The effect of formoterol, alone and in combination with budesonide, upon tumour necrosis factor-a stimulated (10 ng?mL -1 ) human bronchial epithelial cells was investigated.Addition of formoterol (¢10 -10 M) reduced granulocyte macrophage-colony stimulating factor (GM-CSF) levels, as assessed by enzyme-linked immunosorbent assay, by 40 -50% and increased interleukin (IL)-8 levels by y50%. The effects of formoterol were long lasting (23 h). Budesonide (10 -8 M) reduced the amounts of both cytokines (GM-CSF and IL-8) by 40%. Simultaneous addition of formoterol and budesonide reduced GM-CSF levels y75%, while IL-8 levels were decreased y40%, similar to the reduction obtained with budesonide alone. The glucocorticoid receptor (GR) antagonist RU486 did not influence the effect of formoterol, suggesting no involvement of the GR. Formoterol rapidly induced an elevation in intracellular cyclic adenosine monophosphate, which was reduced in the presence of propranolol. In addition, the alterations in cytokine secretion induced by formoterol could be fully blocked by propranolol, demonstrating that these effects are b 2 -receptor mediated.In conclusion, the combination of budesonide and formoterol reduces the secretion of granulocyte macrophage-colony stimulating factor to basal levels and counteracts the capacity of formoterol alone to induce interleukin-8 production, modulations which may facilitate improved asthma control.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in nonregulatory settings, considerable efforts have been made to develop nonanimal approaches to replace current animal testing. Genomic Allergen Rapid Detection (GARD™) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARD™skin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses, and provide a holistic readout of substance-induced DC activation. Here, results from an inter-laboratory ring trial of GARD™skin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1% and 88.9%, whereas the cumulative predictive accuracy across the 3 laboratories was 93.8%. It was concluded that GARD™skin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARD™skin.

In patients with asthma and chronic obstructive pulmonary disease, the addition of long-acting b 2 -agonists (LABA) to glucocorticosteroids (GCS) results in better control than increasing the dose of GCS alone. In smooth muscle cells and fibroblasts, one apparent underlying mechanism involves the ability of LABAs to activate the glucocorticoid receptor (GR).The present study investigates the effects of formoterol (FORM), salmeterol (SALM) and budesonide (BUD) on GR activation in bronchial epithelial cells via tumour necrosis factor-astimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) release, GR nuclear translocation and GR-regulated reporter gene activity.Both BUD and FORM inhibited GM-CSF release by f50%. The combination of these two drugs, in clinically relevant concentrations, inhibited GM-CSF release by 85% down to unstimulated levels. A similar inhibition was obtained when combining BUD and SALM. The ability of FORM to inhibit GM-CSF synthesis was not altered by small interfering RNA-mediated depletion of GR and FORM nor SALM-induced GR translocation into the cell nucleus. In addition, FORM did not activate GR-regulated reporter gene activity (SALM was not tested), in contrast to the clear effect of BUD.It was concluded that in bronchial epithelial cells, inhibition of granulocyte-macrophage colonystimulating factor synthesis by formoterol and salmeterol does not act via previously demonstrated glucocorticoid receptor-related mechanisms, suggesting an alternative pathway in these cells.

Background and purpose: b2-Adrenoceptor agonists (b2-agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, b2-adrenergic agonist stimulation induces desensitization of the b2-adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of b2-agonists in cellular and in vitro tissue models. Experimental approach: b2-Adrenoceptor-induced responses and subsequent loss of receptor responsiveness were studied in primary human airway smooth muscle cells and bronchial epithelial cells by measuring cAMP production. Receptor responsiveness was compared at equi-effective concentrations, either after continuous incubation for 24 h or after a 1 h pulse exposure followed by a 23 h washout. Key findings were confirmed in guinea pig tracheal preparations in vitro. Key results: There were differences in the reduction of receptor responsiveness in human airway cells and in vitro guinea pig trachea by a panel of b2-agonists. When restimulation occurred immediately after continuous incubation, loss of responsiveness correlated with initial effect for all agonists. After the 1 h pulse exposure, differences between agonists emerged, for example isoprenaline and formoterol induced the least reduction of responsiveness. High lipophilicity was, to some extent, predictive of loss of responsiveness, but other factors appeared to be involved in determining the relationships between effect and subsequent loss of responsiveness for individual agonists. Conclusions and implications: There were clear differences in the ability of different b2 agonists to induce loss of receptor responsiveness at equi-effective concentrations.