Protective Effect of L‐Arginine Against Ibuprofen‐induced Gastric Injury in Rats

Pha maceu ical Sciences 1997, 3: 609-612 Recei ed July 21, 1997 Accep ed Oc obe 1, 1997 0 1997 Pha maceu ical Sciences P o ec i e E ec o L-A ginine Agains Ibup o en-induced Gas ic Inju y in Ra s M. J. MARTIN CALERO, M. D. JIMENEZ, c. ALARCON DE LA LASTRA, c. LA CASA, J. M. HERRERIAS*, L.BRUSEGHINI , A. ESTERAS? AND V. MOTILVA Depa men o Pha macology, Facul y o Pha macy, Uni e si y o Se ille, *Depa men o Gas oen e ology, Vi gen Maca ena Hospi al, Uni e si y o Se ille, and TZambon G oup, Ba celona, Spain Abs ac This s udy has been designed o con i m he p o ec i e e ec o di e en single o al doses o L-a ginine in he p esence o equimola doses o ibup o en, and o compa e he esul s wi h hose ob ained a e ea men wi h ibup o en alone. Di e en pa ame e s we e assessed in a s: gas ic damage (mm2 and sco e), a io o lesiona ed s omachs/ o al s omachs e alua ed, and p esence o haemo hage. Six hou s a e dosing, o al adminis a ion o ibup o en (0.3, 0.6 and 1.2 mmol kg-') p oduced a p og essi e dose-dependen inc ease in damage o he gas ic mucosa. All ea men s wi h equimola doses o L-a ginine conside ably educed lesions (mm2 and sco e) and he same endency was obse ed wi h he o he pa ame e s examined. We also e alua ed he gas op o ec i e e ec o L-a ginine agains an i-ulce e e ence d ugs, ani idine and oxa idine ( wo an isec e o y agen s) and misop os ol (a cy op o ec i e d ug). The deg ee o inhibi ion o damage p o ided by L-a ginine was simila o hose ob ained wi h he o he d ugs. Thus, we conclude ha he simul aneous adminis a ion o equimola doses o ibup o en and L-a ginine o e s signi ican p o ec ion compa ed wi h gas olesi e doses o ibup o en alone, wi h an impo an dec ease in he lesiona ed a eas and imp o emen o he ascula s a e. The ex en o his p o ec i e ac ion is compa able wi h ha obse ed wi h an i-ulce e e ence d ugs. Non-s e oidal an i-in lamma o y d ugs (NSAIDs) a e among he mos commonly used d ugs in he wo ld, bu hei use in he ea men o pain, e e and in lamma ion, and in pa icula , in he ea - men o a h i is, is associa ed wi h signi ican un owa d e ec s on he gas oin es inal ac . The bleeding and ulce a ion induced in he s omach by NSAIDs is he mos common ad e se eac ion o medica ion and has a signi ican inancial impac on heal h ca e. Thei gas ic oxici y is ela ed o supp ession o p os aglandin syn hesis, h ough inhibi ion o cyclooxygenase ac i i y, and in addi- ion, se e al NSAIDs ha e opical i i an p ope - ies on gas ic mucosa. In he las ew yea s, a numbe o di e en s a- egies o de eloping NSAlDs wi h educed gas ic Co espondence: M. J. Ma in Cale o, Labo a o io de Fa m- acologia, Facul ad de Fa macia, P o . Ga cia Gonzilez s/n, 41012-Se illa, Spain. oxici y ha e been used. These include o mula ing he agen s wi h an en e ic coa ing o p e en abso p ion in he s omach, he de elopmen o p o- d ugs, which a e no ac i e un il hey ha e unde - gone me abolism by he li e , and co-adminis a- ion o ei he supp esso s o acid (an i-H2 o p o on pump inhibi o s) o exogenous p os aglandins. Recen ly, a new s a egy ela ed o he ole o ni ic oxide (NO) in he gas oin es inal ac has been p oposed. NO is a memb ane-pe meable gas ha se es as a media o o many physiological e en s (Moncada e a1 1991). I is p oduced om L- a ginine by a calcium-dependen NO syn hase. Nume ous s udies ha e implica ed NO as a c i ical media o o mucosal de ence, simila o p os- aglandins (Whi le e a1 1990). NO, o d ugs ha gene a e NO (e.g. sodium ni op usside, glyce yl ini a e) ha e been shown o educe he se e i y o gas ic mucosal inju y in expe imen al models (Whi le e a1 1990; B own e a1 1993) and some 610 M. J. MART~N ET AL au ho s ha e sugges ed ha he gas op o ec i e e ec o L-a ginine could be h ough an NO-pa h- way (Ki agawa e a1 1990; Kon u ek e a1 1993) Ibup o en is a po en NSAID ha ecen ly has been o mula ed wi h equimola doses o L-a ginine in o de o imp o e pha macokine ic pa ame e s (highe and mo e apid abso p ion) wi h quicke and mo e po en analgesic e ec . In a p e ious epo we ound ha adminis a ion o L-a ginine be o e ibup o en ea men induced signi ican inhibi ion o gas ic lesions (Mo il a e a1 1996). Thus, we designed his s udy o con i m he p o- ec i e e ec o he ibup o en and L-a ginine o - mula ion s ibup o en alone, and o compa e his gas op o ec ion wi h o he adi ional he apies such as misop os ol (PGE1 analogue), ani idine (a classic an isec e o y agen ) o oxa idine (a new an i-H2 d ug). Ma e ial and Me hods Animal g oups and compounds Male Wis a a s (supplied by Animal Se ices, Facul y o Medicine, Se ille), 180-250 g, we e used. The animals we e ed a no mal labo a o y die in a con olled oom ( empe a u e 22-24°C and humidi y a 70-75%). They we e placed in single cages wi h wi e-ne loo s o p e en cop ophagy, and we e dep i ed o ood o 18 h be o e he expe imen s bu had ee access o wa e . G oups o 9-10 a s we e ea ed wi h di e en doses o ibup o en (Sigma Chemical Co., MO), ibup o en and L-a ginine (Zambon S.A., Ba celona, Spain), ani idine (Sigma Chemical Co., MO), oxa idine (Zambon S.A., Ba celona, Spain) and misop os ol (CECOFAR, Se ille, Spain). The d ugs we e suspended in dis illed wa e and we e adminis e ed by he in agas ic ou e, in a dose o 1 mL/lOO g body weigh . Con ol g oups ecei ed ehicle in compa able olume. Each expe imen was pe o med a leas wice and esul s we e pooled. Expe imen al p o ocol Di e en g oups o animals we e ea ed wi h ibup o en (0.3, 0.6 and 1-20 mmol kg-') and equimola doses o ibup o en and L-a ginine (0.3/0-3, 0.6/0-6 and 1-20/1.20 mmol kg-'). Six hou s a e ea men he animals we e killed using an o e dose o e hyl e he and he s omachs we e emo ed and cu along he smalle cu a u e. The gas ic lesions we e immedia ely e alua ed and he ollowing pa ame e s ela ed o damage we e assessed. Gas ic damage (sco e): 0, absence o lesions; 1, haemo hagic o nec o ic mucosa; 2, poin ed lesions; 3, poin ed lesions wi h lesions < 3 mm; 4, mainly lesions > 3 mm. A ea o gas- ic damage (mm2): he p oduc o ulce leng h and wid h was calcula ed. Mucosal damage (%): educ ion o damaged a ea o he di e en g oups compa ed wi h he espec i e equimola dose o ibup o en alone (100%). Ra io lesioned s o- machs/ o al s omachs e alua ed. P esence o hae- mo hage (sco e): 0, absence; 1, sligh haemo hage; 2, ma ked haemo hage. The in e media e gas olesi e dose o ibup o en (0.6 mmol kg-') no ed in hese expe imen s was selec ed o examine he p o ec i e e ec o mis- op os ol, ani idine and oxa idine. New g oups o animals ecei ed he ollowing ea men s: ibup o- en alone; equimola dose o ibup o en and L- a ginine (0-6/0.6 mmol kg-'); ibup o en and misop os ol (0-6/0.05 x mmol kg-l); ibu- p o en and ani idine (0.6/0.05 mmol kg-'); and ibup o en and oxa idine (0-6/0.05 mmol kg- '). A e he expe imen al pe iod, he animals we e killed and he s omachs emo ed, opened and assessed as in he p e ious expe imen s. The same pa ame e s we e e alua ed. Analysis o esul s S a is ical analysis was pe o med using con en- ional me hods (a i hme ic mean s.e.). One-way analysis o a iance was used o es o signi ican di e ences o means o ea men g oups. Mul iple compa ison be ween means was pe o med using he Mann-Whi ney U- es and X2- es a a p o ec- ion le el o 0.05. Resul s Six hou s a e dosing, ibup o en gi en o ally in a ious doses (0.3, 0-6 and 1.20 mmol kg-') p o- duced a p og essi e dose-dependen inc ease o damage o he gas ic mucosa (4.12 z 1.01, 17.88 2.74 and 66.41 9.17 mm2, espec i ely). O al ea men o he animals wi h equimola doses o L-a ginine conside ably educed he gas ic lesions (0.6 and 1.20 mmol kg-', P < 0.001 agains ibup o en alone, mm2 and sco e). The same endency was e alua ed wi h he o he pa ame e s, deg ee o haemo hage and pe cen age o mucosal damage (Table 1). Signi ican dec eases in he gas ic lesion pa a- me e s we e ound wi h all he ea men s shown in Table 2. The mos ma ked educ ion o he lesion (mm2 and sco e), as well as haemo hagic sco e and pe cen age o mucosal damage, was obse ed wi h ani idine, oxa idine and misop os ol (P < 0.001). Howe e , L-a ginine ea men also L-ARGININE PROTECTS AGAINST IBUPROFEN-INDUCED GASTRIC INJURY IN RATS 61 1 Table 1. The an i-ulce p o ec ion o di e en doses o L-a ginine in he p esence o equimola doses o ibup o en in a s. T ea men Haemo hage Gas ic damage Mucosal Lesioned s omachs Gas ic damage (sco e) (mm2) damage (%) s omachs e alua ed (sco e) None 0.00 0.00 0.00 0.00 0.0 0120 0.00 0.00 Ibup o en 0.20 0.06 4.12 1.01 100 19/20 2.15 0.20 Ibup o en and 0.08 0.04 2.47 0.68 59.8 19/20 1.95 0.15 (0.3 mmol kg-I) L-a ginine (0.3 mmol kg-I) (0.6 mmol kg- I) L-a ginine (0.6 mmol kg-I) Ibup o en 1.03 0.12 17.88 2.74 100 19/19 3.21 0.10 Ibup o en and 0.32 0.1 1 * 6.29 1.33* 35.2 19/19 2.31 0.11 Ibuu o en 1.82 0.10 66.41 9.17 100 19/19 4.0 0.01 (1.20 mmol kg-') Ibup o en and 0.23 0.09* 17.97 4.15* 27.1 19/20 2.45 0.18* L-a ginine (1.20 mmol kg- I) Resul s a e means * s.e. (n = 19 o 20). *P < 0.001, ibup o en and L-a ginine s ibup o en alone (Mann-Whi ney U- es ). Table 2. The e ec s o di e en ea men s in he p esence o ibup o en (0.06 mmol kg-I): L-a ginine (0.6 mmol kg-I), ani idine (0.05 mmol kg-'), oxa idine (0.05 mmol kg-') and misop os ol (0.05 x mmol kg-') on pa ame e s o gas ic damage in a s. T ea men Haemo hage Gas ic damage Mucosal Lesioned s omachs Gas ic damage (sco e) (mm2) damage(%) s omachs e alua ed (%) (sco e) Ibup o en alone 1.03 0.12 17.88 2.74 100 100 3.21 0.10 + L-A ginine 0.32 * 0.11 * 6.29 1.33* 35.2 100 2.31 0.11* + Misop os ol 0.13 0.06* 2.25 * 0.76* 12.6 89.5 1.95 0.21 * + Rani idine 0.10 0.05* 1.43 0.61 * 8.0 68.41- 1.50 0.29* + Roxa idine 0.10 0.02* 0.08 0.07 0.5 66.7 1 .OO 0.25 Resul s a e means s.e. (n = 19 o 20). *P < 0,001, ea men g oups s ibup o en alone, Mann-Whi ney U es ; P < 0.01, x2 es . induced e y ma ked dec eases in all pa ame e s e alua ed (P < 0.001). Discussion The e is o e whelming e idence ha NO is one o he mos impo an media o s o mucosal de ence, a ec ing ac o s such as mucus sec e ion, mucosal blood low, ulce epai and he ac i i y o a a ie y o mucosal immunocy es. Endogenous NO has he capaci y o down- egula e in lamma o y esponses in he gas oin es inal ac (Kubes & Wallace 1995), and i has been shown o be in ol ed in ce ain ypes o gas op o ec ion such as ha induced by capsaicin, papa e ine (B zozowski e a1 1993), suc al a e and mild i i an s (Kon u ek e a1 1994). In his way, he abili y o NO o educe he se e i y o damage induced by NSAIDs has ecen ly been exploi ed in an a emp o p oduce d ugs which a e no ulce ogenic. By a aching an NO- eleasing moie y o s anda d NSAIDs (by inco po a ion o a ni oxybu yl moie y (e.g. lu bi- p o en, ke op o en and diclo enac ni oxy-bu yl- es e ), he gas oin es inal oxic e ec s o hese compounds was g ea ly educed bu did no in e - e e wi h hei abili y o supp ess in lamma o y p ocesses, inhibi p os aglandin syn hesis o inhibi pla ele agg ega ion (Wallace & Ci ino 1994). O he ecen s a egies such as p ophylac ic he apy wi h he PGEl analogue misop os ol ha e been shown o educe he incidence o NSAID-induced ulce s signi ican ly, bu he use o his d ug is limi ed by i s ad e se e ec s in a conside able numbe o pa ien s. Howe e , he e ec o L-a ginine, he p ecu so o NO, on he gas ic mucosal in eg i y has no 612 M. J. MART~N ET AL been much s udied. Ou da a show ha simul a- neous ea men wi h L-a ginine signi ican ly dec eases ibup o en-induced gas ic damage in a dose-dependen way. This esul is in ag eemen wi h Ki agawa e a1 (1990) who epo ed ha L- a ginine gi en in a enously p e en ed gas ic lesions caused by 0-6 M HC1 o wa e -imme sion s ess. Simila esul s we e ob ained by Takeuchi e a1 (1993) who ound ha o al ea men wi h L- a ginine exhibi s gas ic cy op o ec ion agains 0.6 M HC1-induced lesions in a dose dependen manne . This e ec was mimicked by he enan- iome D-a ginine and was no a ec ed by p e ious adminis a ion o N G-ni o-L-a ginine me hyl es e (L-NAME), a po en inhibi o o NO biosyn hesis. Thus hey sugges ed ha he mucosal p o ec i e ac ion o L-a ginine (p.0.) is un ela ed o he NO pa hway, and i s mechanism may appea h ough adap a i e cy op o ec ion media ed by endogenous p os aglandins. In con as , in a p e ious s udy, we did no obse e any p o ec i e e ec agains diclo enac-induced gas ic inju y a e D-a ginine ea men . Howe e , he p o ec ion induced by L- a ginine was main ained a e adminis a ion o L-NAME sugges ing, a leas in pa , he in ol e- men o NO in he p o ec i e mechanism o o al L- a ginine (Mo il a e a1 1997). Fe az e a1 (1994) ound ha in a-a e ial adminis a ion o L-a ginine be o e he applica ion o 20% e hanol p oduced a dose-dependen inc ease in he ex en o damage. L-A ginine sig- ni ican ly educed gas ic blood low ela i e o con ols, and he au ho s sugges ed ha he inc ease o gas ic inju y was pa ly NO dependen and pa ly NO independen . They sugges ed a pa a- doxical e ec o L-a ginine on gas ic mucosal in eg i y. While small amoun s o NO p oduced cons i u i ely by endo helial cells appea o be c i ical o main enance o mucosal pe usion, la ge amoun s o NO can exace ba e mucosal inju y, p obably as a consequence o he cy o oxic p op- e ies o he NO adical o he pe oxyni i e adical o med h ough he in e ac ion o NO wi h supe - oxide. We conclude ha , unde ou expe imen al con- di ions, in agas ic adminis a ion o L-a ginine p o ec s he gas ic mucosa agains ibup o en- induced inju y, and ha his p o ec i e e ec is compa able wi h hose o an isec e o y agen s o misop os ol. Howe e he mechanism in ol ed in his bene icial ac ion ha e no been s udied. I is possible ha L-a ginine achie es di ec cy op o- ec i e e ec on mucosal issue and ha his p ope y could be ela ed o he NO-pa hway, bu hese supposi ions equi e u he explo a ion. Re e ences B own, J. F., Kea es, A. C., Hanson, P. J., Whi le, B. J. R. (1993) Ni ic oxide gene a o s and cGMP s imula e mucus sec e ion by a gas ic mucosal cells. Am. J. Physiol. 265: B zozowski, T., D ozdowicz, D., Szlachcic, A., Py ko-Polonc- zyk, J., Majka, J., Kon u ek S. J. (1993) Role o ni ic oxide and p os aglandins in gas op o ec ion induced by capsaicin and papa e ine. Diges ion 54: 24-3 1 Fe az, J. G., Tigley, A., Wallace, J. L. (1994) Pa adoxical e ec s o L-a ginine on gas ic mucosal in eg i y. Eu . J. Pha macol. 260: 107-1 11 Ki agawa, H., Takeda, F., Kohei, H. (1990) E ec o endo he- lium-de i ed elaxing ac o on he gas ic lesions induced by HCl in a s. J. Pha macol. Exp. The . 253: 1133-1138 Kon u ek, S. J., B zozowski, T., Majka, J., Py ko-Polonczyc, J., S achu a, J. 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