EP4 Receptor Agonists in the Inhibition of Airway Remodelling

Inhaled prostaglandin E2 (PGE2) has shown beneficial effects in patients with chronic bronchitis and asthma1, however the selective EP2 agonist AH13205 failed to cause bronchodilation in man2. Subsequent data has demonstrated that it is the EP4 and not the EP2 subtype that is responsible for the PGE2-mediated relaxation of human airway smooth muscle3. In addition to bronchoconstriction, there are a number of studies demonstrating severe, persistent asthma is selectively associated with increased airway smooth muscle and fibroblast accumulation4 resulting in pathological airway remodelling. Here we have investigated the potential for EP4 agonists to inhibit processes associated with airway remodelling in addition to causing bronchodilation, and the dependence of this inhibition on cAMP accumulation.

cAMP accumulation (AlphaScreen), ERK phosphorylation (Immunoflourescence) and cell proliferation (DELFIA BrdU incorporation) were monitored in primary human bronchial smooth muscle cells (hBSMc; Promocell) and primary human lung fibroblasts (HLF; Lonza) after treatment with a range of EP receptor agonists. cAMP accumulation: all incubations performed in HBSS (5 mM HEPES, 0.1 % (w/v) HSA, 5 µM rolipram), for 2 hours at room temperature. ERK phosphorylation and proliferation: all incubations were performed in serum-free medium containing 0.1 % HSA. pERK: cells were pre-treated for 30 min with agonists followed by 10 min incubation with 3 ng/mL PDGF-BB, at 37°C. Proliferation: cells were treated with EP receptor agonists for 24 h at 37°C, in the presence of either PDGF (3 ng/mL) or FBS (2 % v/v).

The pan-EP receptor agonists PGE2 and misoprostol, and the EP4-selective agonist AGN205204 (AGN), but not the EP2-selective agonists butaprost or AH13205, were able to promote robust cAMP accumulation, with PGE2 being the most potent and efficacious in both cell types. Despite being a partial agonist with respect to PGE2, AGN was the most efficacious of all the EP receptor agonists in ERK phosphorylation and proliferation assays (see table for selected data). In comparison, the full agonist PGE2 was only partially effective at inhibition proliferation of ERK phosphorylation.

Table 1 Potency and efficacy of EP receptor agonists in a range of primary cell assays (mean ± SEM; n≥3)

Together these data demonstrate that the EP4 receptor has the potential to strongly inhibit processes associated with airway remodelling, making it an interesting new target for the treatment of severe, persistent asthma. From these results it appears that the anti-proliferative effects of the EP4 receptor may not be solely dependent on global cAMP accumulation. This highlights the importance of monitoring the kinetics and localisation of intracellular signals, as well as multiple pathways when profiling novel compounds, as population second messenger assays may not always predict phenotypic outcomes.

1. Pavord et al., (1993) Am Rev Respir Dis 148: 87-90.

2. Nials AT et al., (1993) Cardiovasc Drug Rev 11: 165-179.

3. Buckley et al., (2011) Thorax 66: 1029-1035.

4. Benayoun et al., (2003) Am J Respir Crit Care Med 167: 1360-1368.