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STUDY OF THE ANTISECRETORY AND ANTIULCER MECHANISMS OF A NEW INDENOPIRYDAZINONE DERIVATIVE IN RATS
Pharmacological Research, Vol. 35, No. 5, 1997
STUDY OF THE ANTISECRETORY AND ANTIULCER MECHANISMS OF A NEW INDENOPIRYDAZINONE DERIVATIVE IN RATS E. BAROCELLI, M. CHIAVARINI, V. BALLABENI, D. BARLOCCO†, P. VIANELLO†, V. DAL PIAZ‡ and M. IMPICCIATORE* ` Istituto di Farmacologia e Farmacognosia, Universita degli Studi di Parma, †Istituto di Chimica Farmaceutica e ` Tossicologica, Universita degli Studi di Milano, ‡Dip. Scienze Farmaceutiche, Universita` degli Studi di Firenze, Parma, Italy Accepted 23 April 1997 The present study investigates the antisecretory and antiulcer mechanisms of a new indenopyridazinone derivative previously reported to inhibit gastric acid secretion in pylorusligated rats and to prevent gastric ulcerations induced by indomethacin or ethanol in the same animal species. The new compound was tested on the acid hypersecretion induced by histamine, pentagastrin and bethanechol in in vivo and in vitro experimental models. Furthermore, its influence on the mucosal layer adhering the gastric wall in indomethacintreated rats was considered. Ranitidine was selected as a reference drug. The results obtained demonstrated that the new molecule, at variance with ranitidine, exerts antiulcer activity mainly enhancing the gastric mucosal integrity and simultaneously inhibiting the gastric acid hypersecretion evoked exclusively by cholinergic pulses. Therefore, an involvement of a neuronal rather than an effectorial mechanism has been suggested. Due to these mechanisms of action it clearly differentiates from ranitidine and its possible application in the peptic disease resistant to H2-blockers could be speculated. 1997 The Italian Pharmacological Society KEY WORDS: in vivo–in vitro functional assays, gastric mucosal barrier, antiulcer-antisecretory activity, indenopirydazinone derivative, ranitidine.
INTRODUCTION Available evidence indicates that the disorders in gastric acid secretion as well as the alterations in gastric mucosal integrity may simultaneously contribute to the multifactorial pathogenesis of peptic ulcer [1, 2]. Nowadays the medical treatment for peptic ulcer healing is generally based on the inhibition of gastric acid secretion by H2-blockers, omeprazole and antimuscarinics as well as on the acid-independent therapy provided by sucralfate and bismuth [3]. Obviously, drugs endowed with antisecretory activity coupled with gastroprotective effects could theoretically represent a promising approach for successful treatment of the peptic disease because of potential complementary effects of therapeutic modalities acting by different mechanisms. In the course of our previous studies on tricyclic indenopyridazinones, a new dimethoxy derivative (labelled 4 h) has been proved to exert both antisecretory and antiulcer effects in rats [4, 5]. In fact, the
*Correspondence to: Prof. Mariannina Impicciatore, Istituto di ` Farmacologia e Farmacognosia, Facolta di Farmacia, Viale delle ` Scienze, Campus Univ., Universita degli Studi di Parma, 43100 Parma PR, Italy. 1043–6618/97/050487–06/$25·00/0/fr970168
oral administration of compound 4 h in pylorus-ligated rats induced a significant inhibition of gastric acid secretion comparable to that of ranitidine with a three fold lower potency than the conventional H2antagonist. Furthermore, orally administered in rats, both compound 4 h and ranitidine prevented indomethacin-caused gastric lesions. By contrast, in rat ethanol-induced gastric mucosal damage, the compound under study abolished gastric injury, while ranitidine was completely ineffective. However, the antisecretory and antiulcer mechanisms of this compound are still unclear even if the findings so far accumulated might exclude a significant involvement of the two important elements controlling gastric acid production such as the H2-receptors and the H+/K+ proton pump [5]. Therefore, in this study, we examined the effects of compound 4 h on the gastric hypersecretory response induced by three different stimulants (pentagastrin, bethanechol and histamine) both in anaestethized rats and in mouse isolated perfused stomachs in order to elucidate the mechanism underlying its antisecretory activity. Moreover, we also investigated the gastroprotective effect of such compound by evaluating its ability to preserve the gastric insoluble mucoproteins in indomethacin-treated rats. 1997 The Italian Pharmacological Society
488
MATERIALS AND METHODS Wistar rats of either sex, (200–300 g) young (20–25 g) and adult Swiss mice (30–35 g) (Morini, S. Polo, RE) were used. The rats and the adult mice were fasted for 48 h prior to the experiments with free access to water.
Gastric acid secretion in anaesthetized rats Acid secretion of anaesthetized rats was determined according to Ghosh & Schild [6]. Under urethane anaesthesia (1·25 g kg −1 i.p.), a polyethylene tube for collecting perfusate was introduced into the stomach from a small incision in the duodenum, and fixed at the pyloric portion. A tube cannula for infusing the perfusate was inserted into the esophagus and fixed at cardias level. The stomach was perfused with saline (37°C) at a constant flow rate of 1 ml min−1 through the cannula. Gastric perfusate was collected at 10-min intervals. Acid output was determined by titration (Radiometer, Copenhagen, Denmark) of the flushed perfusate with 0·002 N NaOH to pH 7·0 and expressed as ∆µEq H+ 30 min−1 from the basal value. Basal acid secretion was measured for 60 min, thereafter histamine (2 mg kg−1 h−1), pentagastrin (40 µg kg−1h−1) or bethanechol (0·5 mg kg−1 h−1) dissolved in saline were intravenously infused through a caudal vein throughout the experiment (3 h). The test drug, suspended in 0·5% methoxycellulose, was administered to conscious rats by gavage (1 ml kg−1) at the dose of 30 mg kg−1 2 h before the injection of the secretagogues. Ranitidine 10 mg kg−1 was used as reference compound.
Gastric acid secretion in mouse perfused stomach The mice, anaesthetized by ether inhalation, were killed and the whole stomach was removed according to Szelenyi’s method [7]. The isolated organ was suspended in an organ bath containing 40 ml buffered serosal solution (mM: NaCl 118·1; KCl 4·8; MgSO4·7H2O 1·2; CaCl2·2H2O 0·6; KH2PO4 1·0; NaH2PO4 16·0; glucose 31·6) at 37°C gassed with 95% O2 and 5% CO2. The gastric lumen was continuously perfused at a rate of 1 ml min −1 by means of a peristaltic pump (LKB 2132 Microperpex, Sweden) with an unbuffered mucosal solution (mM: NaCl 135·8; KCl 4·8; MgSO4·7H2O 1·2; CaCl2·2H2O 1·3; glucose 31·6) bubbled with 100% O2 and the perfusate passed over a system adjusted to provide 12 cm H2O pressure to distend the stomach. Samples were collected at interval of 10 min and H+ concentration was measured by means of a pHmeter (Radiometer, Copenhagen, Denmark). The preparation was allowed to stabilize for 60 min. Acid secretion was expressed as ∆pH calculated subtracting the basal pH value, measured immediately prior to drug addition, from stimulated pH value. Single increasing concentrations of the stimulant (histamine 10−7–10 −3 M, pentagastrin
Pharmacological Research, Vol. 35, No. 5, 1997
10−12–10 −6 M or bethanechol 10 −8–10 −3 M) were directly added to the organ bath every 30 min and concentration-effect curves were obtained. The compound under study (10−5–10−4 M) and the reference compound ranitidine 10−4 M were incubated for 30 min before agonist addition. The test compound and ranitidine were dissolved in buffered solution containing dimethylsulphoxide (DMSO) at the maximal final bath concentration of 0·2%. The control curve with agonists were constructed in the presence of DMSO at the same concentration.
Determination of gastric insoluble mucoproteins Groups of 10 rats were orally treated with the vehicle, the test compound 4 h or ranitidine (30 mg kg−1) immediately before indomethacin intraperitoneal injection (40 mg kg−1) and killed by ether inhalation 6 h later. The stomachs were removed and opened along the great curvature. The mucoproteins adhering the gastric mucosal wall were determined according to Corne’s method [8]. The glandular portion of each stomach was isolated and soaked for 2 h in 10 ml of 0·1% Alcian Blue, dissolved in 0·16 M sucrose, buffered with 0·05 M sodium acetate to pH 5·8. The excess dye was removed by two separate rinses with 0·25 M sucrose. Dye complexed with mucus was extracted from each stomach by immersion for 2 h in 10 ml aliquots of 0·5 M MgCl2. The resulting solutions were soaken briefly with equal volume of diethylether and the optical density of the aqueous phase estimated spectrophotometrically at 605 nm (LKB, Ultrospec 4050). The amount of glycoproteins complexed with the dye was expressed in terms of µg of Alcian Blue/stomach by comparison with L-Tyrosine standard curve.
Data analysis All the data were expressed as means±SEM and the statistical significance between treatments was analysed by unpaired Student’s t-test. EC50 values of individual concentration-response curves were calculated by linear regression analysis and the apparent potency of each agonist was indicated as pD2 value. The antagonistic affinity of the tested drugs was determined according to Furchgott’s equation (9): pKB=log([E']/[E]−1)−log[B] [E'] and [E] are the concentrations of the agonist producing the half-maximum effect in the presence and absence of the antagonist, respectively; [B] is the concentration of the antagonist.
Drugs The following drugs were used: Ranitidine hydrochloride (ICN Biomedicals Inc., Ohio, USA); Histamine dihydrochloride, bethanechol chloride, pentagastrin (Sigma Chimica, Italy); Indomethacin meglumine (Chiesi Farmaceutici, Parma, Italy); compound 4 h: 7,8-dimethoxy-5H-indeno[1,2-c]pyridazin-3-(2H)-one
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120–150 min from the beginning of histamine and pentagastrin infusion (∆µEq H+ 30 min−1=21·53±4·01 and 28·56±4·14, respectively) or giving the maximal hypersecretory response after 240 min from the start of bethanechol administration (∆µEq H+ 30 min−1= 26·47±4·14) (Fig. 1). Oral administration of compound 4 h (30 mg kg−1) and ranitidine (10 mg kg−1) as well did not affect basal acid secretion. Unlike ranitidine, which abolished the secretory response evoked by histamine and pentagastrin unaffecting bethanechol evoked response, compound 4 h did not influence histamine induced acid secretion, weakly reduced pentagastrin response and significantly inhibited gastric acid response to bethanechol (about 82%) (P<0·05) (Fig. 1).
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RESULTS
After 1 h of stabilization the outflow pH in mouse isolated stomach preparation was 4·35±0·03 (n=58). The three agonists histamine, pentagastrin and bethanechol produced concentration-dependent increases in basal gastric acid secretion (Fig. 2). Histamine evoked the highest maximal response (∆pH=0·65±0·07) whereas pentagastrin and bethanechol induced 89 and 81%, respectively, of the maximal effect evoked by histamine. Pentagastrin exhibited the greatest potency, its pD2 values being 9·08± 0·13, whereas the pD2 values for bethanechol and histamine were 5·91±0·03 and 5·02±0·06, respectively. Ranitidine (10−4 M) did not modify basal acid secretion. The H2-blocker competitively antagonized histamine induced hypersecretion producing a parallel rightward shift of the agonist concentration-response curve with no change in maximal asymptote (pKB= 5·04±0·15) [Fig. 2(A)]. Pentagastrin-induced secretory responses were greatly depressed in the presence of ranitidine while bethanechol evoked concentration-response curves were reduced in a non-significant way [Fig. 2(B), (C)]. Compound 4 h only at the highest concentration tested (10−4 M), caused a significant (P<0·01) increment in the basal acid secretion while it differently affected the secretory responses evoked by the three stimulants. In fact the compound failed in modifying pentagastrin-induced secretion [Fig. 2(B)] and apparently potentiated bethanechol concentrationresponse curve [Fig. 2(C)]. On the other hand, histamine curves were progressively shifted to the left and amplified by increasing concentrations of compound 4 h [Fig. 2(A)], histamine pD2 value changing from 5·02±0·06 in control curve to 5·77±0·11 in the presence of compound 4 h 10−4 M.
Gastric acid secretion in anaesthetized rats
Determination of gastric insoluble mucoproteins
Basal acid secretion of the anaesthetized rats was about 11·22±0·66 µEqH+ 30 min−1 and was significantly increased by equiactive doses of the three stimulants, reaching a stable maximal value about
When orally administered at the effective antiulcer dose of 30 mg kg−1 in indomethacin-treated rats, compound 4 h, unlike ranitidine which was ineffective, significantly enhanced the amount of mucus adherent
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Fig. 1. Gastric secretory responses to: (A) histamine (B) pentagastrin 40 µg kg−1 h−1; (C) 2 mg kg−1 h−1; bethanechol 0·5 mg kg−1 h−1 in the absence and presence of the compound 4 h (m) 30 mg kg−1, or ranitidine (d) 10 mg kg−1 orally administered 2 h before secretagogues (h) in anaesthetized rats. Each point represents the mean±SEM of 6–8 experiments; *P<0·05, **P<0·01 Student’s t-test.
was synthesised by Prof. D. Barlocco, Ist. Chim. Farm. e Toss., University of Milan, Italy.
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Fig. 3. Effect of compound 4 h (j) and ranitidine ( ), orally administered at 30 mg kg−1, on mucus adhering to the gastric wall in indomethacin-treated rats (40 mg kg−1 i.p.). Control group (h) treated with indomethacin received the vehicle alone . n=8 rats group−1; *P<0·05, **P<0·01 Student’s t-test.
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Fig. 2. Histamine (A), pentagastrin (B) and bethanechol (C) concentration-response curves obtained in mouse isolated stomach following pre-incubation (30 min) with compound 4 h (h, 0; j, 10−5 M; m, 10−4 M) or ranitidine (d, 10−4 M). Each point represents the mean±SEM of 6–8 experiments; *P< 0·05, **P<0·01 Student’s t-test.
to the gastric wall, the µg of Alcian blue dye bound to each stomach increasing from 227·4±19·8 for vehicletreated rats to 348·3±16·9 for indenopyridazinonetreated animals (P<0·01) (Fig. 3).
DISCUSSION The present study showed that compound 4 h, able to inhibit the gastric acid secretion in pylorus-ligated rats, exhibited contradictory effects when tested in in vivo and in vitro experimental models where the acid
hypersecretion was pharmacologically-induced by different secretagogues. In fact, in anaesthetized rats, the compound 4 h significantly inhibited bethanecholevoked acid secretion leaving substantially immodified the secretory response induced by histamine or pentagastrin. On the other hand, in mouse isolated stomachs, it failed to antagonize either bethanechol- or pentagastrin-stimulated acid production and caused an unexpected potentiation of histamine-induced response. These findings suggest that the antisecretory effect of the test compound could be attributed to an inhibitory interference with excitatory cholinergic pathways which actively contribute to gastric acid secretion only in the in vivo model. Indeed, such hypothesis is supported by the ineffectiveness of compound 4 h in antagonizing the in vitro secretory response caused with effectorial mechanisms by histamine, pentagastrin and bethanechol. Unlike compound 4 h, ranitidine exhibited a comparable activity in both experimental models. As expected, it suppressed the hypersecretory response to histamine and pentagastrin in the anaesthetized rat and in the mouse isolated stomach, while unaffecting the secretory responses to the muscarinic agonist bethanechol. The current view on processes regulating acid secretion is that gastrin can act directly on the parietal cell as well as indirectly via endogenous histamine release [10]. This latter mechanism accounts for the inhibition of pentagastrin-induced acid secretion observed with ranitidine in the present investigation. Such behaviour is consistent with previous experimental evidence provided both in anaesthetized rats [11] and mouse isolated stomachs [12].
Pharmacological Research, Vol. 35, No. 5, 1997
With respect to the in vitro assay the compound 4 h at the highest concentration tested significantly increased basal acid secretion acting with a mechanism which remains still undefined. As concerns muscarinic-induced secretory response, the bethanechol curve shift is actually apparent since pD2 values, calculated in the absence and presence of compound 4 h, were superimposable. On the other hand, compound 4 h seems to exert an effective potentiating activity on histamine evoked acid production, causing a leftward shifting of the amine curve with a significant increase in agonist potency. The lack of any facilitating action on pentagastrin response seems to indicate that compound 4 h does not interfere with endogenous histamine suggesting that large amounts of exogenous histamine are required to evidence such overstimulating effect. The mechanism underlying this response is not clear even if it is reasonable to rule out any interference from compound 4 h with the specific receptor or post-receptor events involved in histamine-oxyntic cell interaction. It is likely that compound 4 h can indirectly facilitate histamine secretory response by making more exogenous histamine available to remove inhibitory pathways in target sites other than the parietal cell (e.g. inducing a permissive vasodilation [13] or blocking via H3 receptors somatostatin release [14]). The absence of such potentiation in in vivo test could depend on the limited concentration reached by compound 4 h at gastric level so much so that it did not produce any significative modification of basal secretory activity in the experimental conditions here employed. We demonstrated that compound 4 h inhibits gastric acid secretion exclusively in bethanechol-stimulated and in pylorus-ligated rats. In a preliminary study (unpublished data) we observed that at the antisecretory dosage of 30 mg kg−1 os, compound 4 h failed to affect mice spontaneous motor activity. These findings seem to strengthen the hypothesis that such compound exerts its inhibitory action by modulating specifically the cholinergic transmission rather than generally suppressing the central nervous activity. In a previous work we provided evidence that compound 4 h possesses also antiulcer activity in ethanol and indomethacin-induced experimental gastric lesions [5]. This pilot study seems to indicate that compound 4 h can prevent ulcer formation mainly strengthening the pre-epithelial barrier, which is constituted by the mucus covering the surface of the gastric mucosa, rather than reducing gastric acid secretion. In fact, our data clearly demonstrate that the treatment with the compound 4 h, at the dose (30 mg kg−1 os) giving 84% protection against indomethacin-induced gastric lesions [5], significantly enhanced the amount of mucoproteins adherent to the mucosal epithelium of rat stomachs.
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We reported that the same dose was almost completely effective (97% protection) also in preventing the gastric haemorrhagic damage caused by ethanol [5]. It has been pointed out that, in the pathogenesis of ethanol-induced gastric injury, the initial dislodgement of the superficial mucosal cells and the subsequent perturbation of submucosal microcirculation plays a pivotal role while the involvement of acid secretion has been ruled out [15]. Thereby, an increased production/preservation of mucus (or an enhancement in the defensive factors) seems to be one of the possible mechanisms underlying the gastroprotective actions displayed by compound 4 h. This mucus-related mechanism of action does not seem to be shared by ranitidine. Indeed, this H 2-blocker protected gastric mucosa from indomethacin-caused lesions without affecting the gastric mucoprotein level and failed to prevent ethanol-caused insult [5]. Gastric ulcers induced by indomethacin treatment are usually ascribed to various factors such as the deficiency of endogenous prostaglandins, the occurrence of gastric abnormal contractions and the gastric mucosal blood flow stasis [16]. The antiulcer activity displayed by the H2-antagonist in indomethacin-treated rats, has been widely documented in previous studies [17, 18] and it has been attributed mainly to the blockade of acid secretion and only partially to acid-independent actions [19, 20]. On the basis of the data here reported, we can conclude that the new indenopyridazinone derivative 4 h exerts antiulcer activity enhancing the gastric mucosal integrity and simultaneously it is able to control the gastric acid hypersecretion evoked by nervous cholinergic pulses. Hence this compound, which acts with mechanisms clearly different from ranitidine, suggests new possible approaches for the treatment of peptic ulcers resistant to H2-blockers.
ACKNOWLEDGEMENTS Supported by MURST 40%. The authors would like to thank Dr Giuseppe Domenichini for technical assistance.
REFERENCES 1. Hojgaard L, Nielsen AM, Rune SL. Peptic ulcer pathophysiology: Acid, bicarbonate, and mucosal function. Scanv J Gastroenterol 1996; 31: 10–15. 2. Soll AH. Gastric, duodenal, and stress ulcer. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal disease. Philadelphia: WB Saunders, 1993; 580–679. 3. Soll AH. Medical treatment of peptic ulcer disease: practice guidelines. JAMA 1996; 275: 622–9. 4. Barlocco D, Dal Piaz V, Malandrino S, Cignarella G. Tricyclic indenopyridazinone derivatives as potential antisecretory agents. 4th International symposium on
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the chemistry and pharmacology of pyridazine 1994; Bled, 14. Barlocco D, Cignarella G, Vianello P, Dal Piaz V, Giovannoni MP, Malandrino S, Barocelli E, Chiavarini M, Impicciatore M. Indenopyridazinone derivatives as potential antisecretory and antiulcer agents. Drug Des Disc 1997; (in press). Ghosh MN, Schild HO. Continuous recording of acid secretion in the rat. Br J Pharmacol Chemother 1958; 13: 54–61. Szelenyi I. An isolated mammalian stomach preparation for studying the effect of substances on gastric acid secretion. Arzneim Forsch Drug Res 1981; 31: 998–1000. Corne SJ, Morissey SM, Woods RJ. A method of quantitative estimation of gastric barrier mucus. J Physiol 1974; 242: 116–17. Furchgott RF. The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In: Blanschko H, Muscholl E, eds. Handbook of experimental pharmacology. Berlin Heidelberg New York: Springer, 1972: 33, 283–335. Goldschmiedt M, Feldman M. Gastric secretion in health and disease. In: Sleisenger MH, Fordtran JS eds. Gastrointestinal disease. Philadelphia: WB Saunders, 1993: 524–41. Hayward NJ, Harding M, Lloyd SAC, McKnight AT, Hughes J, Woodruff GN. The effect of CCKB/gastrin antagonists on stimulated gastric acid secretion in the anaesthetized rat. Br J Pharmacol 1991; 104: 973–7. Black JW, Leff P, Shankley NP. Pharmacological
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analysis of the pentagastrin-tiotidine interaction on the mouse isolated stomach. Br J Pharmacol 1985; 86: 589–99. Shankley NP, Welsh NJ, Black JW. Histamine dependence of pentagastrin-stimulated gastric acid secretion in rats. The Yale J Biol Med 1992; 65: 613–19. Vuyyuru L, Schubert ML, Harrington L, Arimura A, Makhlouf GM. Dual inhibitory pathways link antral somatostatin and histamine secretion in human, dog and rat stomach. Gastroenterology 1995; 109: 1566–74. Oates PJ, Hakkinen JP. Studies on the mechanism of ethanol-induced gastric damage in rats. Gastroenterology 1988; 94: 10–21. Ueki S, Takeuchi K, Okabe S. Gastric motility is an important factor in the pathogenesis of indomethacininduced gastric mucosal lesions in rats. Dig Dis Sci 1988; 33: 209–16. Bunce KT, Daly MJ, Humphray JM, Stables R. H2receptor antagonists protect against aspirin-induced gastric lesions in the rat. Agents Actions 1981; 11: 167–200. Guth PH. Mucosal coating agents and other nonantisecretory agents are they cytoprotective? Dig Dis Sci 1987; 32: 647–54. Kauffman KL, Grossman MI. Prostaglandin and cimetidine inhibit the formation of ulcers produced by parenteral salicylates. Gastroenterology 1978; 75: 1099–102. ` ` Nosal’ova V, Navarova J. Indomethacin induced changes in mucosal lysosomal enzyme activity: effect of H2 antagonists. Agents Actions 1994; 41: C95–6.
STUDY OF THE ANTISECRETORY AND ANTIULCER MECHANISMS OF A NEW INDENOPIRYDAZINONE DERIVATIVE IN RATS E. BAROCELLI, M. CHIAVARINI, V. BALLABENI, D. BARLOCCO†, P. VIANELLO†, V. DAL PIAZ‡ and M. IMPICCIATORE* ` Istituto di Farmacologia e Farmacognosia, Universita degli Studi di Parma, †Istituto di Chimica Farmaceutica e ` Tossicologica, Universita degli Studi di Milano, ‡Dip. Scienze Farmaceutiche, Universita` degli Studi di Firenze, Parma, Italy Accepted 23 April 1997 The present study investigates the antisecretory and antiulcer mechanisms of a new indenopyridazinone derivative previously reported to inhibit gastric acid secretion in pylorusligated rats and to prevent gastric ulcerations induced by indomethacin or ethanol in the same animal species. The new compound was tested on the acid hypersecretion induced by histamine, pentagastrin and bethanechol in in vivo and in vitro experimental models. Furthermore, its influence on the mucosal layer adhering the gastric wall in indomethacintreated rats was considered. Ranitidine was selected as a reference drug. The results obtained demonstrated that the new molecule, at variance with ranitidine, exerts antiulcer activity mainly enhancing the gastric mucosal integrity and simultaneously inhibiting the gastric acid hypersecretion evoked exclusively by cholinergic pulses. Therefore, an involvement of a neuronal rather than an effectorial mechanism has been suggested. Due to these mechanisms of action it clearly differentiates from ranitidine and its possible application in the peptic disease resistant to H2-blockers could be speculated. 1997 The Italian Pharmacological Society KEY WORDS: in vivo–in vitro functional assays, gastric mucosal barrier, antiulcer-antisecretory activity, indenopirydazinone derivative, ranitidine.
INTRODUCTION Available evidence indicates that the disorders in gastric acid secretion as well as the alterations in gastric mucosal integrity may simultaneously contribute to the multifactorial pathogenesis of peptic ulcer [1, 2]. Nowadays the medical treatment for peptic ulcer healing is generally based on the inhibition of gastric acid secretion by H2-blockers, omeprazole and antimuscarinics as well as on the acid-independent therapy provided by sucralfate and bismuth [3]. Obviously, drugs endowed with antisecretory activity coupled with gastroprotective effects could theoretically represent a promising approach for successful treatment of the peptic disease because of potential complementary effects of therapeutic modalities acting by different mechanisms. In the course of our previous studies on tricyclic indenopyridazinones, a new dimethoxy derivative (labelled 4 h) has been proved to exert both antisecretory and antiulcer effects in rats [4, 5]. In fact, the
*Correspondence to: Prof. Mariannina Impicciatore, Istituto di ` Farmacologia e Farmacognosia, Facolta di Farmacia, Viale delle ` Scienze, Campus Univ., Universita degli Studi di Parma, 43100 Parma PR, Italy. 1043–6618/97/050487–06/$25·00/0/fr970168
oral administration of compound 4 h in pylorus-ligated rats induced a significant inhibition of gastric acid secretion comparable to that of ranitidine with a three fold lower potency than the conventional H2antagonist. Furthermore, orally administered in rats, both compound 4 h and ranitidine prevented indomethacin-caused gastric lesions. By contrast, in rat ethanol-induced gastric mucosal damage, the compound under study abolished gastric injury, while ranitidine was completely ineffective. However, the antisecretory and antiulcer mechanisms of this compound are still unclear even if the findings so far accumulated might exclude a significant involvement of the two important elements controlling gastric acid production such as the H2-receptors and the H+/K+ proton pump [5]. Therefore, in this study, we examined the effects of compound 4 h on the gastric hypersecretory response induced by three different stimulants (pentagastrin, bethanechol and histamine) both in anaestethized rats and in mouse isolated perfused stomachs in order to elucidate the mechanism underlying its antisecretory activity. Moreover, we also investigated the gastroprotective effect of such compound by evaluating its ability to preserve the gastric insoluble mucoproteins in indomethacin-treated rats. 1997 The Italian Pharmacological Society
488
MATERIALS AND METHODS Wistar rats of either sex, (200–300 g) young (20–25 g) and adult Swiss mice (30–35 g) (Morini, S. Polo, RE) were used. The rats and the adult mice were fasted for 48 h prior to the experiments with free access to water.
Gastric acid secretion in anaesthetized rats Acid secretion of anaesthetized rats was determined according to Ghosh & Schild [6]. Under urethane anaesthesia (1·25 g kg −1 i.p.), a polyethylene tube for collecting perfusate was introduced into the stomach from a small incision in the duodenum, and fixed at the pyloric portion. A tube cannula for infusing the perfusate was inserted into the esophagus and fixed at cardias level. The stomach was perfused with saline (37°C) at a constant flow rate of 1 ml min−1 through the cannula. Gastric perfusate was collected at 10-min intervals. Acid output was determined by titration (Radiometer, Copenhagen, Denmark) of the flushed perfusate with 0·002 N NaOH to pH 7·0 and expressed as ∆µEq H+ 30 min−1 from the basal value. Basal acid secretion was measured for 60 min, thereafter histamine (2 mg kg−1 h−1), pentagastrin (40 µg kg−1h−1) or bethanechol (0·5 mg kg−1 h−1) dissolved in saline were intravenously infused through a caudal vein throughout the experiment (3 h). The test drug, suspended in 0·5% methoxycellulose, was administered to conscious rats by gavage (1 ml kg−1) at the dose of 30 mg kg−1 2 h before the injection of the secretagogues. Ranitidine 10 mg kg−1 was used as reference compound.
Gastric acid secretion in mouse perfused stomach The mice, anaesthetized by ether inhalation, were killed and the whole stomach was removed according to Szelenyi’s method [7]. The isolated organ was suspended in an organ bath containing 40 ml buffered serosal solution (mM: NaCl 118·1; KCl 4·8; MgSO4·7H2O 1·2; CaCl2·2H2O 0·6; KH2PO4 1·0; NaH2PO4 16·0; glucose 31·6) at 37°C gassed with 95% O2 and 5% CO2. The gastric lumen was continuously perfused at a rate of 1 ml min −1 by means of a peristaltic pump (LKB 2132 Microperpex, Sweden) with an unbuffered mucosal solution (mM: NaCl 135·8; KCl 4·8; MgSO4·7H2O 1·2; CaCl2·2H2O 1·3; glucose 31·6) bubbled with 100% O2 and the perfusate passed over a system adjusted to provide 12 cm H2O pressure to distend the stomach. Samples were collected at interval of 10 min and H+ concentration was measured by means of a pHmeter (Radiometer, Copenhagen, Denmark). The preparation was allowed to stabilize for 60 min. Acid secretion was expressed as ∆pH calculated subtracting the basal pH value, measured immediately prior to drug addition, from stimulated pH value. Single increasing concentrations of the stimulant (histamine 10−7–10 −3 M, pentagastrin
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10−12–10 −6 M or bethanechol 10 −8–10 −3 M) were directly added to the organ bath every 30 min and concentration-effect curves were obtained. The compound under study (10−5–10−4 M) and the reference compound ranitidine 10−4 M were incubated for 30 min before agonist addition. The test compound and ranitidine were dissolved in buffered solution containing dimethylsulphoxide (DMSO) at the maximal final bath concentration of 0·2%. The control curve with agonists were constructed in the presence of DMSO at the same concentration.
Determination of gastric insoluble mucoproteins Groups of 10 rats were orally treated with the vehicle, the test compound 4 h or ranitidine (30 mg kg−1) immediately before indomethacin intraperitoneal injection (40 mg kg−1) and killed by ether inhalation 6 h later. The stomachs were removed and opened along the great curvature. The mucoproteins adhering the gastric mucosal wall were determined according to Corne’s method [8]. The glandular portion of each stomach was isolated and soaked for 2 h in 10 ml of 0·1% Alcian Blue, dissolved in 0·16 M sucrose, buffered with 0·05 M sodium acetate to pH 5·8. The excess dye was removed by two separate rinses with 0·25 M sucrose. Dye complexed with mucus was extracted from each stomach by immersion for 2 h in 10 ml aliquots of 0·5 M MgCl2. The resulting solutions were soaken briefly with equal volume of diethylether and the optical density of the aqueous phase estimated spectrophotometrically at 605 nm (LKB, Ultrospec 4050). The amount of glycoproteins complexed with the dye was expressed in terms of µg of Alcian Blue/stomach by comparison with L-Tyrosine standard curve.
Data analysis All the data were expressed as means±SEM and the statistical significance between treatments was analysed by unpaired Student’s t-test. EC50 values of individual concentration-response curves were calculated by linear regression analysis and the apparent potency of each agonist was indicated as pD2 value. The antagonistic affinity of the tested drugs was determined according to Furchgott’s equation (9): pKB=log([E']/[E]−1)−log[B] [E'] and [E] are the concentrations of the agonist producing the half-maximum effect in the presence and absence of the antagonist, respectively; [B] is the concentration of the antagonist.
Drugs The following drugs were used: Ranitidine hydrochloride (ICN Biomedicals Inc., Ohio, USA); Histamine dihydrochloride, bethanechol chloride, pentagastrin (Sigma Chimica, Italy); Indomethacin meglumine (Chiesi Farmaceutici, Parma, Italy); compound 4 h: 7,8-dimethoxy-5H-indeno[1,2-c]pyridazin-3-(2H)-one
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120–150 min from the beginning of histamine and pentagastrin infusion (∆µEq H+ 30 min−1=21·53±4·01 and 28·56±4·14, respectively) or giving the maximal hypersecretory response after 240 min from the start of bethanechol administration (∆µEq H+ 30 min−1= 26·47±4·14) (Fig. 1). Oral administration of compound 4 h (30 mg kg−1) and ranitidine (10 mg kg−1) as well did not affect basal acid secretion. Unlike ranitidine, which abolished the secretory response evoked by histamine and pentagastrin unaffecting bethanechol evoked response, compound 4 h did not influence histamine induced acid secretion, weakly reduced pentagastrin response and significantly inhibited gastric acid response to bethanechol (about 82%) (P<0·05) (Fig. 1).
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RESULTS
After 1 h of stabilization the outflow pH in mouse isolated stomach preparation was 4·35±0·03 (n=58). The three agonists histamine, pentagastrin and bethanechol produced concentration-dependent increases in basal gastric acid secretion (Fig. 2). Histamine evoked the highest maximal response (∆pH=0·65±0·07) whereas pentagastrin and bethanechol induced 89 and 81%, respectively, of the maximal effect evoked by histamine. Pentagastrin exhibited the greatest potency, its pD2 values being 9·08± 0·13, whereas the pD2 values for bethanechol and histamine were 5·91±0·03 and 5·02±0·06, respectively. Ranitidine (10−4 M) did not modify basal acid secretion. The H2-blocker competitively antagonized histamine induced hypersecretion producing a parallel rightward shift of the agonist concentration-response curve with no change in maximal asymptote (pKB= 5·04±0·15) [Fig. 2(A)]. Pentagastrin-induced secretory responses were greatly depressed in the presence of ranitidine while bethanechol evoked concentration-response curves were reduced in a non-significant way [Fig. 2(B), (C)]. Compound 4 h only at the highest concentration tested (10−4 M), caused a significant (P<0·01) increment in the basal acid secretion while it differently affected the secretory responses evoked by the three stimulants. In fact the compound failed in modifying pentagastrin-induced secretion [Fig. 2(B)] and apparently potentiated bethanechol concentrationresponse curve [Fig. 2(C)]. On the other hand, histamine curves were progressively shifted to the left and amplified by increasing concentrations of compound 4 h [Fig. 2(A)], histamine pD2 value changing from 5·02±0·06 in control curve to 5·77±0·11 in the presence of compound 4 h 10−4 M.
Gastric acid secretion in anaesthetized rats
Determination of gastric insoluble mucoproteins
Basal acid secretion of the anaesthetized rats was about 11·22±0·66 µEqH+ 30 min−1 and was significantly increased by equiactive doses of the three stimulants, reaching a stable maximal value about
When orally administered at the effective antiulcer dose of 30 mg kg−1 in indomethacin-treated rats, compound 4 h, unlike ranitidine which was ineffective, significantly enhanced the amount of mucus adherent
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Fig. 1. Gastric secretory responses to: (A) histamine (B) pentagastrin 40 µg kg−1 h−1; (C) 2 mg kg−1 h−1; bethanechol 0·5 mg kg−1 h−1 in the absence and presence of the compound 4 h (m) 30 mg kg−1, or ranitidine (d) 10 mg kg−1 orally administered 2 h before secretagogues (h) in anaesthetized rats. Each point represents the mean±SEM of 6–8 experiments; *P<0·05, **P<0·01 Student’s t-test.
was synthesised by Prof. D. Barlocco, Ist. Chim. Farm. e Toss., University of Milan, Italy.
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Fig. 3. Effect of compound 4 h (j) and ranitidine ( ), orally administered at 30 mg kg−1, on mucus adhering to the gastric wall in indomethacin-treated rats (40 mg kg−1 i.p.). Control group (h) treated with indomethacin received the vehicle alone . n=8 rats group−1; *P<0·05, **P<0·01 Student’s t-test.
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Fig. 2. Histamine (A), pentagastrin (B) and bethanechol (C) concentration-response curves obtained in mouse isolated stomach following pre-incubation (30 min) with compound 4 h (h, 0; j, 10−5 M; m, 10−4 M) or ranitidine (d, 10−4 M). Each point represents the mean±SEM of 6–8 experiments; *P< 0·05, **P<0·01 Student’s t-test.
to the gastric wall, the µg of Alcian blue dye bound to each stomach increasing from 227·4±19·8 for vehicletreated rats to 348·3±16·9 for indenopyridazinonetreated animals (P<0·01) (Fig. 3).
DISCUSSION The present study showed that compound 4 h, able to inhibit the gastric acid secretion in pylorus-ligated rats, exhibited contradictory effects when tested in in vivo and in vitro experimental models where the acid
hypersecretion was pharmacologically-induced by different secretagogues. In fact, in anaesthetized rats, the compound 4 h significantly inhibited bethanecholevoked acid secretion leaving substantially immodified the secretory response induced by histamine or pentagastrin. On the other hand, in mouse isolated stomachs, it failed to antagonize either bethanechol- or pentagastrin-stimulated acid production and caused an unexpected potentiation of histamine-induced response. These findings suggest that the antisecretory effect of the test compound could be attributed to an inhibitory interference with excitatory cholinergic pathways which actively contribute to gastric acid secretion only in the in vivo model. Indeed, such hypothesis is supported by the ineffectiveness of compound 4 h in antagonizing the in vitro secretory response caused with effectorial mechanisms by histamine, pentagastrin and bethanechol. Unlike compound 4 h, ranitidine exhibited a comparable activity in both experimental models. As expected, it suppressed the hypersecretory response to histamine and pentagastrin in the anaesthetized rat and in the mouse isolated stomach, while unaffecting the secretory responses to the muscarinic agonist bethanechol. The current view on processes regulating acid secretion is that gastrin can act directly on the parietal cell as well as indirectly via endogenous histamine release [10]. This latter mechanism accounts for the inhibition of pentagastrin-induced acid secretion observed with ranitidine in the present investigation. Such behaviour is consistent with previous experimental evidence provided both in anaesthetized rats [11] and mouse isolated stomachs [12].
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With respect to the in vitro assay the compound 4 h at the highest concentration tested significantly increased basal acid secretion acting with a mechanism which remains still undefined. As concerns muscarinic-induced secretory response, the bethanechol curve shift is actually apparent since pD2 values, calculated in the absence and presence of compound 4 h, were superimposable. On the other hand, compound 4 h seems to exert an effective potentiating activity on histamine evoked acid production, causing a leftward shifting of the amine curve with a significant increase in agonist potency. The lack of any facilitating action on pentagastrin response seems to indicate that compound 4 h does not interfere with endogenous histamine suggesting that large amounts of exogenous histamine are required to evidence such overstimulating effect. The mechanism underlying this response is not clear even if it is reasonable to rule out any interference from compound 4 h with the specific receptor or post-receptor events involved in histamine-oxyntic cell interaction. It is likely that compound 4 h can indirectly facilitate histamine secretory response by making more exogenous histamine available to remove inhibitory pathways in target sites other than the parietal cell (e.g. inducing a permissive vasodilation [13] or blocking via H3 receptors somatostatin release [14]). The absence of such potentiation in in vivo test could depend on the limited concentration reached by compound 4 h at gastric level so much so that it did not produce any significative modification of basal secretory activity in the experimental conditions here employed. We demonstrated that compound 4 h inhibits gastric acid secretion exclusively in bethanechol-stimulated and in pylorus-ligated rats. In a preliminary study (unpublished data) we observed that at the antisecretory dosage of 30 mg kg−1 os, compound 4 h failed to affect mice spontaneous motor activity. These findings seem to strengthen the hypothesis that such compound exerts its inhibitory action by modulating specifically the cholinergic transmission rather than generally suppressing the central nervous activity. In a previous work we provided evidence that compound 4 h possesses also antiulcer activity in ethanol and indomethacin-induced experimental gastric lesions [5]. This pilot study seems to indicate that compound 4 h can prevent ulcer formation mainly strengthening the pre-epithelial barrier, which is constituted by the mucus covering the surface of the gastric mucosa, rather than reducing gastric acid secretion. In fact, our data clearly demonstrate that the treatment with the compound 4 h, at the dose (30 mg kg−1 os) giving 84% protection against indomethacin-induced gastric lesions [5], significantly enhanced the amount of mucoproteins adherent to the mucosal epithelium of rat stomachs.
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We reported that the same dose was almost completely effective (97% protection) also in preventing the gastric haemorrhagic damage caused by ethanol [5]. It has been pointed out that, in the pathogenesis of ethanol-induced gastric injury, the initial dislodgement of the superficial mucosal cells and the subsequent perturbation of submucosal microcirculation plays a pivotal role while the involvement of acid secretion has been ruled out [15]. Thereby, an increased production/preservation of mucus (or an enhancement in the defensive factors) seems to be one of the possible mechanisms underlying the gastroprotective actions displayed by compound 4 h. This mucus-related mechanism of action does not seem to be shared by ranitidine. Indeed, this H 2-blocker protected gastric mucosa from indomethacin-caused lesions without affecting the gastric mucoprotein level and failed to prevent ethanol-caused insult [5]. Gastric ulcers induced by indomethacin treatment are usually ascribed to various factors such as the deficiency of endogenous prostaglandins, the occurrence of gastric abnormal contractions and the gastric mucosal blood flow stasis [16]. The antiulcer activity displayed by the H2-antagonist in indomethacin-treated rats, has been widely documented in previous studies [17, 18] and it has been attributed mainly to the blockade of acid secretion and only partially to acid-independent actions [19, 20]. On the basis of the data here reported, we can conclude that the new indenopyridazinone derivative 4 h exerts antiulcer activity enhancing the gastric mucosal integrity and simultaneously it is able to control the gastric acid hypersecretion evoked by nervous cholinergic pulses. Hence this compound, which acts with mechanisms clearly different from ranitidine, suggests new possible approaches for the treatment of peptic ulcers resistant to H2-blockers.
ACKNOWLEDGEMENTS Supported by MURST 40%. The authors would like to thank Dr Giuseppe Domenichini for technical assistance.
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