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Full-length article Acta Pharmacologica Sinica 2005 December; 26 (12): 1492-1496 Inhibitory effect of acetamide-45 on airway inflammation and phosphodiesterase 4 in allergic rats1 Kai WANG2, Hua-hao SHEN2,4, Jun-chun CHEN2, Zhong CHEN3 2Respiratory Department, the Second Affiliated Hospital, Medical School of Zhejiang University, Hangzhou 310009; 3Department of Pharmacology, Medical School of Zhejiang University, Hangzhou 310031, China
1 Project supported by Science and Techno-logy department of Zhejiang province
(No 2003C24002).
Aim: To determine the effects of acetamide-45 on respiratory function, airway inflammation, and the activity of
phosphodiesterase 4 (PDE4) in allergic rats.
Methods: Rats were sensitized by a single intramuscular injection with ovalbumin (OVA)
and were challenged with ovalbumin applied by using an aerosol repeatedly for 7 d after 2 weeks. Acetamide-45 at
concentrations of 5, 10, or 30 mg/kg was then administered by intraperitoneal injection. Changes in dynamic lung compliance and
lung resistance, the accumulation of inflammatory cells in bronchoalveolar lavage, PDE4 activity, and the concentration of
interleukin-4 in rat lung tissue were determined.
Results: Seven days of treatment with acetamide-45 prevented eosinophil
accumulation in allergic rats. At doses of 5, 10, and
30 mg/kg, acetamide-45 decreased lung resistance to 0.20±0.04,
0.25±0.07, and 0.22±0.05
cmH2O·s-1·mL-1
, respectively (P<0.05 vs OVA), and it also increased dynamic lung compliance to 0.41±0.07,
0.39±0.06, and 0.42±0.09
mL/cmH2O (P<0.05 vs OVA). After being treated with different doses of acetamide-45, the PDE4
activities in lung tissue were 281±55, 273±57, and 238±36
nmol·g-1·min-1
(P<0.05 vs OVA), and the concentrations of
interleukin-4 in lung tissue were 6.22±1.13,
5.95±1.20, and 5.68±2.20 μg/g protein
(P<0.05 vs OVA).
Conclusions: Acetamide-45 was found to improve respiratory function and inhibit airway inflammation in this animal model, and the PDE4 activity of lung
tissue was obviously inhibited.
Acetamide-45 was an effective anti-inflammatory agent in respiratory inflammation, and the mechanism of its action might
depend on inhibition of PDE4. Key words acetamide-45; 3',5'-cyclic nucleotide phos-phodiesterase; respiratory function tests; eosinophils; asthma Note: Please read the complete
full text with Figures and Tables at
Introduction
Asthma is a chronic inflammatory disease of the airways,
of which lung inflammation and bronchial hyperactivity (BHR)
are two distinct characteristics[1,2]. Eosinophils are
consi-dered to be the principal inflammatory leukocytes involved
in the asthmatic reaction, due in part to the toxic granular
proteins they secrete and the membrane products that induce airway epithelial pathology and mucus
hypersecretion[3,4]. 3กฏ,5กฏ-Cyclic-nucleotide (cAMP) has important
regulatory roles in the process of inflammation.
Phosphodiesterase 4 (PDE4), which catalyzes the hydrolysis of cAMP
to the corresponding nucleotide, 5กฏ-monophosphate, appears
to be an attractive target for anti-inflammatory
drugs[5,6].
Inhibition of PDE4 results in increased levels of cAMP, which
leads to functional inhibition of inflammatory cells such as
eosinophils and lymphocytes, and reduced release of
inflammatory cytokines such as interleukin-4
(IL-4)[7,8]. There has been significant interest in PDE4 inhibitors as potential
therapeutic agents for asthma.
Because asthma has been the focus of much study effort,
treatment of the disease has been improved by the
implementation of management guidelines in recent
years[9]. Such treatments as corticosteroids,
b2-agonists, theophylline and leukotriene antagonists have been used as
anti-inflammatory agents to control
asthma[10]. However, the possible dose-related adverse systemic effects produced by
long-term treatment with these drugs are not acceptable in
clinical practice[11,12]. Therefore, new anti-asthma agents are
required.
A series of new N-(pyridin-4-yl)-(indol-3-yl) alkylamides
(44-84) have been prepared in the search for novel
antiallergic drugs. Initial studies showed that acetamide-45
inhibited IL-4 and IL-5 biosynthesis and histamine
release[13]. We have also previously reported that acetamide-45 has an
inhibitory effect on histamine- and methacholine-induced
contractions of isolated guinea pig trachea, and that it can
inhibit PDE4 activity[14,15]. It was concluded that acetamide-45
was a new antiallergic agent. However, little is known about
its effects on eosinophil infiltration in airway and lung
function in asthmatic rats. There have been no reports regarding
the effect of acetamide-45 on PDE4 activity in the lungs in
any animal model of asthma.
Therefore, in the present study, we examined the
inhibitory effect of acetamide-45 on airway inflammation and lung
function in a rat model of asthma, and indicated a possible
mechanism by which the effect might be exerted.
Materials and methods
Animals Male Sprague-Dawley rats weighing 140-160 g
were obtained from the Laboratory Animal Center of the
Medical School of Zhejiang University (Certificate
No 220010027, conferred by the Zhejiang Medical Laboratory
Animal Administration Committee).
Chemicals Aminophylline, cAMP,
and ovalbumin were purchased from Sigma (St Louis, MO, USA). Acetamide-45
was kindly provided by the Department of Organic
Chemistry and Medical Chemistry, Faculty of Pharmacy (Nantes
University, France). The rat IL-4-specific enzyme-linked
immunosorbent assay (ELISA) kit was purchased from
Jingmei BioTech Co (Shenzhen, China).
Sensitization and challenge procedure Rats were
sensitized by a single intramuscular injection of 10 mg ovalbumin
(OVA) mixed with 100 mg aluminum hydroxide in 1 mL of
saline. After 2 weeks, these animals were then challenged by
exposure for 20 min to aerosolize 1% OVA in saline generated
by a jet nebulizer once a day for 7 d. The drugs were
administered by intraperitoneal injection before challenge. The
doses of acetamide-45 were 5, 10, or 30 mg/kg, respectively,
and the dose of aminophylline was 10 mg/kg per day for 7 d.
At d 21, the animals were killed, and the lung tissues were
immediately removed, frozen in liquid nitrogen, and then
stored at -80 °C until analysis.
Measurement of lung function At 24 h after the last
antigen challenge, rats were anesthetized with urethane
(1 g/kg, ip). The trachea was cannulated and placed in a whole body
plethysmograph for the measurement of lung
resis tance (RL) and dynamic lung compliance
(Cdyn)[16,17].
Cell counts in bronchoalveolar lavage Bronchoalveolar
lavage (BAL) was performed by flushing the airways with 10
mL/kg saline containing 1% bovine serum albumin and 1000
kU/L heparin sodium through a tracheal cannula. The BAL fluid was pooled and immediately centrifuged at
500×g at 4 °C for 10 min. The supernatant was removed and the cells
were resuspended in 1 mL saline containing 10% bovine
serum albumin. Counts of the total number of leukocytes
recovered in the BAL fluid were carried out using a Neubauer
chamber, and differential cell analysis was carried out under
a light microscope after Wright-Giemsa staining.
Determination of IL-4 in lung tissue At a ratio of 1 g to
10 mL, homogenized lung tissue was added to 50
mmol/L potassium phosphate buffer (pH 6.0) containing
0.05% NaN3 and 0.1% 3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHARPS). The homogenates were
centrifuged twice at 19 851×g, each for 30
min[17]. IL-4 in the supernatants was quantified using a rat IL-4-specific ELISA
kit.
PDE4 activity assay Frozen 25-mg pieces of lung tissue
were homogenized and PDE4 activity was determined by high
performance liquid chromatography (HPLC) as described
elsewhere[15,18].
Statistical analysis Data are expressed as mean±SD.
Statistical analysis was performed using one-way analysis
of variance.
Results
Effects of acetamide-45 on inflammatory cells in the
airways of allergic rats In our rat model of allergic asthma,
ovalbumin sensitization and challenge caused an obvious
increase in inflammatory cells in BAL. As shown in Figure 1,
the total number of leukocytes and eosinophils in the OVA
group was significantly greater than that in the
nor mal group (P<0.05), but the number of inflammatory cells was depressed
when aminophylline (Ami) was administered
(P<0.05 vs OVA). Seven days of treatment with acetamide-45 (Ace) at 5, 10, or
30 mg/kg significantly decreased the number of
inflammatory cells in BAL caused by sensitization and
challenge (P<0.05 vs OVA; Figure 1). There was no statistical
difference between the Ace group and the Ami group
(P>0.05; Figure 1).
Effects of acetamide-45 on
RL and
Cdyn in allergic rats After rats were given 7 aerosolized OVA challenges, a
significant decrease in Cdyn was induced. However, through
sensitization and challenge, RL increased to a level that was
significantly greater than that in the normal group. The
administration of aminophylline decreased the
RL and increased the
Cdyn of sensitized and challenged animals. At a dose of 5,
10, or 30 mg/kg, acetamide-45 improved both
Cdyn and RL (Figure 2).
Effect of acetamide-45 on IL-4 in lung tissue in allergic
rats As shown in Figure 3, IL-4 levels in the lungs of rats
from the various groups were measured. Sensitization and
challenge increased the concentration of IL-4 to a
level significantly different from that of the normal group
(P<0.05). Seven days of treatment with acetamide-45 at
concentrations of 5, 10, or 30 mg/kg, or with aminophylline at a
concentration of 10 mg/kg, obviously reduced the increase in the
concentration of IL-4 (P<0.05).
Effect of acetamide-45 on PDE4 activity in lung tissue in
allergic rats Sensitization and challenge increased the PDE4
activity of lung tissue to 503±125
nmol·g-1·min-1, which was greater than that of the normal group
(P<0.05). Acetamide-45 at concentrations of 5, 10, or 30 mg/kg inhibited the
hydrolysis of cAMP by the PDE4 extracted from lung tissue.
The PDE4 activities of the lung tissue were 281±55, 273±57,
and 238±36
nmol·g-1·min-1, respectively (for acetamide-45 at
concentrations of 5, 10, or 30 mg/kg). As a non-selective
inhibitor of PDE4, aminophylline suppressed PDE4 activity
to 341±44
nmol·g-1·min-1 at a dose of 10 mg/kg, which was
weaker than the activity produced by treatment with
acetamide-45 at concentrations of 10 and 30 mg/kg
(P<0.05, Figure 4).
Discussion
Acetamide-45, N-(pyridin-4-yl)-[1-(4-fluorophenyl)
indol-3-yl] acetamide, is a new anti-inflammatory drug that inhibits
the release of cytokines and the activation of
eosinophils[12]. It has also been reported that acetamide-45 inhibits the
contraction of isolated guinea pig trachea induced by histamine
and methacholine[14]. However, the mechanism by which
acetamide-45 acts remains unknown. The present study
shows that acetamide-45 can suppress the accumulation of
inflammatory cells in airways and improve the lung function
of allergic rats. We also found that acetamide-45 could
inhibit PDE4 activity and reduce the IL-4 level in the lung
tissue of allergic rats. To our knowledge, this study represents the first investigation of the effect of acetamide-45 on lung
function and PDE4 concentration in the lung in an animal
model of asthma.
Asthma is a syndrome characterized by eosinophil
infiltration and airway hyperresponsiveness, which occurs via a
mechanism that is very complex. It is well known that
eosinophils play a critical role in airway damage and dysfunction
in asthma. We found that acetamide-45 had an inhibitory
effect on eosinophil infiltration in the airways of allergic rats.
Not only eosinophils but also other inflammatory cells were
decreased in concentration, so that inflammation of the
airway in allergic rats was inhibited by acetamide-45.
Inflammation of the airway leads to spasms of the
bronchial smooth muscles, mucus hypersecretion, and airflow
limitations, which result in pulmonary dysfunction in asthma.
The data obtained by a whole body plethysmograph in this
study indicate that acetamide-45 improves the lung function
of allergic rats produced by sensitization and challenge. Its
effectiveness has been demonstrated in two ways.
First, the Cdyn of allergic rats increased obviously relative to normal
rats at a dose of 5 mg/kg acetamide-45. Second,
the RL of allergic rats was reduced significantly by acetamide-45 in
the same way. The improvement in lung function produced
by acetamide-45 is similar to that produced by aminophylline.
The experiments presented here show that both
acetamide-45 and aminophylline repress IL-4 production in
allergic rats, but that there is no difference between the two
agents. IL-4 is a key cytokine in the development of allergic
inflammation. It is associated with the secretion of IgE by B
lymphocytes and the expression of eotaxin and other
inflammatory cytokines that contribute to inflammation and lung
remodeling in asthma[18,19]. A previous study shows that
acetamide-45 inhibits IL-4 biosynthesis and release, which
is identical to our result[12]. This indicates that acetamide-45
can reduce the release of IL-4 and reduce its concentration
in the lung, which results in the inhibition of airway
inflammation.
Our study of PDE4 activity shows that acetamide-45 has
an inhibitory effect on cAMP hydrolysis of PDE4 in the lung
tissue of allergic rats. In a previous study, we demonstrated
that acetamide-45 could inhibit the hydrolysis of cAMP by
PDE4 extracted from transfected yeast, and that its
inhibitory effect was stronger than that of
theophylline[15]. The present study also shows that at the same dose (10 mg/kg),
the inhibitory effect of acetamide-45 is greater than that of
aminophylline, which is a non-selective PDE inhibitor. PDE4
is specific for cAMP, and is predominantly expressed in
inflammatory cells. It plays an important role in the regulation
of cellular functions in inflammatory and immune cells, and
inhibition of PDE4 may be one of the anti-inflammatory
mechanisms of acetamide-45.
In conclusion, we have shown that acetamide-45 could
improve the lung function of allergic rats produced by
sensitization and challenge, and that production of eosinophils in
those animals could also be repressed. Acetamide-45 had an
inhibitory effect on PDE4 activity in the lung tissue of
allergic rats, and the inhibition was stronger than that produced
by aminophylline. The present research may provide an
experimental basis for further study of this agent.
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