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Experimental and Clinical Pharmacology, 2012, Vol. 75, No 3. – P. 17-21 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), iso- valeroyl-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
three vital interrelated systems: the nervous, the immune and the endocrine systems [4]. Recently, a new approach in Gastropathy, peptic ulcer disease (PUD), hyperacid- and therapy of ulcerous and erosive lesions in GI tract has stress-induced gastritis are common among the able-bodied evolved which implies the use of natural (flavonoids, population, which accounts for the great medical and social phenolic compounds) and synthetic (probucol, etc.) importance of the problem [1]. The complexity of the antioxidants, due to their positive influence on the problem is in the fact that, despite the use of effective "oxidative" stress [1,3] . The role of the immune system in treatments of PUD, the disease relapses in 60 - 100% of the the gastroprotective efficacy of the synthetic analogs of cases. Polypharmacy along with stress is a cause of thymus peptides and melatonin are being currently additional local irritation and alteration in the gastrointestinal investigated [5-7]. (GI) tract among the elderly patients (the use of non-steroidal anti-inflammatory drugs [NSAIDs] for the treatment of The aim of the present study was to assess the efficacy of musculoskeletal disorders, etc.). orally administered glutamyl-tryptophan (EW), and EW analogues: The basic pharmacotherapy of gastropathy includes the nicotinoyl-glutamyl-tryptophan (nEW) and melatonin in following groups of agents: histamine receptor blockers and experimentally modelled stomach ulcer induced by stress proton pump inhibitors, antacids; anti-helicobacter agents; and indometacine as compared to gastroprotective action of and gastroprotective drugs [2]. misoprostol and sucralfate. 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 The experiment involved 274 outbred male Sprague- can only be fruitful if all the existing data about the Dawley rats weighing 180-200 g ("Rappolovo", Leningrad pathogenic mechanisms, as well as a long-term clinical region), which were kept in an animal house under standard experience are used. An ideal solution would be to laboratory conditions. Prior to the study, the animals were synthesize a drug yielding both the systemic and local quarantined for 14 days and randomized. The experiments gastroprotective effect. The gastric and duodenal mucous were carried out in accordance with the Ethics Committee membrane resistance can be improved by activating the requirements [8, 9]. component, and providing the organism with sufficient Two gastropathy models were used: stress-induced ulcer nutrition [1,3]. It is also important to regulate the following by Selye and the indomethacin-induced ulcer. During the 24 h prior to the stress and ulcerogenic exposure the animals 1 State Institute of Toxicology, Federal Medico-Biological Agency, ul. Bekhtereva 1, St. Petersburg, 192019, Russia 2 Cyto-NIR Company, Muchnoi per. 2, St. Petersburg, 191023, Russia COMPOSITE AUTHOR, 2012 T. N. Savateeva-Lyubimova et al.
were deprived of food (with free access to water), as examination of the gastric wall samples was carried out starvation reduces the protective level of the gastric mucosa using the B-350 microscope ("Optika", Italy). due to the activation of anaerobic glycolysis. To model the Statistical analyses were performed using Excel and stomach ulcer by Selye, the animals were immobilized in Statistica 6.0, and the results were presented in the tables as plexiglass boxes for 3 h at 4 °C. The ulcerogenic M ± σ. The differences between the groups were analyzed using the criteria of non-parametric methods for analysis of intramuscularly with a sterile apyrogenic saline solution variance according to the instructions of the data processing during three consecutive days [10, 11]. The below effective software. The results were considered as significant if doses of peptides and drugs of comparison were p ≤ 0.05. administered intragastrically 1 h and 7 days before the modeling of the stress-induced stomach ulcer by Selye (a RESULTS AND DISCUSSION preventive regimen), or were administered intragastrically for 7 days from the time of modeling of indomethacin- EW and melatonin have previously demonstrated stress- induced stomach ulcer (a therapeutic treatment regimen). protective properties [5 - 7]. Therefore, the first stage of the The control animals received the equivolume amount of vehicle in the relevant regimens. gastroprotective activity of these agents on the model of Selye stress-induced ulcer. After the immobilization of the In each series the experimental groups of 10-12 animals animals in the cold condition, the following pathological were as follows: 1 - intact rats, 2 – sick animals receiving changes occurred in the gastric mucosa: hyperemia, saline solution (control group), 3 – sick animals receiving a gastroparesis and dilatation of the gastric muscles, erosions drug of comparison (misoprostol, sucralfate), 4 – sick and ulceration, bile reflux and fibrinous plaque. animals receiving melatonin or one of the investigational EW, melatonin and the drug of comparison misoprostol peptides. Based on the available literature [1 - 3, 5-7] the showed varying degrees of gastroprotective activity when following doses were selected for the study: EW 0.1; 1 and administered as part of preventive treatment. The results are 10 µg/kg; melatonin 0.3 and 3 mg/kg; misoprostol 50 µg/kg; presented in table 1. sucralfate 200 mg/kg. The ivEW and nEW synthetic peptides were investigated in the same doses as EW. A single dosing of melatonin resulted in moderate gastroprotective effect (3 mg/kg, GA = 2), which was Upon termination of immobilization (according to expressed in reduction the number of medium and small- Selye's method) and the experimental treatment, the animals sized ulcers in the gastric mucosa (2-fold and 1.9-fold were sacrificed [9] by instant decapitation, their stomachs decrease, respectively). Melatonin demonstrated dose- were removed, dissected along the greater curvature and dependent gastroprotective activity. However, its efficacy washed with saline solution; following this, the number of was significantly lower than that of misoprostol. EW destructions was determined by microscopic examination administered in a single mode had no gastroprotective effect performed with bright vertical illumination. The destructions and did not reduce the number of animals with ulcers. were differentiated by their surface area and were classified as: small (S) < 1 mm in diameter, medium (M) round or The seven-day preventive treatment with melatonin proved to have a sufficient gastroprotective effect (GA = 3) ≥ 1 mm , and large (L) ≥ 1-2 mm. The size of destructions in the gastric mucosa was determined with an slightly different from that of a single administration of the Expert Prima digital microscope (LOMO, Russia). The hormone. Dosed at 3 mg/kg, melatonin decreased the presence of blood at the bottom of the destructions was number of destructions in the gastric mucosa: practically no determined by a peroxidase reaction with the substrate of large ulcers were present, while the number of medium and orthodianisidin hydroperoxide, with methylene-blue stained small-sized ulcers was significantly lower as compared to mucosa. Thus, the blood-containing surface turned black- the control group. The obtained results confirm the role of melatonin in the development and restriction of a stress brown against the bluish background of intact mucosa. reaction and somatic diseases [5, 15]. The animals in the groups with damaged gastric mucosa The analysis of the obtained data demonstrated that EW were categorized according to the severity of the administered to rats in a preventive mode for one week prior microscopic changes; the Paul's index (PI) was calculated stress had gastroprotective activity similar to that of according to the following formula: (mean number of misoprostol, and when dosed at 1 µg/kg, EW exceeded ulcers × % of animals with ulcers) / 100%, while the misoprostol in its effect. EW showed the highest gastroprotective activity (GA) was the ratio of the control PI gastroprotective activity (GA = 27 when dosed at 0.1 µg/kg , to the experimental PI. The investigational medication was and GA = 50 when dosed at1 µg/kg) surpassing the effect of considered as active if GA was ≥ 2. The level of misoprostol and melatonin. The discovered gastroprotective properties of EW that differed from those of melatonin, as myeloperoxidase (MPO) activity in the 10% homogenates of well as the fact that a single administration of EW had no gastric mucosa were biochemically assessed using the gastroprotective activity, prove that the immune and the standard assay methods [12 - 14]. Following 2 h after antioxidant systems play an important role in the sampling and complete relaxation of the smooth muscles the gastroprotective mechanism of the investigational peptide gastric wall sections were fixed in 10% formalin solution to when the latter is administered repeatedly. perform a histological examination. The sections were stained with hematoxylin and eosin. The microscopic Gastroprotective activity of modified glutamyl-tryptophan dipeptide analogs
In view of the remarkable gastroprotective activity of results. Treatment with sucralfate resulted in the significant EW revealed in the course of preventive treatment, the next decrease in both the number and size of ulcers. Mucosal stage of the experiment consisted in the profound study of hyperemia was observed in 40% of the animals. the efficacy of the peptide and its modified analogs during a The efficacy of the investigational drugs was assessed by therapeutic treatment regimen tested on the model of the number of destructions in the gastric mucosa of the indometacine-induced stomach ulcer. animals (see table 2). It was found that the modeling of an ulcer by the EW introduced into the stomach in different doses introduction of the ulcerogenic dose of indomethacin to rats prevented the formation of gastric ulcers. This effect was is accompanied by significant pathological changes that most obvious with the 0.1 µg/kg dose, judging by both the develop by the 3rd day following inoculation. On the 7th day total number of stomach ulcers and the number of rats with the animals lost up to 30% of body weight as compared to ulcers. Dosed at10 µg/kg, the drug significantly decreased baseline, which was caused both by the loss of blood due to the number of large destructions, with no effect rendered on gastric bleeding, and by increased catabolism. All the control the medium and small-sized ulcers. Melatonin had a animals had severe hyperemia and areas of destruction in the somewhat similar, though lesser, effect as compared to that 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 gastroprotective activity as it reduced the Paul's index as hematest positive (blood: hematin and methemoglobin due compared to the control and drug of comparison (p≤0.05). to the bleeding of impaired mucosal capillaries and the Dosed at10 µg/kg the peptide was similar to EW and caused oxidation of hemoglobin by the stomach hydrochloric acid). a decrease in the mean number of the destructions, and A significant reduction of body weight was recorded in case surpassed sucralfate and other drugs in terms of of the following medication dosing: all control animals that gastroprotective effect. Peptide nEW, comparable with EW received 10 µg/kg of EW and nEW. ivEW administered at in a number of parameters, proved to have better GA than administration of EW and nEW at 0.1 µg/kg gave similar Table 1. The effect of EW and melatonin on ulceration in the Selye stress model (n = 10)
Number of ulcers (М±σ) Single preventive dose 6.6 ± 1.2*
5.7 ± 0.4*
3.0 ± 0.6*
3.2 ± 0.4*
8.0 ± 0.9*
14.3 ± 1.0*
7-Day preventive treatment 0.8 ± 1.2*
12.5 ± 1.4*
13.7 ±0.9*
3.0 ± 1.0*/**
2.9 ±0.2*/**
3.0 ± 1.0*/**
3.2 ± 0.2*/**
1.0 ±0.2*
4.0+ 1.0*/**
5.0 ±0.3*/**
1.8 ±0.8*
22.5 ±4.2*
2.4 ± 0.6*
13.6 ± 1.1*
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.
T. N. Savateeva-Lyubimova et al.
The peptide therapy resulted in the smaller amount of Sucralfate reduced the level of lipid peroxidation products peroxidation products in the gastric mucosa and lower by 1.6 times as compared to the control, the MPO activity, by 1.5 times, restored the decreased glutathione level by increased. The established positive changes in the 1.3 times mainly due to the binding and adsorption on the biochemical parameters characterizing the function of the surface of the gastric mucosa. EW demonstrated the activity gastric mucosa were associated with the use of experimental similar to that of sucralfate (reduced the level of malonic agents and showed the role of the antioxidant system in the dialdehyde by 2.2 times [0.1 µg / kg]), and was comparable gastroprotective activity of the selected compounds [6, 7]. with sucralfate in terms of influence on MPO and glutathione The results are presented in table 3. levels (0.1 µg / kg). However, this was probably due to another mechanism which was different from the effects of the drug In the control group the level of malonic dialdehyde of comparison. Dosed at 10 µg/kg, EW was somewhat (MDA) increased significantly, by 2.9 times, the inferior to sucralfate. myeloperoxidase activity increased by 1.9 times, and The greatest positive changes were observed in rats glutathione levels fell by 2.1 times as compared to intact treated with the ivEW peptide. Thus, the 0.1 µg/kg dose lead rats (p≤0.05). It testifies to the acceleration of oxidation to the significant normalization of the lipid peroxidation caused by free radicals, as well as the depletion of processes and MPO activity, as well as the recovery of the antioxidant pools (due to inhibition of the synthesis of glutathione level (p≤0.05). However, the glutathione level in protective factors), and the inflow of polymorphonuclear the gastric mucosal homogenate still did not reach the levels leukocytes activated by chloride medium, which resulted in reported for the intact animals. In terms of activity, the ivEW the increased activity of myeloperoxidase. 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) Group, dose, µg/kg 5.0 ± 2.0*
8.0 ± 2.0*
17.0 ±4.0*
3.0 ± 1.0*
9.0 ± 1.0*
17.0 + 2.0*
4.0 ± 1.0*
21.0 ±3.0*
3.0 ± 1.0*
14.0 ± 3.0*
5.0 ± 1.0*
19.0 ±2.0*
6.0 ± 1.0*
8.0 ± 2.0*
22.0 ±4.0*
4.0 ± 2.0*
9.0 ± 2.0*
20.0 ±2.0*
4.0 ± 2.0*
22.0 + 3.0*
2.0 ± 1.0*
3.0 ± 1.0*
25.0 + 2.0*
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)
Glutathione, µ mol/g 56.8 ± 1.4
0.80 + 0.07
Sucralfate, 200 mg/kg 34.6+ 1.1 *
57.2 ± 1.4
1.03 ±0.07 *
25.7 ± 1.0 */**
59.3 ± 2.2 *
1.10 + 0.11 *
31.9±0.8 *
0.87 ± 0.09
Gastroprotective activity of modified glutamyl-tryptophan dipeptide analogs
ivEW, 0.1 µg/kg 44.6+ 1.9*/**
1.29±0.08 */**
Glutathione, µ mol/g 22.6 ± 1.3 */**
69.6 + 2.5 *
1.17 + 0.06 *
36.5 ± 1.0 *
85.2 ± 1.4
1.02 ±0.09
34.9 ± 2.6 *
82.3 ± 2.2
1.06±0.10 *
37.1 ±3.3 *
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 1. VG Podoprigorova, Oxidative Stress and Ulcer Disease, mucosa. The presence of deep ulcers exposing the muscle Moscow (2004). plate, superficial ulcers (up to glandular epithelium), and numerous surface erosions were recorded in the control 2. Russian Register of Medicines, Drug Encyclopedia, group. The animals treated with sucralfate had no deep ulcers Vol. 12, Moscow (2005). and a small number of superficial erosions. Treatment with 3. V. N. Shatalov, D.B. Korman, T.V. Krutova et al., melatonin resulted in a lower number of ulcers, however the Pharmacol. and Toxicol., 3, 60 - 63 (1988).
depth of ulcerization was similar to that of the control group. 4. E.E. Lesiovskaya, Metaprot in Extreme Conditions, EW contributed more to the treatment of the damaged gastric St. Petersburg (2010). mucosa than melatonin. In the ivEW and nEW treatment groups only minimal pathological changes of the gastric 5. T.V. Kvetnaya, I.V. Knyazkin, Melatonin: Role and mucosa were found, the majority of them involving Importance in the Diseases of the Elderly, Military superficial erosion and small punctuate areas of necrosis in Medical Academy, St. Petersburg (2003). Thus, investigational drugs used for the treatment of the Antihypoxic Agent (Thymogenum), (2001). indometacine-induced ulcer can be rated by their gastroprotective activity from the most active to the least 7. Clinical Pharmacology of Thymogenum, V.S. Smirnov (ed.), St. Petersburg. (2003). effective: ivEW > nEW > EW ≈ melatonin. 8. Public Health Service on Human Care and Use of Laboratory Animals. Washington. — D. C: U. S. Department of Health and Human Services, 28 (1996). 1. Dipeptide glutamyl-tryptophan has no gastroprotective activity when administered as a single preventive treatment 9. Report of the AVMA panel on euthanasia, American on the model of the stress-induced ulcer. Sevenfold Veterinary Medical Association, 202(2), 229 - 249 (1993).
preventive administration of EW leads to a significant 10. M. G. Derelanko, G. T. Long, Digestive Diseases and decrease in the number of gastric ulcerous destructions in rats Science, 25(11), 830-838 (1980).
previously stressed by immobilization according to the Selye 11. F.P. Trinus, N.A. Mokhort, B.M. Klebanov, Non-steroid method, and this effect is significantly different from that of Anti-inflammatory Agents, Zdorovya, Kiev (1975). 12. Medical Laboratory Technologies 2. Glutamyl-tryptophan, isovaleroyl-glutamyl-tryptophan, A.I. Karpischenko (ed.), Intermedica, St. Petersburg nicotinoyl-glutamyl-tryptophan and melatonin demonstrate varying degrees of gastroprotective activity when tested on 13. G. Aykac, М. Uysal, A. S. Yalcin, et al., Toxicology, 36,
the model of the indomethacin-induced stomach ulcer in 14. P. P. Bradley, D. A. Priebat, R. D. Christerser, G. 3. As compared to other agents, isovaleroyl-glutamyl- Rothstein, J. Invest. Dermatol., 78, 206- 109 (1982).
tryptophan proved to have the greatest gastroprotective 15. D. Melchiorri, E. Sewerynek, R. J. Reiter, et al., Br. J. activity when administered together with a cyclooxygenase Pharmacol., 121, 264 - 70 (1997).
inhibitor. The experimental treatment with the peptide resulted in the significant increase of antioxidant levels, and Received on 28 October 2011 less severe lipid peroxidation in the gastric mucosa.

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