levels of inhibition by both active and inactive WIN 55,212 stereoisomers. Blockade of the adenylate cyclase/cAMP intracellular messaging system by 8-Br-cAMP did not affect CP 55,940 inhibition. Results suggest that cannabinoids may act through both CBI receptor dependent and independent mechanisms to inhibit nicotine evoked contraction in the myenteric (' plexus. The mechanisms through which cannabinoids act demonstrated some degree of selectivity dependent upon the level of stimulus evoked by nicotine, with contraction evoked by 100 IlM nicotine demonstrating CBI receptor independence whilst contraction evoked by 10 IlM nicotine showed SR141716 sensitivity. Blockade of action potential propagation by TTX showed a CB1 receptor independent inhibition of contraction occurring within the nerve endings of motor neurones. Whole cell recording confirmed previous work with the lack of WIN 55.212 stereoselectivity corroborating CBI receptor independent inhibition. The inhibition by cannabinoids of electrically induced contraction of the guinea-pig is well accepted. However, the intracellular mechanisms through which cannabinoid receptors act have yet to be established. The effects of cannabinoid receptor ligands on Ca2+ currents were examined in primary cultures of guinea-pig ileum myenteric AH neurones using the whole-cell patch clamp technique. Membrane depolarisation evoked a high-voltage activated calcium current (lca) that was reduced by 65.6% by c.o-conotoxin GVIA (100 nM). Exposure to the cannabinoid agonists CP 55,940 (10 JlM) and WIN 55,212-2 (10 JlM) reduced peak 1ca by 79.7% and 74.7% respectively. The inhibitory effects of CP 55,940 were concentration . dependent over the range 0.3 - 10 JlM with an ICso of 0.54 JlM, and blocked by pertussis toxin (lOO ng.mrl , 18 hr equilibration). WIN 55,212-3 (lO JlM), the (-)- enantiomer of WIN 55,212-2, evoked significantly less inhibition tha~ that produced by WIN 55,212-2, reducing peak lea by 43.1 %. The CBl receptor selective antagonist, SR141716 (l and 10 JlM), had no effect on CP 55,940 (l and 10 JlM) inhibition, whilst another CBl receptor selective antagonist, AM251 (lO JlM), and the CB2 receptor selective antagonist SR144528 (1 Jl~) produced only slight blockade of CP 55,940 (1 JlM) inhibition. In combination, SR141716 (1 JlM) and SR144528 (0.1 JlM) produced complete blockade of CP 55,940 (1 JlM) inhibition. When tested on their own, SR141716 (lO JlM) and the putative 'silent antagonist' 0-2050 (lO JlM) reduced lea by 53.0% and 82.3% respectively. Results suggest that in primary cultures of guinea-pig ileum myenteric AH neurones, cannabinoids inhibit N-type calcium channels through Gilo coupled receptors, comprising of either a combination of CBt and CB2 receptors, or involve a novel ,. cannabinoid receptor. Cannabinoids and opioids have been shown to possess similar pharmacological properties, including analgesia, addiction and euphoria. The powerful analgesic effects of opioids make them vital in the treatment of patients, but their undesirable effects limit their use. The interaction of cannabinoids and opioids opens the possibility of achieving similar or greater beneficial effects whilst reducing undesirable effects through the use oflower amounts ofdrug. The interaction between cannabinoid and opioid receptor ligands.was examined on electrically evoked contractions of the guinea-pig ileum myenteric plexus-longitudinal muscle preparation, and their action in both primary myenteric and NG108-15 neuronal cell cultures investigated. Further examination of the action of cannabinoids on opioidwithdrawal evoked contractions ofthe whole ileum was performed. Both morphine and CP 55,940, when applied independently, produced a concentration-dependent inhibition of electrically evoked contraction of the MPLM preparation. With prior application of CP 55,940, at concentrations of 1 and 10 nM which produced inhibition of 12.0 ± 2.9% (n = 7) and 26.8 ± 5.6% (n = 11) respectively, morphine displayed similar co?~entration-dependent characteristics as displayed in the absence of CP 55,940, suggesting a purely additive interaction. 100 nM CP 55,940 inhibited all morphine evoked inhibition. An inhibition of 40.8± 7.5% (n = 9) was produced by 100 nM CP 55,940 in the absence of morphine, whilst in combination with 1 JlM morphine, which produced maximum inhibition, an inhibition of 46.8 ± 6.8% (n = 9) was observed. Maximal inhibition produced by morphine in the presence of 100 nM CP 55,940 (46.8 ± 6.8%, n = 9) was smaller that that produced in the presence of 10 nM CP 55,940 (62.7 ± 3.4%, n = 11). Prior application of 100 nM and 1 JlM morphine prevented 1 JlM WIN 55,212-2 inducing inhibition of contraction. SR141716 (100 nM) significantly increased the size of electrically evoked contraction and increased inhibition produced by morphine. Opioidwithdrawal contractions of whole ileum were not inhibited by CP 55,940. Both potassium and calcium currents were not affected by morphine or DADLE. In NG108-15 neurones calcium currents were inhibited by DADLE and WIN 55,212-2 in a concentration-dependent manner. In the presence of a concentration of WIN 55,212-2 that produced maximal inhibition, DADLE, also at a concentration producing maximal inhibition, produced a significantly reduced inhibition

A multichannel electron detection system for use in a stabilized magnetic spectrometer

Multichannel counting systems facilitate the determination of differential cross sections in electron scattering experiments. The need for a high resolution counting system capable of handling fast counting rates has led to the construction of a transistorized, multichannel system for use with the 100 Mev linear electron accelerator being constructed at the U.S. Naval Postgraduate School. Design and operation of the counting system are discussed, and testing procedures are described. Since accurate measurement and stability of the spectrometer magnetic field is of fundamental importance in the operation of the multichannel detector, an accurate rotating coil fluxmeter for measuring and regulating the spectrometer magnetic field has also been built and is described. More familiar techniques of magnetic field measurement are not suitable for this application. Measurement of magnet current does not yield sufficient accuracy due to variations in field strength not linearly related to magnet current, and nuclear­ magnetic resonance devices are impractical where there is an inhomogeneous field such as exists in a double focusing spec­trometer magnet. The fluxmeter measures and regulates field strength to an accuracy of one part in 1000 or one gauss, whichever is greater.http://hdl.handle.net/10945/12696http://www.archive.org/details/multichannelelec00kenaLieutenant, United States NavyLieutenant, United States NavyLieutenant, United States NavyApproved for public release; distribution is unlimited

Choosing the target difference ('effect size') for a randomised controlled trial - DELTA(2) guidance protocol

BACKGROUND: A key step in the design of a randomised controlled trial (RCT) is the estimation of the number of participants needed. By far the most common approach is to specify a target difference and then estimate the corresponding sample size; this sample size is chosen to provide reassurance that the trial will have high statistical power to detect such a difference between the randomised groups (at the planned statistical significance level). The sample size has many implications for the conduct of the study, as well as carrying scientific and ethical aspects to its choice. Despite the critical role of the target difference for the primary outcome in the design of an RCT, the manner in which it is determined has received little attention. This article reports the protocol of the Difference ELicitation in TriAls (DELTA(2)) project, which will produce guidance on the specification and reporting of the target difference for the primary outcome in a sample size calculation for RCTs. METHODS/DESIGN: The DELTA(2) project has five components: systematic literature reviews of recent methodological developments (stage 1) and existing funder guidance (stage 2); a Delphi study (stage 3); a 2-day consensus meeting bringing together researchers, funders and patient representatives, as well as one-off engagement sessions at relevant stakeholder meetings (stage 4); and the preparation and dissemination of a guidance document (stage 5). DISCUSSION: Specification of the target difference for the primary outcome is a key component of the design of an RCT. There is a need for better guidance for researchers and funders regarding specification and reporting of this aspect of trial design. The aim of this project is to produce consensus based guidance for researchers and funders

Anti-tumour necrosis factor therapy for Dupuytren's Disease: a randomised dose response proof of concept phase 2a clinical trial

Background Dupuytren's disease is a common fibrotic condition of the hand that causes irreversible flexion contractures of the fingers, with no approved therapy for early stage disease. Our previous analysis of surgically-excised tissue defined tumour necrosis factor (TNF) as a potential therapeutic target. Here we assessed the efficacy of injecting nodules of Dupuytren's disease with a TNF inhibitor. Methods Patients were randomised to receive adalimumab on one occasion in dose cohorts of 15 mg in 0.3 ml, 35 mg in 0.7 ml, or 40 mg in 0.4 ml, or an equivalent volume of placebo in a 3:1 ratio. Two weeks later the injected tissue was surgically excised and analysed. The primary outcome measure was levels of mRNA expression for α-smooth muscle actin (ACTA2). Secondary outcomes included levels of α-SMA and collagen proteins. The trial was registered with ClinicalTrial.gov (NCT03180957) and the EudraCT (2015-001780-40). Findings We recruited 28 patients, 8 assigned to the 15 mg, 12 to the 35 mg and 8 to the 40 mg adalimumab cohorts. There was no change in mRNA levels for ACTA2, COL1A1, COL3A1 and CDH11. Levels of α-SMA protein expression in patients treated with 40 mg adalimumab (1.09 ± 0.09 ng per μg of total protein) were significantly lower (p = 0.006) compared to placebo treated patients (1.51 ± 0.09 ng/μg). The levels of procollagen type I protein expression were also significantly lower (p < 0.019) in the sub group treated with 40 mg adalimumab (474 ± 84 pg/μg total protein) compared with placebo (817 ± 78 pg/μg). There were two serious adverse events, both considered unrelated to the study drug. Interpretation In this dose-ranging study, injection of 40 mg of adalimumab in 0.4 ml resulted in down regulation of the myofibroblast phenotype as evidenced by reduction in expression of α-SMA and type I procollagen proteins at 2 weeks. These data form the basis of an ongoing phase 2b clinical trial assessing the efficacy of intranodular injection of 40 mg adalimumab in 0.4 ml compared to an equivalent volume of placebo in patients with early stage Dupuytren's disease

Clarithromycin and endoscopic sinus surgery for adults with chronic rhinosinusitis with and without nasal polyps: study protocol for the MACRO randomised controlled trial.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common source of ill health; 11% of UK adults reported CRS symptoms in a worldwide population study. Guidelines are conflicting regarding whether antibiotics should be included in primary medical management, reflecting the lack of evidence in systematic reviews. Insufficient evidence to inform the role of surgery contributes to a fivefold variation in UK intervention rates. The objective of this trial is to establish the comparative effectiveness of endoscopic sinus surgery (ESS) or a prolonged course of antibiotics (clarithromycin) in adult patients with CRS in terms of symptomatic improvement and costs to the National Health Service compared with standard medical care (intranasal medication) at 6 months. METHODS/DESIGN: A three-arm parallel-group trial will be conducted with patients who remain symptomatic after receiving appropriate medical therapy (either in primary or secondary care). They will be randomised to receive: (1) intranasal medication plus ESS, (2) intranasal medication plus clarithromycin (250 mg) or (3) intranasal medication plus a placebo. Intranasal medication (current standard medical care) is defined as a spray or drops of intranasal corticosteroids and saline irrigations. The primary outcome measure is the SNOT-22 questionnaire, which assesses disease-specific health-related quality of life. The study sample size is 600. Principal analyses will be according to the randomised groups irrespective of compliance. The trial will be conducted in at least 16 secondary or tertiary care centres with an internal pilot at six sites for 6 months. DISCUSSION: The potential cardiovascular side effects of macrolide antibiotics have been recently highlighted. The effectiveness of antibiotics will be established through this trial, which may help to reduce unnecessary usage and potential morbidity. If ESS is shown to be clinically effective and cost-effective, the trial may encourage earlier intervention. In contrast, if it is shown to be ineffective, then there should be a significant reduction in surgery rates. The trial results will feed into the other components of the MACRO research programme to establish best practice for the management of adults with CRS and design the ideal patient pathway across primary and secondary care. TRIAL REGISTRATION: ISRCTN36962030 . Registered on 17 October 2018

Practical help for specifying the target difference in sample size calculations for RCTs: the DELTA2 five-stage study, including a workshop

BACKGROUND: The randomised controlled trial is widely considered to be the gold standard study for comparing the effectiveness of health interventions. Central to its design is a calculation of the number of participants needed (the sample size) for the trial. The sample size is typically calculated by specifying the magnitude of the difference in the primary outcome between the intervention effects for the population of interest. This difference is called the 'target difference' and should be appropriate for the principal estimand of interest and determined by the primary aim of the study. The target difference between treatments should be considered realistic and/or important by one or more key stakeholder groups. OBJECTIVE: The objective of the report is to provide practical help on the choice of target difference used in the sample size calculation for a randomised controlled trial for researchers and funder representatives. METHODS: The Difference ELicitation in TriAls2 (DELTA2) recommendations and advice were developed through a five-stage process, which included two literature reviews of existing funder guidance and recent methodological literature; a Delphi process to engage with a wider group of stakeholders; a 2-day workshop; and finalising the core document. RESULTS: Advice is provided for definitive trials (Phase III/IV studies). Methods for choosing the target difference are reviewed. To aid those new to the topic, and to encourage better practice, 10 recommendations are made regarding choosing the target difference and undertaking a sample size calculation. Recommended reporting items for trial proposal, protocols and results papers under the conventional approach are also provided. Case studies reflecting different trial designs and covering different conditions are provided. Alternative trial designs and methods for choosing the sample size are also briefly considered. CONCLUSIONS: Choosing an appropriate sample size is crucial if a study is to inform clinical practice. The number of patients recruited into the trial needs to be sufficient to answer the objectives; however, the number should not be higher than necessary to avoid unnecessary burden on patients and wasting precious resources. The choice of the target difference is a key part of this process under the conventional approach to sample size calculations. This document provides advice and recommendations to improve practice and reporting regarding this aspect of trial design. Future work could extend the work to address other less common approaches to the sample size calculations, particularly in terms of appropriate reporting items. FUNDING: Funded by the Medical Research Council (MRC) UK and the National Institute for Health Research as part of the MRC-National Institute for Health Research Methodology Research programme

GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.We thank David Wiggins for excellent technical assistance. This work was supported by the Medical Research Council, Diabetes UK (to R. Ramracheya ), Oxford Biomedical Research Centre (to A. Tarasov), the Wellcome Trust (Senior Investigator Awards to A. Galione and P. Rorsman), the Warwick Impact Fund (to C. Weston and G. Ladds), the Biotechnology and Biological Sciences Research Council (to G. Ladds), the Knut and Alice Wallenberg Foundation (to P. Rorsman), and the Swedish Research Council (to P. Rorsman). The initial stages of M. Shigeto’s stay in Oxford were supported by a fellowship from Kawasaki Medical School.This is the final version of the article. It was first available from the American Society for Clinical Investigation via http://dx.doi.org/10.1172/JCI8197

DELTA2 guidance on choosing the target difference and undertaking and reporting the sample size calculation for a randomised controlled trial.

BACKGROUND: A key step in the design of a RCT is the estimation of the number of participants needed in the study. The most common approach is to specify a target difference between the treatments for the primary outcome and then calculate the required sample size. The sample size is chosen to ensure that the trial will have a high probability (adequate statistical power) of detecting a target difference between the treatments should one exist. The sample size has many implications for the conduct and interpretation of the study. Despite the critical role that the target difference has in the design of a RCT, the way in which it is determined has received little attention. In this article, we summarise the key considerations and messages from new guidance for researchers and funders on specifying the target difference, and undertaking and reporting a RCT sample size calculation. This article on choosing the target difference for a randomised controlled trial (RCT) and undertaking and reporting the sample size calculation has been dual published in the BMJ and BMC Trials journals METHODS: The DELTA2 (Difference ELicitation in TriAls) project comprised five major components: systematic literature reviews of recent methodological developments (stage 1) and existing funder guidance (stage 2); a Delphi study (stage 3); a two-day consensus meeting bringing together researchers, funders and patient representatives (stage 4); and the preparation and dissemination of a guidance document (stage 5). RESULTS AND DISCUSSION: The key messages from the DELTA2 guidance on determining the target difference and sample size calculation for a randomised caontrolled trial are presented. Recommendations for the subsequent reporting of the sample size calculation are also provided

Choosing the target difference and undertaking and reporting the sample size calculation for a randomised controlled trial – the development of the DELTA2 guidance

Background A key step in the design of a randomised controlled trial is the estimation of the number of participants needed. The most common approach is to specify a target difference in the primary outcome between the randomised groups and then estimate the corresponding sample size. The sample size is chosen to provide reassurance that the trial will have high statistical power to detect the target difference at the planned statistical significance level. Alternative approaches are also available, though most still require specification of a target difference. The sample size has many implications for the conduct of the study, as well as incurring scientific and ethical aspects. Despite the critical role of the target difference for the primary outcome in the design of a randomised controlled trial (RCT), the manner in which it is determined has received little attention. This article reports the development of the DELTA2 guidance on the specification and reporting of the target difference for the primary outcome in a sample size calculation for a RCT. Methods The DELTA2 (Difference ELicitation in TriAls) project has five components comprising systematic literature reviews of recent methodological developments (stage 1) and existing funder guidance (stage 2), a Delphi study (stage 3), a 2-day consensus meeting bringing together researchers, funders and patient representatives (stage 4), and the preparation and dissemination of a guidance document (stage 5). Results The project started in April 2016. The literature search identified 28 articles of methodological developments relevant to a method for specifying a target difference. A Delphi study involving 69 participants, along with a 2-day consensus meeting were conducted. In addition, further engagement sessions were held at two international conferences. The main guidance text was finalised on April 18, 2018, after revision informed by feedback gathered from stages 2 and 3 and from funder representatives. Discussion The DELTA2 Delphi study identified a number of areas (such as practical recommendations and examples, greater coverage of different trial designs and statistical approaches) of particular interest amongst stakeholders which new guidance was desired to meet. New relevant references were identified by the review. Such findings influenced the scope, drafting and revision of the guidance. While not all suggestions could be accommodated, it is hoped that the process has led to a more useful and practical document. Keywords Target difference Clinically important difference Sample size Guidance Randomised tria