Download OS002 EP4 Receptor Agonists in the Inhibition of Airway

OS002
EP4 Receptor Agonists in the Inhibition of Airway Remodelling
1,2
1,2
EM Rosethorne , SJ Charlton
1
2
University of Nottingham, Nottingham, UK, Novartis Institutes for Biomedical Research,
Horsham, UK
Inhaled prostaglandin E2 (PGE2) has shown beneficial effects in patients with chronic
1
bronchitis and asthma , however the selective EP2 agonist AH13205 failed to cause
2
bronchodilation in man . 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
3
muscle . In addition to bronchoconstriction, there are a number of studies demonstrating
severe, persistent asthma is selectively associated with increased airway smooth muscle and
4
fibroblast accumulation 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 serumfree 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.
PGE
2
cAMP EC50 (µM)
pERK IC50 (µM)
Proliferation IC50 (µM)
(% max)
(% inhibition)
(% inhibition)
hBSMc
HLF
hBSMc
HLF
hBSMc
HLF
serum
v
0.01 ± 0.01
(100 ± 2.9)
0.05 ± 0.01
(101 ± 8.6)
0.01 ± 0.01
(56.5
±
4.7)
0.01 ± 0.01
(79.7
±
8.4)
0.41 ± 0.12
(67.6
±
7.9)
0.13 ± 0.05
(43.1
±
6.1)
HLF
PDGF
v
0.05 ± 0.04
(75.7
±
5.5)
0.04 ± 0.01 0.38 ± 0.16 0.02 ± 0.01 0.01 ± 0.01 0.67 ± 0.26 0.13 ± 0.03
0.12 ± 0.01
(66.3
± (45.1
± (60.7
± (91.9
± (74.7
(69.1
±
(108 ± 3.1)
4.5)
8.8)
5.1)
4.7)
±12.5)
9.7)
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 antiproliferative 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.
AGN
1. Pavord et al., (1993) Am Rev Respir Dis 148:87-90.
Drug Rev 11:165-179.
2. Nials AT et al., (1993) Cardiovasc
3. Buckley et al., (2011) Thorax 66:1029-1035.4. Benayoun et al., (2003) Am J Respir Crit
Care Med 167:1360-1368.