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THE INFLUENCE OF LEUKOTRIENE RECEPTOR ANTAGONISTS ON EXPERIMENTAL

INDUCED ULCER IN RATS (Abstract): Aim: Gastric mucosal cells synthesize a large number of

eicosanoids (including leukotrienes) which are distinctively involved in ulcerogenesis. Thisexperimental study investigated the effect of 4 leukotriene receptors’ antagonists onindomethacin(IND)-induced ulcer in rats. Material and methods: Animals were divided into six

groups (of 8 rats each) which received as follows: group I (control) - saline; group II - IND20mg/kg p.o.; group III - montelukast sodium 10mg/kg p.o. and IND 20mg/kg p.o.; group IV - aquinolinic derivative (19363) 20μM/kg p.o. and IND 20mg/kg p.o.; group V - a phenethylamidoderivative (20599) 20μM/kg p.o. and IND 20mg/kg p.o.; group VI - a resatophenone derivative(19072) 20μM/kg p.o. and IND 20mg/kg p.o. Animals were sacrificed eight hours after the lastadministration. The gastric index (UI), gastric pH and histopathological exam were performed on

the removed stomachs. Results: UI was 25.8±6.3 in group II, 10.20±2.3 in group III (p


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Adult, male Wistar rats (n=48), weighing 160-180 g, were housed in groups of four in pPlexiglascages (65 x 40 x30 cm) with the floor covered with sawdust. Animals (obtained from LaboratoryAnimal Center of Cantacuzino Institute of Research, Bucharest) were maintained in a controlled

environment (12-h light/dark cycle with lights on at 07.00, temperature of 20 ± 1ºC) before and

throughout the experimental period. The rats had free access to food (standard lab rat chow) except24 hours before the experiment but tap water ad libitum. All experiments involving animals were

approved by the University’s Committee for Bioethics and animal experimentation and , accordingto international rules of conduct.

After a adaptation habituation period of one week, the animals were randomly dividedasignedassigned into six groups of 8 Wistar male adults rats each. Group I was for control andtreated with saline, group II received indomethacin 20mg/kg p.o., group III received MK 10mg/kg

p.o. and indomethacin 20mg/kg p.o., to group IV was given derivative 19363 20μM/kg p.o. andindomethacin 20mg/kg p.o. , to group V was administered 20559 derivative 20μM/kg p.o. andindomethacin 20mg/kg p.o. and group VI received 19072 20μM/kg p.o. and indomethacin 20mg/kg p.o. The lLeukotrieneT antagonists were administered 3 hours before indomethacin. The

iIndomethacin solution was prepared by dissolving the drug in NaHCO3 0.84‰ solution at 40350C.

Montelukast was purchased from Merck Sharp Dohme, Bucharest, Romania. The LTleukotrienes

antagonists have been synthesized by prof. PhD. Miroslav Kuchar from The Research Institute forPharmacy and Biochemistry (VUFB), Prague, Czech Republic (table 1).

Under deep ether anesthesia, the animals were sacrificed at 8 hours post-drugs administration. ThesStomachs were opened along the greater curvature. We evaluated the ulcer index, the gastric pH

and the histopathological lesions.

Table 1. Inhibitory concentrations 50% (IC50) of the tested antagonists for R LTB4 and R LTD4.

Inhibition values (%) are determined for the concentration of 20μM for each antagonist.

Results were expressed as means ± standard deviation. Statistical analysis was carried out using theStudent’s t -test. The rR esults were considered statistically significant when if p < 0.05.

For the histopathological exam, the stomachs were fixed in 10% buffered formalin. The areas andthe processed . t presenting the most severe macroscopic lesions were sectioned and these fragmentswere embedded in paraffin blocks. The specimens, standard stained with haematoxylin eosin, weremicroscopically examined for morphological changes such as congestion, hemorrhage, edema,

erosions and ulcers. The gastric index was determined by Szelenyi and şi Thiemer method (8) as follows: Gastric index

UI= (n lesions type I) + (n lesions II)x2 + (n lesions III)x3. Edema, hyperemia, patches areconsidered type I lesions, haemorragichemorrhagic erosions in submucoasaa are considered type IIlesions and profound ulcerations and invasive lesions are considered type III lesions.

RESULTS

Compounda –at 20μM b- μM

R LTB4 R LTD4 Molecularweight %

inhibitiona

IC50 b %

inhibitiona

IC50 b

Quinoline derivative19363 45 16.6 100 98 387.5

Phenethylamino derivative

20559 66 6.6 41 48 505.6

Resatophenone derivative

19072 48 11 100 100 467.0

http://www.ncbi.nlm.nih.gov/pubmed/718423?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/718423?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/718423?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/718423?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumhttp://www.ncbi.nlm.nih.gov/pubmed/718423?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum


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Histopathological exam showeds that, compared to control group (fig. 1), indomethacin administrationcaused severe gastric lesions. Group II presenteds hemorrhagic erosion, inflammatory infiltrate inlamina propria and profound ulceration thatwhich extend eds till muscularis mucosae (fig. 2).

MK co-administration reducdecreaseeds the the severity of indomethacin -induced gastric lesions,

small erosion areas alternating with large zones of normal mucoasa (fig. 3). Compared to groupIIgroup II, group III presenteds statistically significant lower UI (25.8±6.3 in group II versus10.20±2.3 in group III) (fig. 4) and higher pH values (2.2±0.3 in group II versus 3.4±01.91 in group III,

p


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treatment with montelukast (10 mg/kg) administrated by gavage for 4 days shown protection againstalendronat-induced gastric damage (.20). Vaananen et al. (21) indicated that two LTD4 receptor antagonists (MK-571 and ICI-204.219)

significantly reduced the permeability changes induced by indomethacin, while two LTB4 antagonists

(SC-41930 and LY-255.283) were without significant effect. In our study, 20559 phenethylamidoderivative 20μM/kg (thatwhich inhibates 41% of CysLT R and 66% of BLT R ) determines the bestgastric protection among the leukotrine LT antagonists synthetised by Prof. Kuchar. Our data point to

the direction that LTB4 acts as an ulcerogenic agent in this experimental model. In this context, it isimportant to note that gastro-protective effect of MK is mainly due to antagonizing CysLT effects ( on

CysLT1 R). We consider that MK’s effect is only partially realized performed through changes ingastric acidity. Still, other mechanisms than antagonizing antagonism of CysLTs may be involved inMK’s mechanism of action. As an example, MK directly inhibits 5-LO and possesses has antioxidant (22) and anti-inflammatory properties (.23). As regarding 20559 phenethylamido derivative, its

protective activity is attributed to antagonizing both CysLTs and LTB4 effects.

CONCLUSIONSLeukotrieneT antagonists present gastro-protective activity in different degrees. In our study, MK and

phenethylamido derivative 20559 demonstrated gastro-protective effects. Compound 20559 (antagonist both for BLT R and CysLT1 R) displayed a lesser protective intensity than MK.

BIBLIOGRAPHYREFERENCES 1. Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 2001;

294(5548):1871-1875.

2. Brink C, Dahlén SE, Drazen J. et al. International Union of Pharmacology XXXVII. Nomenclaturefor leukotriene and lipoxin receptors. Pharmacol Rev 2003; 55(1):195-227

3. Bisgaard H. Role of leukotrienes in asthma pathophysiology. Pediatr Pulmonol 2000; 30(2):166-76.

4. Drazen J. Clinical pharmacology of leukotriene receptor antagonists and 5-lipoxygenase inhibitors.

Am J Respir Crit Care Med 1998; 157: S233-723.5. Subbarao K, Jala VR, Mathis S. et al. Role of leukotriene B4 receptors in the development of

atherosclerosis: potential mechanisms. Arterioscler Thromb Vasc Biol 2004; 24(2): 369-375.6. Okazaki M, Shimizu I, Ishikawa M. et al. Gastric mucosal levels of prostaglandins and leukotrienes in

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healing: topics of this decade. J Clin Gastroenterol 1995; 21 Suppl: S12-7.8. Szelenyi I, Thiemer K. Distention ulcer as a model for testing of drugs for ulcerogenic side effects.

Arch Toxicol 1978; 41(1):99-105.9. Kapui Z, Boér K, Rózsa I, Blaskó G, Hermecz I. Investigations of indomethacin-induced gastric ulcer

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from eicosapentaenoic acid and arachidonic acid on the rat stomach. J Exp Med 1990; 171(5):1827-32.

11. Wallace JL, McKnight GW, Keenan CM, Byles NI, MacNaughton WK. Effects of leukotrienes onsusceptibility of the rat stomach to damage and investigation of the mechanism of action.Gastroenterol 1990; 98: 1178-1186.

12. Szabo S. Mechanisms of gastric mucosal injury and protection. J Clin Gastroenterol 1991; 13(Suppl2): S21-34.

Formatted: Bullets and Numbering

Formatted: Bullets and Numbering


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13. Filaretova L, Tanaka A, Miyazawa T, Kato S, Takeuchi K. Mechanisms by which endogenousglucocorticoid protects against indomethacin-induced gastric injury in rats. Am J Physiol Gastrointest

Liver Physiol 2002; 283(5): G1082-1089.

14. Suzuki K, Araki H, Komoike Y, Takeuchi K. Permissive role of neutrophils in pathogenesis of

indomethacin-induced gastric lesions in rats. Med Sci Monit 2000; 6(5): 908-914.15. Swarnakar S, Ganguly K, Kundu P, Banerjee A, Maity P, Sharma AV. Curcumin regulates expression

and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer. J Biol Chem 2005; 280(10): 9409-9415.

16. Gyömber E, Vattay P, Szabo S, Rainsford KD. Effect of lipoxygenase inhibitors and leukotrieneantagonists on acute and chronic gastric haemorrhagic mucosal lesions in ulcer models in the rat. JGastroenterol Hepatol 1996; 11(10): 922-927.

17. Rainsford KD. Inhibition by leukotriene inhibitors, and calcium and platelet-activating factorantagonists, of acute gastric and intestinal damage in arthritic rats and in cholinomimetic-treated mice.

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18. Nechifor M, Neughebauer IB, Costuleanu M. et al. The influence of an PGF2x analogue and of some peptidoleukotrienes and platelet activating factor antagonists in experimentally induced gastric ulcer

in rats. Rom J Physiol 1993; 30(3-4):155-159.

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Konturek SJ, Brzozowski T, Drozdowicz D, Beck G. Role of leukotrienes in acute gastric lesionsinduced by ethanol, taurocholate, aspirin, platelet-activating factor and stress in rats. Dig Dis Sci

1988; 33(7):806-813.20. Sener G, Kapucu C, Cetinel S, Cikler E, Ayanoğlu-Dülger G. Gastroprotective effect of leukotriene

receptor blocker montelukast in alendronat-induced lesions of the rat gastric mucosa. Prostaglandins Leukot Essent Fatty Acids 2005; 72(1): 1-11.

21. Vaananen PM, Keenan CM, Grisham MB, Wallace JL. Pharmacological investigation of the role of

leukotrienes in the pathogenesis of experimental NSAID gastropathy. Inflammation 1992; 16(3): 227-240.

22. Ramires R, Caiaffa MF, Tursi A, Haeggström JZ, Macchia L. Novel inhibitory effect on 5-

lipoxygenase activity by the anti-asthma drug montelukast. Biochem Biophys Res Commun 2004;324(2): 815-821.

23. Dengiz GO, Odabasoglu F, Halici Z, Cadirci E, Suleyman H. Gastroprotective and antioxidant effects

of montelukast on indomethacin-induced gastric ulcer in rats. J Pharmacol Sci 2007; 105(1): 94-102.

Figure legends

Fig. 1. Group I - Normal mucosa (HE x10)

Fig. 2. Group II - Hemorrhagic erosion (HE x4)Fig. 3. Group III – Large areas of normal mucosa (HE, x 10)Fig. 4. Ulcer index- * Group III şi and group V versus group II, * p


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Fig. 1. Group I - Normal mucosa (HE x10) Fig. 2. Group II - Hemorrhagic erosion (HE x4)

Fig. 3. Group III – Large areas of normal

mucosa (HE, x 10)

Fig. 6. Group IV - Ulceration with fibrino-inflammatory

exudate and profound fibrinoid necrosis(HE x10)

Fig. 7. Group V - Superficial non-hemorrhagic

erosion (HE x4)

Fig. 8. Group VI - Profound non- hemorrhagic necrosis

(HE x10)


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Fig. 4. Gastric pH - * Group III versus group II, * p

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