Comparing the re-assertion profile of β2-adrenoceptor agonists that exhibit a range of clinical duration of action

Michelle Bradley, Luke Bevan, Gareth Williams, Zhengjin Jiang, John Reilly, Paul Nicklin, Steven Charlton

Novartis Institutes of Biomedical Research, West Sussex, UK

The β2-adrenoceptor agonists currently used in the management of asthma and COPD can be classified as either short acting e.g. salbutamol (4-6h) or long acting e.g. formoterol and salmeterol(∼12h). However, preclinical data for indacaterol suggests that its duration of action after a single inhalation is 24 hours (Beeh et al., 2007). It is now widely accepted that agonists with a long duration of action have the capacity to “reassert” airway smooth muscle relaxation in vitro, despite repeated washing (Anderson et al., 1994). The aim of this study was investigate the reassertion profile of indacaterol compared to the marketed inhaled β2-adrenoceptor agonists salbutamol, salmeterol and formoterol in CHO-β2A cells.

CHO-β2A cells, seeded overnight at 40000 cells/well, were incubated with a range of concentrations of each agonist for 30 minutes. They were then subjected to 10 washes, incubated with assay buffer for 30 minutes and lysed. Levels of cAMP were determined using AlphaScreen™ technology. The same experiment was performed using un-transfected CHO-WT cells, however, after washing, CHO-β2A cells (80 000 cells/well), were added to each well and incubated for 30 minutes before the cells were lysed. Finally, following incubation with agonist and washing, CHO-β2A cells were solubilised by the addition of acetonitrile, 0.1% formic acid, and concentrations of each agonist were measured using LC-MS.

By comparing pre- and post-wash pEC50 values, the rank order of reassertion was salmeterol > indacaterol > formoterol, with ∼11, 115 and 1411 fold reduction in potency, respectively. Salbutamol demonstrated negligible reassertion. pEC50 values determined following incubation with CHO-WT cells were comparable to post-wash values, suggesting that these agonists can migrate from CHO-WT cell membranes to activate receptors on CHO-β2A cells. Finally, LC-MS data confirmed that the rank order to retention of agonist in the cell membrane correlated with the magnitude of reassertion.

The rank order of reassertion was consistent with the clinical duration of action, with the exception of salmeterol which appeared more persistent than would be predicted. This suggests that duration of action of β2 agonists may not be governed solely by the ability of an agonist to reassert. It is possible that the lower clinical dose combined with poor intrinsic efficacy means salmeterol does not perform as effectively in the clinical setting.

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Beeh KM, Derom E, Kanniess F, et al (2007) Eur Respir J.29:871-8