New patient-reported experience measure for children with allergic disease: development, validation and results from integrated care.

OBJECTIVES: To develop and validate a new allergy-specific patient-reported experience measure (PREM) for children and their parents, and to collect feedback in an integrated care setting. DESIGN: Two allergy-specific PREMs were produced using focus groups, cognitive testing, two prospective validation studies (collaboration: Royal College of Paediatrics and Child Health, Picker Institute Europe, Imperial College/London): 'Your Allergy Care', for children aged 8-16 years; 'Your Child's Allergy Care', for parents of children aged 0-7 years. SETTING: Community event, primary/secondary/tertiary allergy care settings. MAIN OUTCOME MEASURES: Performance of PREMs in validation study; reported experience of allergy care. PARTICIPANTS: 687 children with allergic conditions and their parents/carers. RESULTS: In total, 687 questionnaires were completed; 503/687 (253 child; 250 parent) for the final survey. In both surveys, demographic variations were not associated with differences in results. Although 71% of patients reported one or more allergic conditions (food allergy/eczema/hay fever/asthma), 62% required multiple visits before receiving final diagnosis. Overall, patient experience was good for communication with patient/parent, competence and confidence in ability, and 73% felt looked after 'very well' and 23% 'quite well'. Areas for improvement included communication with nurseries/schools, more information on side effects, allergic conditions and allergen/irritant avoidance. Allergy care in primary/emergency care settings was associated with higher problem-scores (worse experience) than in specialist clinics. CONCLUSIONS: These new PREMs will allow allergy-specific patient experience reporting for children and parents and help identification of priority areas for improvement and commissioning of care. Efforts towards better allergy care provision must be targeted at primary and emergency care settings and underpinned by improving communication between healthcare providers and the community.

Behavioural satiety sequence (BSS): separating wheat from chaff in the behavioural pharmacology of appetite.

The history of anti-obesity drug development is far from glorious, with transient magic bullets and only a handful of agents currently licensed for clinical use. In view of recent progress in our understanding of the multiplicity of signalling pathways involved in appetite regulation, and the resultant deluge of reports on the anorectic efficacy of novel therapies, it seems timely to stress the need to differentiate treatments that suppress intake by primary means from those that only indirectly achieve this endpoint. The current article reviews the conceptual history of the behavioural satiety sequence (BSS), also known as the behavioural sequence of satiety, post-ingestive satiety, and the postprandial satiety sequence. Early research confirmed that natural satiation, produced by a caloric load on the gut, is associated with a predictable transition from feeding through grooming to resting. Although many less naturalistic manipulations are also capable of reducing food intake, very few do so without disrupting the normal structure of this feeding cycle. Thus, while CCK and d-fenfluramine reduce intake by accelerating but otherwise maintaining the integrity of the BSS, other anorectic interventions disrupt the BSS through response competition (e.g. d-amphetamine), nausea/discomfort (e.g. lithium chloride) and/or interference with taste-mediated positive feedback (e.g. quinine adulteration of the diet). A substantial literature now strongly supports the specific involvement of serotonin 5-HT(1B) and 5-HT(2C) receptor subtypes in satiety and in the anorectic effect of agents such as fenfluramine and fluoxetine. Recent BSS analyses have also identified rather selective anorectic profiles for the dual noradrenaline and 5-HT reuptake inhibitor sibutramine, the orexin-1 receptor antagonist SB-334867, and the broad spectrum opioid receptor antagonist naloxone. However, similar analyses have offered little/no support for the anorectic potential of the gut peptide PYY(3-36) while the acute anorectic efficacy of cannabinoid CB1 receptor antagonist/inverse agonists appears largely to be secondary to response competition. In contrast, studies with low-dose combinations of naloxone and CB1 receptor antagonist/inverse agonists have very recently confirmed the potential of drug polytherapies not only in appetite suppression but also in attenuating/eliminating unwanted side-effects. In sum, as BSS analysis offers a reliable means of differentiating the wheat (primary anorectics) from the chaff (secondary anorectics), it should form an integral part of early phase testing in any anti-obesity drug screening programme.

Sibutramine & naloxone: infra-additive interaction in the regulation of appetite?

Sibutramine is one of a small number of clinically approved anti-obesity agents while naloxone not only has intrinsic anorectic efficacy but, in low doses, also produces additive/synergistic anorectic effects in combination with other compounds. In view of the potential advantages of drug polytherapy over conventional monotherapy, the present study explored the effects of acute low dose combinations of sibutramine (0.125, 0.25 mg/kg) and naloxone (0.1 mg/kg) on food intake, feeding and non-feeding behaviour, and post-treatment weight gain in male rats. Neither drug, alone or in combination, significantly affected weight gain. Naloxone per se modestly though significantly suppressed both food intake and time spent feeding without disrupting the behavioural satiety sequence (BSS). However, neither dose of sibutramine affected these measures nor did they further enhance the anorectic response to the opioid receptor antagonist. Indeed, the combination of naloxone and 0.25 mg/kg sibutramine produced effects on intake and feeding behaviour that were substantially lower than those predicted on the basis of the sum of the individual drug effects (i.e. an infra-additive profile). These data, which contrast directly with reported positive interactions between opioid receptor antagonists and other anorectic agents (e.g. rimonabant, bupropion), would not support naloxone-sibutramine polytherapy in the clinical management of obesity.

Effects of acute low-dose combined treatment with rimonabant and sibutramine on appetite and weight gain in rats.

In view of its potential advantages, drug polytherapy is currently attracting significant interest in the field of obesity research. In this context, concurrent manipulation of serotonergic and cannabinoid pathways in rodents has been found to reduce food and fluid intake in both an additive or synergistic manner. To further assess the value of this polytherapeutic approach, the current study examined the acute effects of low-dose combinations of the cannabinoid CB1 receptor antagonist/inverse agonist rimonabant (0.5 mg/kg) and the dual serotonin- and noradrenaline-reuptake inhibitor sibutramine (0.125 and 0.25 mg/kg) in male rats. Ethological analysis was used to generate comprehensive behavioural profiles, including the behavioural satiety sequence (BSS). Findings confirmed that, although neither drug given alone significantly altered food intake, feeding behaviour or weight gain, rimonabant per se tended to reduce consumption and time spent feeding while significantly increasing scratching and grooming responses. However, none of these effects of the CB1 receptor antagonist/inverse agonist was significantly altered by the presence of either dose of sibutramine. In striking contrast to recent reports of acute low-dose interactions (enhanced appetite suppression and reduced side-effects) between rimonabant and naloxone, present results would not appear to support the clinical potential of rimonabant/sibutramine polytherapy for obesity.

Night and day: diurnal differences in the behavioural satiety sequence in male rats.

The behavioural satiety sequence (BSS) is an important method for assessing the selectivity of treatment (physiological, pharmacological and/or genetic) effects on food intake in rodents. The concept describes the natural progression from feeding to resting, with the transition between the two a useful biomarker of behavioural satiety. Although treatments can accelerate (anorexigenics) or delay (orexigenics) this transition without disrupting behavioural structure, the detection of such changes depends upon the timing of the transition under control conditions. Fasting and presatiation are known to affect this timing. However, recent observations in our laboratory have suggested that phase of testing (light or dark) might also be an important consideration. The present study therefore directly compared food intake and the BSS in thoroughly habituated male rats maintained either on a normal light cycle and tested during the light phase or on a reversed light cycle and tested during the dark phase. The results show that phase of testing had relatively little impact on food intake or diverse measures of ingestive and non-ingestive behaviour. Although modest differences were detected in locomotion, grooming and scratching (higher in dark phase), by far the largest difference concerned resting behaviour which had both a later onset and a much lower peak level during dark phase testing. Importantly, these behavioural differences delayed the transition between eating and resting. The potential contribution of diurnal differences in rate of eating is discussed as are the implications of these findings for future studies on the neurobiology of feeding behaviour.

Effects of acute low-dose combined treatment with naloxone and AM 251 on food intake, feeding behaviour and weight gain in rats.

Low-dose combinations of naloxone and rimonabant produce additive effects on food intake and feeding behaviour, yet abolish the scratching syndrome typically induced by rimonabant per se. To assess the generality of these findings, we have examined the acute effects of low-dose combinations of naloxone (0.1 mg/kg) and the rimonabant derivative AM 251 (0.5 and 1.0 mg/kg) on food intake, feeding behaviour and weight gain in non-deprived male rats. Although ineffective when given alone, combined treatment with naloxone and 0.5 mg/kg AM 251 significantly and selectively suppressed mash intake and time spent feeding. By itself, 1.0 mg/kg AM 251 failed to alter any measure of feeding behaviour but did reduce food consumption and induce scratching behaviour. Co-administration of naloxone with 1.0 mg/kg AM 251 not only significantly suppressed both food intake and feeding behaviour but also simultaneously attenuated AM 251-induced scratching. This profile mirrors earlier findings with naloxone/rimonabant and is consistent with the reported diversity of opioid-cannabinoid system interactions at a more molecular level. Although further studies are required (e.g. 'neutral' CB1 receptor antagonists), current data constitute further proof of concept regarding the anorectic efficacy, selectivity and added value of low-dose polytherapy with opioid and CB1 receptor antagonists.

Sibutramine-induced anorexia: potent, dose-dependent and behaviourally-selective profile in male rats.

The serotonin and noradrenaline reuptake inhibitor sibutramine has been licensed as an anti-obesity treatment for more than a decade. However, while inhibitory effects on food intake and weight gain are well documented, surprisingly little published detail exists regarding its influence on feeding and related behaviours. The present study was therefore designed to assess the effects of acute sibutramine treatment on food intake, the behavioural satiety sequence (BSS) and post-treatment weight gain. Subjects were 10 non-deprived adult male Lister hooded rats, tested with 0.5-3.0 mg/kg sibutramine hydrochloride during 1-h DVD-recorded test sessions with palatable mash. Our results show that sibutramine dose-dependently reduced food intake, an effect significant at all doses tested. Ethological analysis revealed very few behavioural effects, except for a dose-dependent reduction in time spent feeding and an increase in the frequency of resting. Behavioural specificity was further supported by time-bin analysis which confirmed both the structural integrity and dose-dependent acceleration of the BSS. Single dosing with sibutramine (3.0 mg/kg) also suppressed daily weight gain over the 24-72 h period post-dosing. Current data support the conclusion that the acute anorectic and weight loss efficacy of sibutramine in adult male rats is not secondary to behavioural disruption but, instead, is due largely to an acceleration in behavioural satiety.

Endogenous opioids and cannabinoids: system interactions in the regulation of appetite, grooming and scratching.

Growing evidence suggests substantial crosstalk between endogenous opioid and cannabinoid systems in the regulation of appetite. Not only is cannabinoid-induced hyperphagia abolished by opioid receptor antagonists (and vice versa), but several laboratories have reported supra-additive anorectic responses following co-administration of opioid and CB1 receptor antagonists. In the present study, videoanalysis has been used to characterise the acute effects of sub-anorectic doses of rimonabant (0.25, 0.75 mg/kg) and naloxone (0.1 mg/kg), alone and in combination, on mash intake, ingestive and non-ingestive behaviour, and post-treatment weight gain in male rats. The results confirmed that, when administered alone, none of these treatments significantly altered mash consumption, various measures of feeding behaviour, or weight gain. Although most non-ingestive behaviours were also unaffected, 0.75 mg/kg rimonabant induced compulsive scratching and grooming. However, when naloxone was given in combination with either dose of rimonabant, both food intake and time spent feeding were significantly decreased while the behavioural satiety sequence (BSS) was accelerated. On further analysis, the co-treatment reductions in food intake and feeding behaviour were found to be of an additive rather than supra-additive nature. Intriguingly, the co-administration of naloxone also virtually abolished the compulsive scratching response to the higher dose of rimonabant. Findings are discussed in relation to current views on the molecular bases of opioid-cannabinoid system interactions and the unexpected 'dual' advantage (reduction in appetite plus attenuation of side-effect) of low-dose combinations of opioid and cannabinoid CB1 receptor antagonists.

Behaviourally-selective hypophagic effects of naloxone in non-deprived male rats presented with palatable food.

Endogenous opioids have long been implicated in mechanisms of appetite control. A significant strand in the evidence base has been the hypophagic action of broad-spectrum opioid receptor antagonists (such as naloxone) in opiate-naïve animals. However, while much has been learned about sites of action, underlying receptor mechanisms and the role of taste hedonics, surprisingly little is known about the behavioural selectivity of naloxone-induced hypophagia. As such, two experiments employed detailed video analysis to profile the behavioural effects of naloxone (Experiment 1: 1.0-5.0 mg/kg; Experiment 2: 0.01-1.0 mg/kg) in non-deprived male rats during 1 h free-feeding tests with palatable mash. Results confirmed that, at doses > or =1.0 mg/kg, naloxone consistently suppresses food consumption and feeding behaviour but, congruent with its short biological half-life, had no carryover effects on post-treatment weight gain. Crucially, the anorectic doses of naloxone did not alter the time taken to find food or to commence feeding, the time spent feeding in the initial phase of testing, or the rate at which food was consumed. Furthermore, they neither interfered with non-ingestive components of the behavioural repertoire (e.g. locomotion, rearing) nor did they disrupt the normal structure of feeding behaviour (the behavioural satiety sequence, BSS). Rather, the principal effect of naloxone was to produce a shift to the left in (i.e. accelerate) the BSS. Findings are discussed in relation to the role of (mu) opioid receptor mechanisms in taste hedonics and the likelihood of a naloxone-induced reduction in the orosensory reward that would normally accompany/follow the ingestion of palatable food.

Grooming, scratching and feeding: role of response competition in acute anorectic response to rimonabant in male rats.

RATIONALE: Although the CB1 receptor antagonist/inverse agonist rimonabant acutely suppresses food intake in rodents, the behavioural specificity of this effect remains unclear. OBJECTIVES: To profile the behavioural effects of rimonabant in a free-feeding context. MATERIALS AND METHODS: Videoanalysis was employed to characterise the effects of acute rimonabant (1.5 and 3.0 mg/kg, IP) on the behaviour of non-deprived male rats exposed to palatable mash. Data were also collected on post-treatment weight gain, and, as prolonged appetite suppression has been found after single dosing with compounds of this series, rats were reassessed (drug-free) for food intake 7 days after initial testing. RESULTS: Both doses of rimonabant not only decreased mash consumption (44-55%) but also reduced 24-h weight gain. Although videoanalysis confirmed the inhibitory effects of rimonabant on feeding behaviour, it also revealed concurrent reductions in locomotion, rearing and sniffing as well as substantial (up to tenfold) and dose-dependent increases in grooming and scratching. Timecourse analyses further revealed that rimonabant dose-dependently induced frequent episodes of atypical scratching that waned over the test but which were succeeded by prolonged and behaviourally disruptive grooming. Finally, as groups did not differ in mash consumption on retest, any prolonged anorectic effect of acute rimonabant dissipates within 7 days of treatment. CONCLUSIONS: The anorectic response to rimonabant in male rats would appear to be due largely to response competition. This parsimonious conclusion is supported by the less profound (although still significant) increases in scratching and grooming observed in rats treated with a sub-anorectic dose (0.5 mg/kg) of the compound.

Atypical anxiolytic-like response to naloxone in benzodiazepine-resistant 129S2/SvHsd mice: role of opioid receptor subtypes.

RATIONALE: Mice of many 129 substrains respond to environmental novelty with behavioural suppression and high levels of anxiety-like behaviour. Although resistant to conventional anxiolytics, this behavioural phenotype may involve stress-induced release of endogenous opioids. OBJECTIVES: To assess the effects of opioid receptor blockade on behavioural reactions to novelty stress in a chlordiazepoxide-resistant 129 substrain. MATERIALS AND METHODS: Experiment 1 contrasted the effects of the broad-spectrum opioid receptor antagonist naloxone (1.0-10.0 mg/kg) in C57BL/6JOlaHsd and 129S2/SvHsd mice exposed to the elevated plus-maze. Experiments 2-4 examined the responses of 129S2/SvHsd mice to the mu-selective opioid receptor antagonist beta-funaltrexamine (2.5-10.0 mg/kg), the delta-selective antagonist naltrindole (2.5-10.0 mg/kg) and the kappa-selective antagonist nor-binaltorphimine (2.5-5.0 mg/kg). RESULTS: 129 mice displayed higher levels of anxiety-like behaviour and lower levels of general exploration relative to their C57 counterparts. Although naloxone failed to alter the behaviour of C57 mice, both doses of this antagonist produced behaviourally selective reductions in open-arm avoidance in 129 mice. Surprisingly, none of the more selective opioid receptor antagonists replicated this effect of naloxone: beta-funaltrexamine was devoid of behavioural activity, naltrindole suppressed rearing (all doses) and increased immobility (10 mg/kg), while nor-binaltorphimine (5 mg/kg) nonspecifically increased percent open arm entries. CONCLUSIONS: Recent evidence suggests differential involvement of opioid receptor subtypes in the anxiolytic efficacy of diverse compounds including conventional benzodiazepines. The insensitivity of 129 mice to the anxiolytic action of chlordiazepoxide, coupled with their atypical anxiolytic response to naloxone (but not more selective opioid receptor antagonists), suggests an abnormality in anxiety-related neurocircuitry involving opioid-GABA interactions.

Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells.

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.

Neuropeptides stimulate tyrosine phosphorylation and tyrosine kinase activity in small cell lung cancer cell lines.

Stimulation of small cell lung cancer (SCLC) cells with neuropeptides bombesin, bradykinin, gastrin, and neurotensin resulted in increased tyrosine kinase activity and tyrosine phosphorylation of a number of polypeptides including a p120 kDa polypeptide identified by immunoblotting as focal adhesion kinase (p125FAK). The neuropeptides stimulated a rapid, concentration-dependent phosphorylation of p125FAK (EC50 of 1 nM, 5 nM, and 2 nM for bombesin, bradykinin, and gastrin, respectively), which was receptor mediated and inhibited by both specific and broad-spectrum neuropeptide receptor antagonists. Specific inhibition of protein tyrosine kinase activity by tyrphostin-25 inhibited both basal and neuropeptide-stimulated SCLC cell growth. These results identify a novel neuropeptide-stimulated growth signaling event in SCLC cells.

Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit.

The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family. In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+. Glu-129 was previously shown to be critical for both enzymatic activities. In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129. The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses. Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1. This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way. In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1. These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates.

Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping.

Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.