| pA2 online © Copyright 2004 The British Pharmacological Society |
100P
GKT, University of London Winter Meeting December 2003 |
|
The effect
of roflumilast N-oxide on human leukocyte function in-vitro |
|
Print abstractRoflumilast, an orally
active, selective PDE4 inhibitor, currently in phase III clinical development
for Chronic Obstructive Pulmonary Disease and asthma, can suppress both
myeloperoxidase (MPO) activity and neutrophil elastase (NE) release from
fMLP-stimulated neutrophils and also TNF-
,
but not IL-8 from LPS-stimulated mononuclear cells (Boswell et al 2002).
The aim of this study was to investigate whether the pharmacologically
active metabolite of roflumilast, roflumilast N-oxide, also suppressed
these parameters.
Neutrophils and mononuclear
cells were isolated from blood of healthy, non-smoking volunteers (n=6)
by density dependent centrifugation. Mononuclear cells (2 x106/well)
were incubated with roflumilast N-oxide (10-10-10-6M)
or 0.1% DMSO vehicle control, and stimulated for 18 hours with LPS (10ngml-1)
(37oC, 5% CO2).
TNF- and IL-8 release
was quantified by ELISA. Neutrophils (2.5 x 10-6
/well) were incubated with roflumilast N-oxide (10-9-10-5M)
or vehicle control, along with TNF-
(100Uml-1) or HBSS vehicle control and
cytochalasin B (5µgml-1), for 30 minutes prior to activation with
fMLP (10-7M) (Room temperature). After
45 minutes the cells were removed by centrifugation and NE release and
MPO activity were assessed colorimetrically. Inhibitor data were expressed
as % of control, mean ± SEM (n=6). Statistical significance was
assessed by ANOVA followed by Dunnett's test.
LPS induced significant
TNF- release (control,
25.6 ± 8.4 absorbance units (AU) vs. LPS, 417 ± 88.1 AU;
P<0.01) and IL-8 release (control, 26.01 ± 10.4 AU vs. LPS,
811.7 ± 137.3 AU; P<0.01) from human mononuclear cells Roflumilast
N-oxide suppressed LPS-induced TNF-
release (max % inhibition: 92.5 ± 4.5, P<0.01), but not IL-8
release from human mononuclear cells.Roflumilast N-oxide also suppressed
NE release and MPO activity from stimulated neutrophils in both the absence
(max. % inhibition, NE: 58.4 ± 15.7; MPO: 75.2 ± 10.1, P<0.05)
and presence of TNF-
(max. % inhibition NE: 62.6 ± 10.3; MPO: 63.29 ± 11.60,
P<0.05).
These results demonstrate
that the pharmacologically active metabolite of roflumilast, roflumilast
N-oxide can significantly suppress neutrophil degranulation and TNF-
release from mononuclear cells.
Boswell et al., 2003. Am. J. Resp. Crit. Care. Med. 167, A310.
This research is funded by the MRC/Altana LTD.