Clinical Science (2001) 100, 411–417 (Printed in Great Britain)
Mucosal protective effects of ecabet sodium:
pepsin inhibition and interaction with mucus
J. P. PEARSON* and N. B. ROBERTS†
*Department of Physiological Sciences, The Medical School, Framlington Place, University of Newcastle upon Tyne, Newcastle
upon Tyne NE2 4HH, U.K., and †Department of Clinical Chemistry, Royal Liverpool University Hospital, Liverpool L69 3BX, U.K.
A
B
S
T
R
A
C
T
Pepsin, acid and Helicobacter pylori are major factors in the pathophysiology of peptic ulcer
disease and reflux oesophagitis. Ecabet sodium reduces the survival of H. pylori in the stomach
and inhibits pepsin activity in the gastric juice of experimental animals. Here we have
investigated the effects of ecabet sodium on some of the factors involved in the dynamics of the
mucosal barrier, i.e. pepsins and mucins. This study used gastric juice obtained from 12 nonsymptomatic volunteers and nine patients with reflux oesophagitis. Ecabet sodium significantly
inhibited pepsin activity in human gastric juice, with a maximum inhibition of 78 %. Pepsin 1, the
ulcer-associated pepsin, was inhibited to the greatest extent. The ability of gastric juice to digest
mucin was significantly inhibited by ecabet. As with gastric juice proteolytic activity, the
inhibitory effect of ecabet on mucolysis was greater in gastric juice from patients with reflux
oesophagitis than in that from controls. Ecabet sodium showed a positive interaction with gastric
mucin, as assessed by an increase in viscosity. Thus ecabet sodium may reduce the aggressive
potential of gastric juice towards the mucosa, which may be relevant in the treatment of reflux
oesophagitis and peptic ulcer disease. In addition, it may strengthen the mucus barrier in peptic
ulcer disease and gastritis.
protection and repair. Ecabet stimulates re-epithelization
after mucosal damage [9] and binds strongly to the gastric
mucus layer [10]. (3) Inhibition of pepsin activity. Ecabet
binds to and precipitates pig pepsin [11] and, by coating
the mucus gel, can prevent its mucolysis by pepsin [12].
The protective adherent gastroduodenal mucus\
bicarbonate barrier is the first line of defence, protecting
the underlying epithelial cells from damage by luminal
aggressors (e.g. acid and pepsin). In the healthy human
stomach, mucus gel is present as a continuous layer
omean (S.D.) thickness 144 (52) µm [13]q, which is
maintained by a dynamic balance between secretion from
the epithelial cells and erosion from the surface into the
lumen. The adherent gastric mucus barrier has been
shown to be structurally weaker in patients with peptic
ulcer [14]. This structural weakness is caused by a
decrease in the content of polymeric, gel-forming
INTRODUCTION
Ecabet sodium, a dehydroabietic acid derivative from
pine resin (Figure 1), has been used clinically for the
treatment of gastritis and gastric ulcer [1]. Ecabet is a
locally acting anti-ulcer agent with a high affinity for the
stomach wall [2]. It has also been shown, in animal
experimental models of colorectal cancer, that ecabet
sodium may slow the development of the mucosal
dysplasia–invasive adenocarcinoma sequence [3]. Ecabet
is believed to exert its effects through a series of
mechanisms. (1) Anti-bacterial effects. Triple therapy
with amoxicillin, ecabet and a proton pump inhibitor
effectively eradicated H. pylori [4,5]. Ecabet prevents the
survival of H. pylori in the stomach via inhibition of
essential urease activity [6,7], and inhibits the adhesion
of H. pylori to gastric epithelia [8]. (2) Enhanced mucosal
Key words : gastric juice, mucolysis, proteolysis.
Correspondence : Dr J. P. Pearson (e-mail j.p.pearson!ncl.ac.uk).
#
2001 The Biochemical Society and the Medical Research Society
411
412
J. P. Pearson and N. B. Roberts
obtained : a basal sample collected over 30 min before
pentagastrin stimulation (6 µg\kg subcutaneously), and a
60 min post-pentagastrin sample. The subjects had fasted
overnight, and had stopped anti-secretory treatment 5
days before collection. The juice samples were dialysed
twice against 2 litres of 0.05 M sodium acetate buffer,
pH 4.1, at 4 mC for 24 h. Each juice sample was divided
into 8 ml aliquots and stored at either 4 mC or k20 mC.
Measurement of proteolytic activity
Figure 1
Chemical structure of ecabet sodium
Ecabet sodium is [(j)-(1R,4aS,10aR )-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-6-sulpho-1-phenanthrenecarboxylic acid 6-sodium salt
pentahydrate].
mucins : decreases of 48 % in patients with gastric ulcer
and 18 % in patients with H. pylori-induced gastritis
have been recorded compared with controls [14,15].
Associated with the decrease in the strength of the gel in
peptic ulcer disease is a change in the pepsin isoenzyme
profile in the gastric juice. Seven pepsins (1, 2, 3, 3a, 4, 5
and 6) have been identified in human gastric juice, with
pepsin 3 being the most abundant [16]. The pepsin 1
content increases from 3.6 % of total pepsin activity in
pentagastrin-stimulated gastric juice from non-ulcer
controls to between 16.5 % and 23 % in patients with
peptic ulcer [17]. This pepsin has been shown to be up to
6 times more mucolytic than pepsin 3 [18]. Therefore
gastric juice from peptic ulcer patients could be mucolytic
enough to tip the balance in favour of erosion of the
mucus barrier, leading to mucosal damage and ulceration.
It is therefore important to determine the ability of ecabet
to inhibit individual pepsins. Here we report the first
study on the inhibition of pepsin activity by ecabet in
basal and pentagastrin-stimulated gastric juice, and the
effects of ecabet on individual human pepsins 1, 3 and 5.
MATERIALS AND METHODS
Materials
Ecabet sodium was obtained from Tanabe Seiyaku Co.
Ltd (Osaka, Japan). All other reagents were of analytical
grade, and were obtained from Sigma Chemical Co.
(Poole, Dorset, U.K.).
Human gastric juice
Gastric juice was obtained via a naso-gastric tube from
nine patients investigated for reflux oesophagitis, and
from 12 non-symptomatic volunteers. Two samples were
#
2001 The Biochemical Society and the Medical Research Society
The proteolytic activity of human gastric juice samples
was measured : (1) using bovine haemoglobin as a
substrate at pH 2.0 [19] ; and (2) using succinyl albumin
as a substrate at pH 2.2, a modification of the method of
Lin et al. [20,21]. Both of these assays used pig pepsin as
a standard.
Effect of ecabet on pepsin proteolytic
activity
A 200 µl sample of pig pepsin or pepsinogen solution
(1 mg\ml) was incubated with 200 µl of ecabet, to give a
final concentration of 0–10 mg\ml ecabet, for 30 min at
37 mC at pH 1.6 (0.1 M glycine\HCl and 0.05 M NaCl)
or pH 5.0 (0.05 M sodium acetate buffer containing 0.1 M
NaCl) for pepsin solutions, or at pH 7.4 (0.067 M sodium
phosphate buffer) for pepsinogen solutions. The suspensions were then centrifuged for 10 min at 1200 g in a
bench centrifuge. Pepsin activity in the supernatant was
measured using bovine haemoglobin as a substrate.
For investigations of pepsin activity in gastric juice,
1 ml of gastric juice (pepsin activity range 20–260 µg\ml)
was mixed with ecabet to give a final concentration of
5 mg\ml ecabet, and incubated for 30 min at 37 mC at
pH 1.6 or pH 5.0. A 0.5 ml portion of human pepsin
isoenzymes 1, 3 and 5 (containing 200 or 400 µg\ml
pepsin) was mixed with ecabet to give a final ecabet concentration of 5 mg\ml, followed by incubation for 30 min
at 37 mC at pH 1.6 or pH 5.0. The suspensions were
then processed as above, with the exception that measurements of enzyme activity involved the production of a
new N-terminal peptide from succinyl albumin.
The statistical test used to compare the different groups
was a Mann–Whitney U test, with significance set at P
0.05.
Preparation of human pepsin isoenzymes
Pepsins in gastric juice were fractionated by HPLC using
an anion-exchange TSK DEAE-5PW gel with a linear
gradient of 50–300 mM NaCl in 50 mM acetate buffer,
pH 4.1, over 30 min, followed by a single step to 1 M
NaCl in the same buffer. Pepsin 5 was eluted between 50
and 100 mM NaCl, pepsin 3 between 100 and 200 mM
NaCl, and pepsin 1 between 450 and 650 mM NaCl [22].
Inhibition of pepsin by ecabet sodium
Table 1
Effects of ecabet on pepsin activity in pentagastrin-stimulated gastric juice
Pepsin activity is expressed in µg/ml, and was measured using the N-terminal assay (n l 3 for each assay). The symbols j and k represent the presence and absence
respectively of a preincubation step with 5 mg/ml ecabet. The prefix P denotes a patient with reflux oesophagitis, and the prefix V denotes a non-symptomatic volunteer.
‘ Activity (%) ’ is the percentage activity remaining after ecabet treatment. (The supernatant produced from centrifugation of 5 mg/ml ecabet alone had no effect in
the N-terminal assay.)
j
k
pH 1.6
Subject
Activity ( µg/ml)
Activity ( µg/ml)
P1
P2
P3
P4
P5
P6
P7
P8
102.1
123.1
124.2
118.4
120.7
21.8
121.9
124
37.1
68.4
60.8
47.4
28.2
8.9
15.3
46.2
37
55
49
40
23
41
13
37
8.5
38
7.8
5.3
49.7
3.6
78.6
6.7
8
31
6
4
41
16
64
5
V1
V2
V3
V4
V5
V6
V7
215.6
227.3
234.4
227.5
241.7
244.5
228.5
129.7
161.7
138.5
80.1
152.8
57.7
147
60
71
59
35
63
24
65
147.3
160.6
157.7
142.5
111.2
86.7
149.7
68
71
68
63
46
36
66
The isoenzyme solutions were dialysed exhaustively
against distilled water and then freeze-dried.
Effect of ecabet on the mucolytic activity of
pepsin
Gastric juice at pH 4.0 was incubated at 37 mC for 30 min
with or without ecabet (5 mg\ml). The samples were then
centrifuged at 1200 g, and the supernatant was dialysed
against 0.1 M glycine\HCl buffer, pH 1.6, containing
0.05 M NaCl. A 0.5 ml sample of the supernatant was
incubated with a 2 ml solution of purified pig gastric
mucin at pH 1.6 [21] (final mucin concentration 4–
6 mg\ml). Mucolytic activity was measured at 37 mC
against time, using a Contraves low-shear 30 viscometer
[23]. The results were expressed as the percentage
decrease in specific viscosity after 1 h of incubation. The
percentage decreases were corrected for any non-pepsininduced changes in the viscosity of the mucin solution.
The statistical comparison used was a Wilcoxon paired
test, with significance set at P 0.05.
Mucin–ecabet interaction studies
Solutions of pig gastric mucin (10 mg\ml), at pH 1.6 and
pH 5.0 (buffers as in the proteolytic activity section),
purified by CsCl equilibrium density centrifugation
followed by exclusion on Sepharose CL-2B, were mixed
pH 5.0
Activity ( µg/ml)
Activity (%)
Activity (%)
with solid ecabet sodium to give final ecabet concentrations of between 0.5 and 10 mg\ml. The mixture was
preincubated at 37 mC for 3 h in a shaking water bath, and
viscosity was measured using a Contraves low-shear
viscometer at 37 mC. The results were expressed as
percentage synergism, calculated using the equation :
Synergism (%) l (MEkMjE)\(MjE)
where M is mucin viscosity, E is ecabet viscosity and ME
is the measured viscosity of the mixture. A positive
synergism indicates an interaction between mucin and
ecabet that enhances viscosity.
RESULTS
Inhibition of pepsin and of pepsin activity
derived from pepsinogen
Preincubation of pig pepsin with ecabet at concentrations
up to 10 mg\ml at pH 1.6 or pH 5.0 had no effect on the
pepsin activity measured at pH 2.0 using bovine haemoglobin as substrate (e.g. only 3–6 % of the activity was
lost following a 30 min preincubation at pH 5.0 with the
highest concentrations of ecabet). However, a marked
decrease in pepsin activity from pepsinogen was
observed : 85 % and 90 % decreases were measured
#
2001 The Biochemical Society and the Medical Research Society
413
414
J. P. Pearson and N. B. Roberts
following preincubation with 5 and 10 mg\ml ecabet
respectively at pH 7.4.
When ecabet sodium was preincubated with pentagastrin-stimulated gastric juice from patients with
reflux oesophagitis, and the pepsin activity remaining in
the supernatant was assessed by new N-terminal development using succinyl albumin as the substrate,
significant inhibition occurred at both preincubation pH
values (Table 1). The mean activity remaining was
36.9p4.8 % (pS.E.M. ; n l 8) and 21.7p7.7 % after
preincubation with 5 mg\ml ecabet at pH 1.6 and pH 5.0
respectively. The inhibition data, when plotted, showed a
skewed distribution, and therefore a non-parametric
statistical test was used. The effect of ecabet was not
significantly different between the two pH values. Pepsin
activity in pentagastrin-stimulated gastric juice from nonsymptomatic volunteers also showed a significant decrease after treatment with 5 mg\ml ecabet : the mean
activity remaining was 53.9p6.6 % and 59.8p5.0 % (n l
7) after preincubation at pH 1.6 and pH 5.0 respectively.
As with the patient samples, the effect of ecabet was not
significantly different between pH 1.6 and pH 5.0. However, there were significant differences between the
patient and volunteer groups : (1) pentagastrin-stimulated
gastric juice from patients showed significantly more
inhibition by ecabet at pH 1.6 than did volunteer juice at
pH 5.0 ; and (2) pentagastrin-stimulated gastric juice from
patients showed significantly more inhibition by ecabet
at pH 5.0 than did volunteer juice at pH 1.6 or pH 5.0.
Ecabet also inhibited pepsin activity in the basal gastric
juice samples, with 55p20 % and 56.7p15.8 % of activity
remaining in gastric juice from patients at pretreatment
pH values of 1.6 and 5.0 respectively. Pepsin activity in
basal gastric juice samples from healthy volunteers was
also inhibited by preincubation with 5 mg\ml ecabet,
with 74p8.2 % and 76.3p10.9 % of pepsin activity
remaining at pH 1.6 and pH 5.0 respectively. However,
because only three basal samples were available for each
group, no statistical analysis was carried out.
Inhibition of individual pepsin isoenzymes
by ecabet
Table 2 shows the percentage inhibition of isoenzyme
proteolytic activities by 5 mg\ml ecabet at incubation pH
values of 1.6 and 5.0. Pepsin 1 showed the greatest
inhibition by ecabet at pH 5.0 (66 %), followed by pepsin
3 (47 %) and pepsin 5 (42 %). Ecabet inhibited a
400 µg\ml pepsin solution by 264.7p31.4 µg\ml for
pepsin 1, by 189p33.7 µg\ml for pepsin 3 and by
169p12.4 µg\ml for pepsin 5 (n l 3). Ecabet inhibited
pepsin 1 to a significantly greater extent than pepsin 5 at
pH 5.0 (P l 0.04), but there was no significant difference
compared with pepsin 3. Preincubation of the pepsins
with 5 mg\ml ecabet at pH 1.6 again showed most
inhibition of pepsin 1 (100 %), compared with 88 % and
#
2001 The Biochemical Society and the Medical Research Society
Table 2 Treatment of human pepsin isoenzymes 1, 3 and 5
with ecabet sodium
Pepsin solutions containing 200 or 400 µg were incubated with or without ecabet
sodium (5 mg/ml) at pH 1.6 or pH 5.0 (final volume 1 ml). Pepsin activity was
measured using the N-terminal assay, and levels were calculated using a pig pepsin
standard curve. The studies were carried out with three different preparations of
each isoenzyme, and each preparation was measured in triplicate.
pH
Pepsin
5.0
1
71
51
77
3
50
60
32
5
37
48
42
1
100
100
100
3
91
85
88
5
99
95
96
1.6
Inhibition (%)
97 % for pepsins 3 and 5 respectively. Ecabet inhibited a
200 µg\ml pepsin solution by 200 µg\ml for pepsin 1, by
175.7p3.5 µg\ml for pepsin 3 and by 193p2.1 µg\ml
for pepsin 5 (n l 3).
HPLC data confirmed that pepsin 1 interacts strongly
with ecabet. When gastric juice was preincubated with
ecabet and chromatographed, it showed a 50 % loss of the
pepsin 1 peak (results not shown).
Inhibition of mucolytic activity of gastric
juice
Table 3 shows that mucolytic activity measured at pH 1.6
was significantly inhibited in the gastric juice from reflux
oesophagitis patients, but not in that from controls.
Mucolytic activity in both pentagastrin-stimulated and
basal juice samples from patients was significantly
decreased by preincubation with ecabet sodium (onetailed Wilcoxon paired analysis ; P l 0.004 and P l 0.03
respectively). These data confirm that ecabet can bind to
and inhibit pepsin in human gastric juice.
Mucin–ecabet interactions
Viscosity studies demonstrated that ecabet and mucin
form complexes in solution, as their measured viscosity is
Inhibition of pepsin by ecabet sodium
Table 3
Effects of ecabet on mucolytic activity
Gastric juice (pH 4.0) was incubated with ecabet, and mucolytic activity was assessed at pH 1.6 by the decrease in viscosity over the first 1 h of a solution of 4–6 mg/ml
pig gastric mucin. Results were corrected for controls containing mucin alone. The prefix P denotes a patient with reflux oesophagitis, and the prefix V denotes a nonsymptomatic volunteer. N.E.S., not enough sample.
Decrease in viscosity at pH 1.6 (%)
Pentagastrin-stimulated gastric juice
Basal gastric juice
Subject
Before ecabet treatment
After ecabet treatment
Before ecabet treatment
After ecabet treatment
P1
P2
P3
P4
P5
P6
P7
P8
P9
17.4
31.1
16.6
12.8
14.4
10.5
7.5
31
0
0
26.6
8.8
2.2
0
0
3.4
10.2
0
6.7
N.E.S.
2.8
4.9
19
N.E.S.
9.3
3.7
N.E.S.
0
–
2.8
1.6
9.5
–
0
0
–
V1
V2
V3
V4
V5
V6
V7
V8
V9
17.8
28
0
22.6
36
9.6
2.3
5
5.4
33.3
11.3
3.2
5
6
0
0
0
5.4
0
4.3
22.1
0
N.E.S.
17.6
N.E.S.
N.E.S.
24.8
0
0.2
18.4
0
–
0
–
–
6.6
maximum (19.8 %) at 5 mg\ml ecabet, and then fell to
7.7 % at 10 mg\ml ecabet.
DISCUSSION
Figure 2
Mucin–ecabet interaction studies
Evidence of an interaction was assessed by measuring the specific viscosity at
37 mC of a 5 mg/ml gastric mucin solution mixed with ecabet sodium at
concentrations between 0.5 and 10 mg/ml.
greater than that of the individual components (Figure 2).
At 5 mg\ml mucin and 5 mg\ml ecabet, there was 28.8 %
and 19.8 % synergism at pH 5.0 and pH 1.6 respectively.
The interaction was dependent on the ecabet concentration and the pH. At pH 5.0, the amount of interaction
increased with increasing ecabet concentrations, to a
maximum of 32.2 % at 10 mg\ml (the highest concentration used). At pH 1.6 the interaction increased to a
Pepsin, acid and probably Helicobacter pylori are major
factors in peptic ulcer disease and reflux oesophagitis
[24,25]. Therefore any agent that inhibits the action of
these factors would be useful in the treatment of upper
gastrointestinal disease. Ecabet sodium has already been
shown to inhibit Helicobacter pylori mucosal adhesion
and urease activity [6–8], and to inhibit pig pepsin\
pepsinogen activity and pepsin activity in rat gastric juice.
If ecabet could also be shown to be an effective inhibitor
of human pepsins in gastric juice, in particular the ulcerassociated pepsin 1, and to prevent mucolysis of the
protective mucus barrier in the stomach and duodenum,
the rationale for its use would be strengthened. The
situation pertaining to the oesophagus in inflammation
caused by the reflux of gastric juice is different from that
in the gastroduodenal mucosa, because the oesophagus is
not protected by a dynamic mucus layer [26]. The
#
2001 The Biochemical Society and the Medical Research Society
415
416
J. P. Pearson and N. B. Roberts
obvious treatment would be to prevent reflux, but if this
cannot be achieved, then inhibition of pepsin activity\
secretion and neutralization of acid\inhibition of
acid secretion would be the next best approach.
In the present study we have investigated the potential
of ecabet sodium to inhibit proteolytic and mucolytic
activities in gastric juice, and its ability to strengthen the
gastric mucus barrier. Previously it has been shown that
ecabet can inhibit enzyme activity by binding to enzyme
protein and precipitating it, thus preventing interaction
with its substrate [11]. The greatest degree of binding
occurs below pH 2.0. It did, however, depend on the
interacting species, i.e. binding was greater and more pHdependent with human serum albumin and fibrinogen
than with degraded pig mucin and pepsin [27]. This pH
effect was why we used pH values of 1.6 and 5.0 to
investigate inhibition and binding in the present study.
Ito et al. [11] showed 96 % inhibition of pig pepsinogen
pepsin activity, compared with a value of 85 % reported
here, following preincubation with 5 mg\ml ecabet at
neutral pH. There was little or no effect on a pepsin
solution preincubated with ecabet at concentrations up to
10 mg\ml. This was again in agreement with Ito et al.
[11], who measured 10 % inhibition following preincubation with 10 mg\ml ecabet at pH 1.6. These results
show that ecabet binds much more efficiently to pepsinogen than to pepsin, precipitating it from solution.
The reason for this difference in binding could be
twofold : (1) the peptide cleaved off when pepsinogen is
converted into pepsin has a high affinity for ecabet,
and\or (2) pepsinogen has a different conformation at
pH 7.4 to pepsin at pH 1.6, and the change occurs during
acid-induced activation. The differences in conformation
may explain differences in binding.
Using the more sensitive N-terminal proteolysis assay,
ecabet significantly inhibited proteolytic activity in the
gastric juice from both patients with reflux oesophagitis
and non-symptomatic volunteers. The inhibition of
pepsin activity in gastric juice was independent of the
pH, and activity in pentagastrin-stimulated gastric juice
from patients was inhibited to a significantly greater
extent than that in pentagastrin-stimulated juice from
volunteers. This trend was also seen with the unstimulated basal gastric juice samples. These results
demonstrate a difference in the type of pepsin activity in
reflux gastric juice compared with normal juice, and
imply that ecabet has the potential to reduce the
oesophagal-mucosa-damaging effects of gastric juice in
reflux disease. Human gastric juice contains at least seven
different pepsins : 1, 2, 3, 3a, 4, 5 and 6 [16]. The majority
of the proteolytic activity is due to pepsin 3 (70 %) and
pepsin 5 (17 %) [28]. Pepsin 1, a complex of protein
and proteoglycan, constitutes only 3.6 % of the total
pepsin activity in stimulated gastric juice from normal
subjects, but its levels are 4–5 times greater in stimulated
gastric juice from patients with peptic ulcer [17,22].
#
2001 The Biochemical Society and the Medical Research Society
Therefore pepsin 1 has been referred to by some authors
as the ‘ ulcer-associated pepsin ’, and gastric juice containing increased levels of this enzyme has an enhanced ability
to degrade the protective mucosal barrier [18]. It is therefore pertinent to determine the ability of ecabet to inhibit
the different pepsins. Ecabet at 5 mg\ml resulted in major
inhibition of the three isoenzymes, with values ranging
from 42 % to 100 %. These substantial inhibitions occurred at concentrations of pepsin (0.2–0.4 mg\ml)
within the range demonstrated to be present in human
gastric juice. A statistical comparison of the inhibition of
pepsins 3 and 5 by ecabet can be made between the two
pH values, even though the pepsin concentrations varied
2-fold, if absolute amounts of pepsin inhibited are
compared. The degree of inhibition of pepsins 3 and 5
was unaffected by the pH of the incubation with ecabet.
This means that ecabet should be effective in inhibiting
human gastric juice pepsin activity, irrespective of the
isoenzyme composition or pH. Pepsin 1 was inhibited to
the greatest extent by ecabet at both pH values, and this
was significant compared with the inhibition of pepsin 5
at pH 5.0. A statistical comparison of the inhibition of
pepsin 1 by ecabet at pH 1.6 with that of the other
pepsins could not be carried out, because complete
inhibition occurred with 200 µg of the enzyme, and more
enzyme may have been inhibited by 5 mg\ml ecabet if
the pepsin concentration had been raised. For the same
reason, a comparison of the inhibition of pepsin 1 by
ecabet between the two pH values could not be made.
Further experiments with pepsin 1 could not be undertaken, as not enough was available, and its isolation from
gastric juice in milligram quantities is an extensive
undertaking.
The greater inhibition of pepsin 1 could be explained
by the composition of this isoenzyme, which contains
50 % carbohydrate and one or two phosphate groups
which may enhance ecabet binding. The binding of
ecabet to other biomolecules varies depending on their
net charge. This enhanced inhibition of pepsins, and in
particular pepsin 1, could go some way to explaining why
combination therapy with ecabet and ranitidine resulted
in a lower rate of recurrence of peptic ulcer than treatment
with ranitidine alone in H. pylori-positive patients [29].
Mucus is the first line of defence in the stomach and
duodenum against the damaging effects of gastric juices.
The mucus layer is maintained by a balance between
secretion by the epithelial cells and its erosion from the
surface. This balance is swung in favour of degradation
and erosion in peptic ulcer disease [18]. If ecabet is to
enhance protection of the mucosa, it must be able to
strengthen the mucus barrier and reduce its breakdown
by mucolysis. Here we have shown that ecabet can
indeed inhibit proteolytic activity in gastric juice from
patients with reflux oesophagitis, and it can also
strengthen the mucus barrier in the stomach and duodenum via an interaction with mucin. In addition,
Inhibition of pepsin by ecabet sodium
intragastric administration of ecabet has been shown
recently to stimulate mucin biosynthesis and secretion in
the rat stomach [30]. The results presented here show that
ecabet may be useful in the treatment of peptic ulcer
disease and reflux oesophagitis, in that it will effectively
inhibit pepsin and pepsinogen at the pH values likely to
be present in the stomach, duodenum and oesophagus,
and thereby reduce damage to the gastroduodenal mucus
barrier and to the oesophagal mucosa. Ecabet may be of
particular relevance in the treatment of reflux oesophagitis, where pepsin and acid are major aggressive
factors. In addition, further work is needed to investigate
why ecabet is better at inhibiting proteolytic and mucolytic activity in gastric juice from patients with reflux
oesophagitis than in that from healthy volunteers. One
possibility is that an H. pylori infection in the oesophagitis patients may modify pepsin secretion, and that
this or another mechanism may have resulted in a
different pepsin isoenzyme profile. Unfortunately, the
H. pylori status of the patients was not measured in
the present study.
REFERENCES
1 Waba, H., Kadato, S., Kawamoi, M. et al. (1985) Antiulcer
activity of dehydroabietic acid derivatives. Chem. Pharm.
Bull. 33, 1472–1487
2 Ito, Y., Sugawara, Y., Takaiti, O. and Nakamura, S. (1991)
Metabolic fate of a new anti-ulcer drug (j)(1R,4aS,10aR)-1,2,3,4,4a,9,10,10a-Octahydro-1,4adimethyl-7-(1-methylethyl)-6-sulfo-1phenanthrenecarboxylic Acid 6-Sodium Salt Pentahydrate
(TA-2711). II. Distribution in the Rat Stomach.
J. Pharmacobio-Dyn. 14, 547–554
3 Yarimizu, T., Mitamura, T., Suzuki, S. and Sakamoto, S.
(1998) Protective effects of an antiulcer agent, ecabet
sodium, on colorectal carcinogenesis in rodents. Oncol.
Rep. 5, 1103–1107
4 Ohkusa, T., Takashimizu, I., Fujiki, K. et al. (1998)
Disappearance of hyperplastic polyps in the stomach after
eradication of Helicobacter pylori. Ann. Intern. Med. 129,
712–715
5 Kagaya, H., Kato, M., Kobayashi, T. et al. (1998) Study on
the effect of a new antiulcer agent, Ecabet sodium, on the
dual therapy for eradication of Helicobacter pylori in
randomized patients. Gastroenterology 114, G0672
6 Ito, Y., Shibata, K., Hongo, A. and Kinoshita, M. (1998)
Ecabet sodium, a locally acting antiulcer drug, inhibits
urease activity of Helicobacter pylori. Eur. J. Pharmacol.
345, 193–198
7 Shibata, K., Ito, Y., Hongo, A., Yasoshima, A., Endo, T.
and Ohashi, M. (1995) Bactericidal activity of a new
antiulcer agent, ecabet sodium, against Helicobacter pylori
under acidic conditions. Antimicrob. Agents Chemother.
39, 1295–1299
8 Hayashi, S., Sugiyama, T., Asaka, M. et al. (1998)
Modification of Helicobacter pylori adhesion to human
gastric epithelial cells by antiadhesion agents. Dig. Dis. Sci.
43 (suppl. 9), 56S–60S
9 Furukawa, O., Kitmura, M., Suzuki, K. and Takeuchi, K.
(1998) Effect of ecabet sodium, a novel antiulcer drug, on
epithelial restitution following injury by hyperosmolar
sodium chloride in bullfrog stomach in vitro.
Gastroenterology 114, G0516
10 Kinoshita, M., Yamasaki, K., Kokusenya, Y. and Tamaki,
H. (1995) Relationship between gastroprotective effect of
locally acting ecabet sodium and its binding to gastric
mucosa in rats. Dig. Dis. Sci. 40, 661–667
11 Ito, Y., Nakamura, S., Onoda, Y. et al. (1993) Effects of
the new anti-ulcer drug ecabet sodium (TA-2711) on
pepsin activity. I. Inactivation of enzyme protein. Jpn.
J. Pharmacol. 62, 169–174
12 Kinoshita, M., Endo, M., Yasoshima, A. et al. (1999)
Ecabet sodium, a novel locally-acting anti-ulcer agent,
protects the integrity of the gastric mucosal gel layer from
pepsin-induced disruption in the rat. Aliment. Pharmacol.
Ther. 13, 687–694
13 Jordan, N., Newton, J., Pearson, J. P. and Allen, A. (1998)
A novel method for the visualization of the in situ mucus
layer in rat and man. Clin. Sci. 95, 97–106
14 Younan, F., Pearson, J. P., Allen, A. et al. (1982) Changes
in the structure of the mucus gel on the mucosal surface of
the stomach in association with peptic ulcer disease.
Gastroenterology 82, 827–831
15 Newton, J. L., Jordan, N., Oliver, L. et al. (1998)
Helicobacter pylori in vivo causes structural changes in the
adherent gastric mucus layer but barrier thickness is not
compromised. Gut 43, 470–475
16 Roberts, N. B., Sheers, R. and Taylor, W. H. (1981) Pepsin
1 secretion in normal human subjects. Clin. Sci. 61, 37
17 Walker, V. and Taylor, W. H. (1980) Pepsin 1 secretion in
chronic peptic ulceration. Gut 21, 766–771
18 Pearson, J. P., Ward, R., Allen, A. et al. (1986) Mucus
degradation by pepsin : comparison of mucolytic activity
of human pepsin 1 and pepsin 3 : implications in peptic
ulceration. Gut 27, 243–248
19 Anson, M. L. (1938) The estimation of pepsin, trypsin,
papain and cathepsin with haemoglobin. J. Gen. Physiol.
22, 79–89
20 Lin, Y., Means, G. E. and Feaney, R. E. (1969) The action
of proteolytic enzymes on N,N-dimethyl proteins. J. Biol.
Chem. 244, 789–793
21 Hutton, D. A., Pearson, J. P., Allen, A. and Foster, S. N.
E. (1990) Mucolysis of the colonic mucus barrier by faecal
proteinases : inhibition by interacting polyacrylate. Clin.
Sci. 78, 265–271
22 Pearson, J. P., Blackburn, A., Lynn, S. et al. (1992) Human
pepsin 1, a complex of proteoglycan and protein. Biochem.
Soc. Trans. 20, 355S–357S
23 Foster, S. N. E., Pearson, J. P., Hutton, D. A. et al. (1994)
Interaction of polyacylates with porcine pepsin and the
gastric mucus barrier : a mechanism for mucosal
protection. Clin. Sci. 87, 719–726
24 Marshall, B. J. (1994) Helicobacter pylori. Am.
J. Gastroenterol. 89, S116–S128
25 Dent, J. (1994) Roles of gastric acid and pH in the
pathogenesis of gastroesophageal reflux disease. Scand.
J. Gastroenterol. 29, 55–61
26 Pearson, J. P. and Hutton, D. A. (1998) Structure and
function of the stomach. In The Encyclopedia of Human
Nutrition (Sadler, M. J., Strain, J. J. and Caballero, B.,
eds.), pp. 929–938, Academic Press, London
27 Ito, Y., Onoda, Y., Nakamura, S. et al. (1993) Effects of
the new anti-ulcer drug ecabet sodium (TA-2711) on
pepsin activity II. Interaction with substrate protein. Jpn.
J. Pharmacol. 62, 175–181
28 Pearson, J. P., Blackburn, A., Allen, A. and Venables,
C. W. (1990) Pepsin 5 (Gastricsin) : atypical pH profile of
mucolytic activity. Biochem. Soc. Trans. 18, 1255
29 Koizumi, W., Tanabe, S., Ohida, M., Imaizumi, H., Kida,
M. and Saigenji, K. (1999) A prospective randomized study
of relapse in peptic ulcers on maintenance therapy with
ecabetjranitidine or ranitidine alone. Gastroenterology
116, A218
30 Ichikawa, T., Ishihara, K., Hayashida, H., Hiruma, H.,
Saigenji, K. and Hotta, K. (2000) Effects of ecabet sodium,
a novel gastroprotective agent, on mucin metabolism in
rat gastric mucosa. Dig. Dis. Sci. 45, 606–613
Received 7 August 2000/13 October 2000; accepted 11 December 2000
#
2001 The Biochemical Society and the Medical Research Society
417