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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 17 18 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. 19 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. REFERENCES 1. VG Podoprigorova, Oxidative Stress and Ulcer Disease, Moscow (2004). 2. Russian Register of Medicines, Drug Encyclopedia, Vol. 12, Moscow (2005). 3. V. N. Shatalov, D.B. Korman, T.V. Krutova et al., Pharmacol. and Toxicol., 3, 60 - 63 (1988). 4. E.E. Lesiovskaya, Metaprot in Extreme Conditions, St. Petersburg (2010). 5. T.V. Kvetnaya, I.V. Knyazkin, Melatonin: Role and Importance in the Diseases of the Elderly, Military Medical Academy, St. Petersburg (2003). 6. RU 2213571, 30.11.2001, p. 6, left O.V. Filippova, D.V. Zolotarev, E.L. Antihypoxic Agent (Thymogenum), (2001). column, Karpova, 7. Clinical Pharmacology of Thymogenum, V.S. 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