Download pharmacology of gastrointestinal tract

2012, Vol. 75, No 3. – P. 17-21
Experimental and Clinical Pharmacology,
PHARMACOLOGY OF GASTROINTESTINAL TRACT
GASTROPROTECTIVE ACTIVITY OF MODIFIED GLUTAMYL-TRYPTOPHAN
DIPEPTIDE ANALOGS AND MELATONIN
T. N. Savateeva-Lyubimova1, К. V. Sivak1, and V. V. Malinin2
The gastroprotective action of synthetic dipeptides glutamyl-tryptophan (EW), isovaleroyl-glutamyl-tryptophan (ivEW) and nicotinoyl-glutamyl-tryptophan (nEW) in
comparison to melatonin was studied in experiments on male with the model
indometacine and starvation stress-induced stomach ulcers. It was shown, that EW and
melatonin exhibit antiulcer activity upon preventive intragastric administration on the
model of starvation stress stomach ulcers. At the same time, ivEW, nEW, EW and
melatonin exhibit antiulcer activity during the treatment of model indometacine-induced
stomach ulcers. The maximum gastroprotective action was observed for ivEW on the
model of indomethacin-induced stomach ulcer. The therapeutic efficiency of substances
studied is based on their cytoprotective, antioxidant and immunomodulating action.
_______________________________________________________________________________
Key words: Dipeptides, glutamyl-tryptophan, iso-valeroyl-glutamyl-tryptophan,
nicotinoyl-glutamyl-tryptophan, melatonin, misoprostol, sucralfate, Selye’s stress,
indomethacin, stomach ulcer, gastroprotective action
INTRODUCTION
Gastropathy, peptic ulcer disease (PUD), hyperacid- and
stress-induced gastritis are common among the able-bodied
population, which accounts for the great medical and social
importance of the problem [1]. The complexity of the
problem is in the fact that, despite the use of effective
treatments of PUD, the disease relapses in 60 - 100% of the
cases. Polypharmacy along with stress is a cause of
additional local irritation and alteration in the gastrointestinal
(GI) tract among the elderly patients (the use of non-steroidal
anti-inflammatory drugs [NSAIDs] for the treatment of
musculoskeletal disorders, etc.).
The basic pharmacotherapy of gastropathy includes the
following groups of agents: histamine receptor blockers and
proton pump inhibitors, antacids; anti-helicobacter agents;
and gastroprotective drugs [2].
Since the pharmacotherapy of PUD is associated with
great difficulties, the search of the drugs with more
sophisticated mechanisms of action and better clinical results
can only be fruitful if all the existing data about the
pathogenic mechanisms, as well as a long-term clinical
experience are used. An ideal solution would be to
synthesize a drug yielding both the systemic and local
gastroprotective effect. The gastric and duodenal mucous
membrane resistance can be improved by activating the
antioxidant system, normalizing the psychosomatic
component, and providing the organism with sufficient
nutrition [1,3]. It is also important to regulate the following
1
2
three vital interrelated systems: the nervous, the immune and
the endocrine systems [4]. Recently, a new approach in
therapy of ulcerous and erosive lesions in GI tract has
evolved which implies the use of natural (flavonoids,
phenolic compounds) and synthetic (probucol, etc.)
antioxidants, due to their positive influence on the
“oxidative” stress [1,3] . The role of the immune system in
the gastroprotective efficacy of the synthetic analogs of
thymus peptides and melatonin are being currently
investigated [5-7].
The aim of the present study was to assess the efficacy of
orally administered glutamyl-tryptophan (EW), and EW
analogues:
iso-valeroyl-glutamyl-tryptophan
(ivEW),
nicotinoyl-glutamyl-tryptophan (nEW) and melatonin in
experimentally modelled stomach ulcer induced by stress
and indometacine as compared to gastroprotective action of
misoprostol and sucralfate.
METHODS
The experiment involved 274 outbred male SpragueDawley rats weighing 180-200 g (“Rappolovo”, Leningrad
region), which were kept in an animal house under standard
laboratory conditions. Prior to the study, the animals were
quarantined for 14 days and randomized. The experiments
were carried out in accordance with the Ethics Committee
requirements [8, 9].
Two gastropathy models were used: stress-induced ulcer
by Selye and the indomethacin-induced ulcer. During the 24
h prior to the stress and ulcerogenic exposure the animals
State Institute of Toxicology, Federal Medico-Biological Agency, ul. Bekhtereva 1, St. Petersburg, 192019, Russia
Cyto-NIR Company, Muchnoi per. 2, St. Petersburg, 191023, Russia
© COMPOSITE AUTHOR, 2012
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were deprived of food (with free access to water), as
starvation reduces the protective level of the gastric mucosa
due to the activation of anaerobic glycolysis. To model the
stomach ulcer by Selye, the animals were immobilized in
plexiglass boxes for 3 h at 4 °C. The ulcerogenic
indometacine dose (10 mg/kg) was administered
intramuscularly with a sterile apyrogenic saline solution
during three consecutive days [10, 11]. The below effective
doses of peptides and drugs of comparison were
administered intragastrically 1 h and 7 days before the
modeling of the stress-induced stomach ulcer by Selye (a
preventive regimen), or were administered intragastrically
for 7 days from the time of modeling of indomethacininduced stomach ulcer (a therapeutic treatment regimen).
The control animals received the equivolume amount of
vehicle in the relevant regimens.
In each series the experimental groups of 10-12 animals
were as follows: 1 - intact rats, 2 – sick animals receiving
saline solution (control group), 3 – sick animals receiving a
drug of comparison (misoprostol, sucralfate), 4 – sick
animals receiving melatonin or one of the investigational
peptides. Based on the available literature [1 - 3, 5-7] the
following doses were selected for the study: EW 0.1; 1 and
10 µg/kg; melatonin 0.3 and 3 mg/kg; misoprostol 50 µg/kg;
sucralfate 200 mg/kg. The ivEW and nEW synthetic
peptides were investigated in the same doses as EW.
Upon termination of immobilization (according to
Selye’s method) and the experimental treatment, the animals
were sacrificed [9] by instant decapitation, their stomachs
were removed, dissected along the greater curvature and
washed with saline solution; following this, the number of
destructions was determined by microscopic examination
performed with bright vertical illumination. The destructions
were differentiated by their surface area and were classified
as: small (S) < 1 mm in diameter, medium (M) round or
linear ≥ 1 mm , and large (L) ≥ 1-2 mm. The size of
destructions in the gastric mucosa was determined with an
Expert Prima digital microscope (LOMO, Russia). The
presence of blood at the bottom of the destructions was
determined by a peroxidase reaction with the substrate of
orthodianisidin hydroperoxide, with methylene-blue stained
mucosa. Thus, the blood-containing surface turned blackbrown against the bluish background of intact mucosa.
The animals in the groups with damaged gastric mucosa
were categorized according to the severity of the
microscopic changes; the Paul’s index (PI) was calculated
according to the following formula: (mean number of
ulcers × % of animals with ulcers) / 100%, while the
gastroprotective activity (GA) was the ratio of the control PI
to the experimental PI. The investigational medication was
considered as active if GA was ≥ 2. The level of
malondialdehyde
(MDA)
and
glutathione
and
myeloperoxidase (MPO) activity in the 10% homogenates of
gastric mucosa were biochemically assessed using the
standard assay methods [12 - 14]. Following 2 h after
sampling and complete relaxation of the smooth muscles the
gastric wall sections were fixed in 10% formalin solution to
perform a histological examination. The sections were
stained with hematoxylin and eosin. The microscopic
T. N. Savateeva-Lyubimova et al.
examination of the gastric wall samples was carried out
using the B-350 microscope (“Optika”, Italy).
Statistical analyses were performed using Excel and
Statistica 6.0, and the results were presented in the tables as
M ± σ. The differences between the groups were analyzed
using the criteria of non-parametric methods for analysis of
variance according to the instructions of the data processing
software. The results were considered as significant if
p ≤ 0.05.
RESULTS AND DISCUSSION
EW and melatonin have previously demonstrated stressprotective properties [5 - 7]. Therefore, the first stage of the
experiment was devoted to investigation of the
gastroprotective activity of these agents on the model of
Selye stress-induced ulcer. After the immobilization of the
animals in the cold condition, the following pathological
changes occurred in the gastric mucosa: hyperemia,
gastroparesis and dilatation of the gastric muscles, erosions
and ulceration, bile reflux and fibrinous plaque.
EW, melatonin and the drug of comparison misoprostol
showed varying degrees of gastroprotective activity when
administered as part of preventive treatment. The results are
presented in table 1.
A single dosing of melatonin resulted in moderate
gastroprotective effect (3 mg/kg, GA = 2), which was
expressed in reduction the number of medium and smallsized ulcers in the gastric mucosa (2-fold and 1.9-fold
decrease, respectively). Melatonin demonstrated dosedependent gastroprotective activity. However, its efficacy
was significantly lower than that of misoprostol. EW
administered in a single mode had no gastroprotective effect
and did not reduce the number of animals with ulcers.
The seven-day preventive treatment with melatonin
proved to have a sufficient gastroprotective effect (GA = 3)
slightly different from that of a single administration of the
hormone. Dosed at 3 mg/kg, melatonin decreased the
number of destructions in the gastric mucosa: practically no
large ulcers were present, while the number of medium and
small-sized ulcers was significantly lower as compared to
the control group. The obtained results confirm the role of
melatonin in the development and restriction of a stress
reaction and somatic diseases [5, 15].
The analysis of the obtained data demonstrated that EW
administered to rats in a preventive mode for one week prior
stress had gastroprotective activity similar to that of
misoprostol, and when dosed at 1 µg/kg, EW exceeded
misoprostol in its effect. EW showed the highest
gastroprotective activity (GA = 27 when dosed at 0.1 µg/kg ,
and GA = 50 when dosed at1 µg/kg) surpassing the effect of
misoprostol and melatonin. The discovered gastroprotective
properties of EW that differed from those of melatonin, as
well as the fact that a single administration of EW had no
gastroprotective activity, prove that the immune and the
antioxidant systems play an important role in the
gastroprotective mechanism of the investigational peptide
when the latter is administered repeatedly.
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Gastroprotective activity of modified glutamyl-tryptophan dipeptide analogs
In view of the remarkable gastroprotective activity of
EW revealed in the course of preventive treatment, the next
stage of the experiment consisted in the profound study of
the efficacy of the peptide and its modified analogs during a
therapeutic treatment regimen tested on the model of
indometacine-induced stomach ulcer.
results. Treatment with sucralfate resulted in the significant
decrease in both the number and size of ulcers. Mucosal
hyperemia was observed in 40% of the animals.
It was found that the modeling of an ulcer by the
introduction of the ulcerogenic dose of indomethacin to rats
is accompanied by significant pathological changes that
develop by the 3rd day following inoculation. On the 7th day
the animals lost up to 30% of body weight as compared to
baseline, which was caused both by the loss of blood due to
gastric bleeding, and by increased catabolism. All the control
animals had severe hyperemia and areas of destruction in the
gastric mucosa, including a significant number of superficial
and deep erosions, as well as the edema of the surrounding
tissues. The bottom of the majority of destructions was
hematest positive (blood: hematin and methemoglobin due
to the bleeding of impaired mucosal capillaries and the
oxidation of hemoglobin by the stomach hydrochloric acid).
A significant reduction of body weight was recorded in case
of the following medication dosing: all control animals that
received 10 µg/kg of EW and nEW. ivEW administered at
0.1 µg/kg showed the highest protective activity;
administration of EW and nEW at 0.1 µg/kg gave similar
EW introduced into the stomach in different doses
prevented the formation of gastric ulcers. This effect was
most obvious with the 0.1 µg/kg dose, judging by both the
total number of stomach ulcers and the number of rats with
ulcers. Dosed at10 µg/kg, the drug significantly decreased
the number of large destructions, with no effect rendered on
the medium and small-sized ulcers. Melatonin had a
somewhat similar, though lesser, effect as compared to that
of EW.
The efficacy of the investigational drugs was assessed by
the number of destructions in the gastric mucosa of the
animals (see table 2).
The
ivEW
peptide
demonstrated
remarkable
gastroprotective activity as it reduced the Paul’s index as
compared to the control and drug of comparison (p≤0.05).
Dosed at10 µg/kg the peptide was similar to EW and caused
a decrease in the mean number of the destructions, and
surpassed sucralfate and other drugs in terms of
gastroprotective effect. Peptide nEW, comparable with EW
in a number of parameters, proved to have better GA than
sucralfate.
Table 1. The effect of EW and melatonin on ulceration in the Selye stress model (n = 10)
Group, dose,
µg/kg
Animals
with
ulcers, %
Number of ulcers (М±σ)
L
M
S
Total number
(М±σ)
PI
GA
Single preventive dose
Control
100
2.7 ± 1.4
6.0 ± 1.1
16.3 ± 1.4
25.0 + 3.6
25.00
-
Misoprostol, 50
40*
0*
0*
6.6 ± 1.2*
5.7 ± 0.4*
2.28*
10.96*
ЕW, 0.1
100
2.5 ± 1.1
5.8 ±0.9
17.1 ± 1.1
25.4 + 3.0
25.20
0
EW, 1
100
2.2 ± 1.0
5.5 ± 1.3
16.9 ± 1.6
24.6 ± 2.8
25.01
0
EW, 10
100
2.8 + 1.2
6.3 ± 1.0
18.0 ± 1.9
27.1 ±3.3
26.72
0
М, 0.3
100
2.0 ± 0.6
3.0 ± 0.6*
12.7 ±3.2
17.7 + 3.2
17.70*
1.41
М, 3
80*
1.1 ±0.9
3.2 ± 0.4*
8.0 ± 0.9*
14.3 ± 1.0*
11.44*
2.18*
7-Day preventive treatment
Control
100
4.4 ± 0.8
9.6 ± 1.4
17.8 ±3.6
31.8 ±3.7
31.80
-
Misoprostol, 50
30*
0*
0.8 ± 1.2*
12.5 ± 1.4*
13.7 ±0.9*
4.11*
7.74*
Е W, 0.1
40*
0*
0*
3.0 ± 1.0*/**
2.9 ±0.2*/**
1.16*/**
27.41*/**
EW, 1
20*
0*
0*
3.0 ± 1.0*/**
3.2 ± 0.2*/**
0.64*/**
49.69*/**
EW, 10
60*
0*
1.0 ±0.2*
4.0+ 1.0*/**
5.0 ±0.3*/**
3.0*/**
10.6*/**
М, 0.3
100
1.8 ±0.8*
6.0+ 1.8
14.7 ±2.6
22.5 ±4.2*
22.50*
1.41
М, 3
80*
0*
2.4 ± 0.6*
11.0± 1.5
13.6 ± 1.1*
10.88*
2.92*
Comment. * — Significant differences as compared to the control, p ≤0.05, ** - Significant positive differences as compared to
misoprostol, p ≤0.05. M - melatonin, L - large ulcers, M - medium-sized ulcers, S – small-sized ulcers; IP – Paul’s index; GA gastroprotective activity.
20
T. N. Savateeva-Lyubimova et al.
The peptide therapy resulted in the smaller amount of
peroxidation products in the gastric mucosa and lower
myeloperoxidase activity, while glutathione levels
increased. The established positive changes in the
biochemical parameters characterizing the function of the
gastric mucosa were associated with the use of experimental
agents and showed the role of the antioxidant system in the
gastroprotective activity of the selected compounds [6, 7].
The results are presented in table 3.
In the control group the level of malonic dialdehyde
(MDA) increased significantly, by 2.9 times, the
myeloperoxidase activity increased by 1.9 times, and
glutathione levels fell by 2.1 times as compared to intact
rats (p≤0.05). It testifies to the acceleration of oxidation
caused by free radicals, as well as the depletion of
antioxidant pools (due to inhibition of the synthesis of
protective factors), and the inflow of polymorphonuclear
leukocytes activated by chloride medium, which resulted in
the increased activity of myeloperoxidase.
Sucralfate reduced the level of lipid peroxidation products
by 1.6 times as compared to the control, the MPO activity, by
1.5 times, restored the decreased glutathione level by
1.3 times mainly due to the binding and adsorption on the
surface of the gastric mucosa. EW demonstrated the activity
similar to that of sucralfate (reduced the level of malonic
dialdehyde by 2.2 times [0.1 µg / kg]), and was comparable
with sucralfate in terms of influence on MPO and glutathione
levels (0.1 µg / kg). However, this was probably due to another
mechanism which was different from the effects of the drug
of comparison. Dosed at 10 µg/kg, EW was somewhat
inferior to sucralfate.
The greatest positive changes were observed in rats
treated with the ivEW peptide. Thus, the 0.1 µg/kg dose lead
to the significant normalization of the lipid peroxidation
processes and MPO activity, as well as the recovery of the
glutathione level (p≤0.05). However, the glutathione level in
the gastric mucosal homogenate still did not reach the levels
reported for the intact animals. In terms of activity, the ivEW
peptide significantly surpassed the EW, nEW, melatonin and
sucralfate.
Table 2. Effect of modified analogs of EW on the process of ulceration in animal model of indometacine-induced
stomach ulcer (n = 12)
Number of ulcers (М±σ)
Animals
with ulcers, %
S
Total number
(М±σ)
L
M
PI
GA
0
0
0
0
0
-
-
Control
100
11.0 ±2.0
7.0 ± 1.0
16.0 + 3.0
35.0 ±3.0
34.7
-
Sucralfate, 200
100
5.0 ± 2.0*
4.0 ± 1.0
8.0 ± 2.0*
17.0 ±4.0*
16.9
2.05
EW,0.1
67*
3.0 ± 1.0*
5.0 ±2.0
9.0 ± 1.0*
17.0 + 2.0*
11.4*/**
3.40**
EW, 10
100
4.0 ± 1.0*
7.0 + 2.0
11.0± 3.0
21.0 ±3.0*
21.7*
1.60
ivEW,0.l
42*/**
0*/**
3.0 ± 1.0*
12.0 ±2.0
14.0 ± 3.0*
5.9*/**
6.19**
ivEW, 10
67*
5.0 ± 1.0*
3.0 ± 2.0
10.0 ±3.0
19.0 ±2.0*
12.7*/**
3.04**
nEW, 0.1
42*/**
6.0 ± 1.0*
7.0 ± 1.0
8.0 ± 2.0*
22.0 ±4.0*
9.2*/**
3.94**
nEW, 10
84*
4.0 ± 2.0*
6.0 ± 1.0
9.0 ± 2.0*
20.0 ±2.0*
16.8*
2.17
M, 0.3
100
4.0 ± 2.0*
7.1 ±1.0
12.1 ±2.0
22.0 + 3.0*
21.9*
1.58
M, 3
84*
2.0 ± 1.0*
3.0 ± 1.0*
19.0 ±3.0
25.0 + 2.0*
21.0*
1.65
Group, dose, µg/kg
Intact
Comment. * — Significant differences as compared to the control, р ≤ 0.05; ** — Positive changes that are significant
as compared to sucralfate, р ≤ 0.05. M - melatonin, L - large ulcers, M - medium-sized ulcers, S – small-sized ulcers; IP
– Paul’s index; GA - gastroprotective activity.
Table 3. Effect of modified analogs of EW on the biochemical parameters of the gastric mucosa during ulceration
in animal model of indomethacin-induced stomach ulcer (n = 12)
Group, dose
MDA, nmol/g
MPO, IU/g
Glutathione, µmol/g
Intact
19.6 + 0.9
45.0+1.3
1.68 ±0.14
Control
56.8 ± 1.4^
85.9 + 2.6^
0.80 + 0.07^
Sucralfate, 200 mg/kg
34.6+ 1.1^*
57.2 ± 1.4^
1.03 ±0.07^*
EW, 0.1 µg/kg
25.7 ± 1.0^*/**
59.3 ± 2.2^*
1.10 + 0.11^*
ЕW, 10 µg/kg
31.9±0.8^*
83.1 + 1.7^
0.87 ± 0.09 ^
21
Gastroprotective activity of modified glutamyl-tryptophan dipeptide analogs
ivEW, 0.1 µg/kg
Group, dose
18.9+1.0*/**
44.6+ 1.9*/**
MDA, nmol/g
MPO, IU/g
1.29±0.08 ^*/**
Glutathione, µmol/g
ivEW, 10 µg/kg
22.6 ± 1.3^*/**
69.6 + 2.5^*
1.17 + 0.06^*
nEW, 0.1 µg/kg
36.5 ± 1.0^*
85.2 ± 1.4^
1.02 ±0.09^
nEW, 10 µg/kg
34.9 ± 2.6^*
82.3 ± 2.2^
1.06±0.10^*
М, 0.3 mg/kg
37.1 ±3.3^*
84.6+ 1.3^
0.91 +0.06^
М, 3 mg/kg
36.4 ± 1.7^*
55.6 ± 2.0^*
1.10±0.11^*
Comment. ^- the differences are significant as compared to intact rats, р ≤ 0.05, * - significant difference as compared to
the control group, р ≤ 0.05, ** - positive changes that are significant as compared to sucralfate, p ≤ 0.05. M - melatonin,
MDA - malonic dialdehyde, MPO - myeloperoxidase.
Microscopic examination of the stained sections of the
murine gastric wall revealed pathological changes in the
mucosa. The presence of deep ulcers exposing the muscle
plate, superficial ulcers (up to glandular epithelium), and
numerous surface erosions were recorded in the control
group. The animals treated with sucralfate had no deep ulcers
and a small number of superficial erosions. Treatment with
melatonin resulted in a lower number of ulcers, however the
depth of ulcerization was similar to that of the control group.
EW contributed more to the treatment of the damaged gastric
mucosa than melatonin. In the ivEW and nEW treatment
groups only minimal pathological changes of the gastric
mucosa were found, the majority of them involving
superficial erosion and small punctuate areas of necrosis in
the epithelium.
Thus, investigational drugs used for the treatment of the
indometacine-induced ulcer can be rated by their
gastroprotective activity from the most active to the least
effective: ivEW > nEW > EW ≈ melatonin.
CONCLUSIONS
1. Dipeptide glutamyl-tryptophan has no gastroprotective
activity when administered as a single preventive treatment
on the model of the stress-induced ulcer. Sevenfold
preventive administration of EW leads to a significant
decrease in the number of gastric ulcerous destructions in rats
previously stressed by immobilization according to the Selye
method, and this effect is significantly different from that of
melatonin.
2. Glutamyl-tryptophan, isovaleroyl-glutamyl-tryptophan,
nicotinoyl-glutamyl-tryptophan and melatonin demonstrate
varying degrees of gastroprotective activity when tested on
the model of the indomethacin-induced stomach ulcer in
rats.
3. As compared to other agents, isovaleroyl-glutamyltryptophan proved to have the greatest gastroprotective
activity when administered together with a cyclooxygenase
inhibitor. The experimental treatment with the peptide
resulted in the significant increase of antioxidant levels, and
less severe lipid peroxidation in the gastric mucosa.
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Received on 28 October 2011