The pharmacological properties of the novel selective 5-HT3 receptor antagonist, alosetron, and its effects on normal and perturbed small intestinal transit in the fasted rat.

The purpose of this study was to investigate the pharmacological properties of the novel, selective 5-HT3 receptor antagonist, alosetron, and its effects on transit time in both the normal and perturbed small intestine of the rat. Alosetron concentration-dependently inhibited radioligand binding in membranes containing rat and human 5-HT3 receptors with estimated pKi values of 9.8 (n = 3) and 9.4 (n = 6), respectively. In selectivity studies alosetron had little or no significant affinity for any of the many other receptors and ion channels studied. Alosetron potently antagonized the depolarization produced by 5-HT in the rat vagus nerve (estimated pKB value of 9.8, n = 25). In anaesthetized rats, i. v. administration of alosetron inhibited 2-methyl-5-HT induced bradycardia (Bezold Jarisch index) at 1 and 3 microg kg-1, with an agonist dose ratio of approximately 3.0 at 1.0 microg kg-1, = 3-5). Alosetron administered via the duodenum also inhibited this reflex, with duration of action that was significantly longer than that seen with ondansetron (120-60 min, respectively, n = 6). Alosetron had no significant effect on normal small intestinal propulsion in the rat, but fully reversed the increase in intestinal propulsion (96%, n = 3) produced by egg albumin challenge. Alosetron is a highly selective 5-HT3 antagonist which normalizes perturbed small intestinal propulsion. Previous clinical data in IBS patients together with the transit data provide a good rationale for further studies with alosetron in IBS patients.

The role of acid in the pathogenesis of indomethacin-induced gastric antral ulcers in the rat.

BACKGROUND: The role of acid in the pathogenesis of indomethacin-induced ulcers of the rat gastric antrum was studied by comparing the effects of pretreating animals with both long-acting (loxtidine, AH22216) and short-acting (ranitidine and cimetidine) inhibitors of acid secretion. RESULTS: Ranitidine and cimetidine were much weaker at inhibiting antral damage when compared to their reported potencies as antisecretory agents. In marked contrast, loxtidine and AH22216 inhibited indomethacin-induced antral ulcers at doses similar to their reported potencies as inhibitors of acid secretion. Histological analysis at doses causing near maximal inhibition of macroscopic damage revealed an almost complete absence of ulcers but a large and significant increase in mucosal damage due to superficial erosions. Hourly dosing with hydrochloric acid reversed the protective effect of ranitidine, cimetidine and loxtidine on macroscopic damage and, histologically, this was associated with the widespread appearance of antral ulcers and a reduction in the proportion of mucosal damage caused by superficial erosions. CONCLUSIONS: The results of this study suggest that the pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced antral ulcers involves at least two stages: (1) an initial acid-independent formation of mucosal erosions followed by (2) an acid-dependent conversion of erosions to frank ulcers. Clinically, drugs that suppress acid completely for long periods may be very effective in preventing NSAID-induced gastric antral ulcers.

5-HT4 receptors are not involved in the control of small intestinal transit in the fasted conscious rat.

Cisapride and metoclopramide are used clinically in the treatment of gastro-oesophageal reflux disease and also in a variety of motility disorders of the gastrointestinal tract. Their prokinetic effect is thought to be due to the augmentation of acetylcholine release from the myenteric plexus, an effect likely to be mediated through the stimulation of 5-HT4 receptors. The role of 5-HT4 receptors in the control of intestinal motility in man and animals is not clear, therefore we have investigated their role in the control of small intestinal transit in the rat. Radioactive microspheres were administered into the proximal duodenum of fasted conscious rats through an indwelling cannula. The extent of small intestinal transit was examined by determining the distribution of the microspheres within the intestine. Following i.p. injection small intestinal transit was inhibited (78%) by atropine (3 mg/kg), suggesting the presence of a basal cholinergic influence. Furthermore, in the presence of p-amino clonidine intestinal transit was stimulated (126%) by bethanechol (3 mg/kg). The 5-HT4 receptor agonists cisapride (1.0 mg/kg) and zacopride (1.0 mg/kg) failed to increase small intestinal transit. The 5-HT4 receptor selective antagonist GR125487 (1 mg/kg) was also without effect. These data suggest that 5-HT4 receptors are not involved in the control of small intestinal transit in the fasted conscious rat.

Comparison of the profiles of agonists as stimulants of the beta 3-adrenoceptor in vitro with their gastroprotective effects in the conscious rat.

1. This paper compares the activity of a range of agonists as stimulants of the beta 3-adrenoceptor in rat isolated oesophagus with their ability to afford protection against indomethacin-induced gastric damage in the conscious rat. 2. The beta 3-adrenoceptor agonists, CL 316243 and BRL 37344, the non-selective beta-adrenoceptor agonist, isoprenaline and the selective beta 2-adrenoceptor agonist, salmeterol, all evoked concentration-dependent relaxation of precontracted muscularis mucosa from rat oesophagus. The rank order of agonist potency was BRL 37344 > CL 316243 > isoprenaline >> salmeterol. The selective beta 1-adrenoceptor agonist, denopamine, did not relax the preparation. 3. The relaxant responses to all agonists were resistant to blockade by atenolol (10 microM), and ICI 118551 (1 microM) thus suggesting that they were not mediated by either beta 1- or beta 2-adrenoceptor stimulation. In contrast, cyanopindolol and propranolol did inhibit responses to BRL 37344, CL 316243 and isoprenaline, giving pA2 values or pKB estimates which were consistent with an interaction at beta 3-adrenoceptors (i.e. approximately 8.0 and 6.5 respectively). However, responses to salmeterol were resistant to blockade by all the antagonists tested, which suggests that the high (> 1 microM) concentrations of salmeterol used exerted non-specific relaxant effects. 4. The agonist effects of CL 316243 and BRL 37344 on beta 1- and beta 2-adrenoceptors were assessed on guinea-pig right atrium and precontracted trachea respectively. Both agonists had minimal activity as stimulants of heart rate, but did relax trachea, being 380 (CL 316243) and 21 (BRL 37344) fold less potent than isoprenaline. 5. CL 316243 and BRL 37344 were potent inhibitors of indomethacin-induced gastric antral ulceration in the conscious rat (ED50 values = 0.24 and 0.09 mumol kg-1, p.o.) Salmeterol was approximately 100 times less potent than BRL 37344 as a gastroprotective agent and denopamine was without effect. 6. The gastroprotective effects of CL 316243 and BRL 37344 were resistant to blockade by ICI 118551 (10 mg kg-1, p.o.) and propranolol (10 mg kg-1, p.o.). In contrast, both antagonists caused dose-related inhibition of the protective action of salmeterol (10 mg kg-1, p.o.). Cyanopindolol was not assessed as an antagonist in vivo because preliminary experiments revealed that it exacerbated indomethacin-induced gastric damage in its own right. 7. In conclusion, the beta 3-adrenoceptor agonists CL 316243 and BRL 37344 were potent inhibitors of indomethacin-induced gastric antral ulceration in the rat. These data suggest that an agonist which is potent and selective for the human beta 3-adrenoceptor may confer mucosal protection in man.

Non-steroidal anti-inflammatory drug-induced gastric damage in experimental animals: underlying pathological mechanisms.

A major impetus to experimental studies examining the pathogenesis of NSAID-induced gastric damage is the hope that key mechanisms can be identified that may lead to the design of "safer" NSAIDs or to the development of novel cytoprotectives to co-administer with current NSAIDs. Virtually every hypothesis proposed to explain the pathogenesis of NSAID-induced gastric damage has arisen from studies examining fundic lesions. Our results suggest that not only is the pathogenesis of gastric damage in the fundus and antrum of the rat potentially very different but that it is NSAID-induced damage to the rat gastric antrum (and not the fundus) that more closely resembles that of humans. Thus, hypotheses constructed from experimental studies examining fundic damage may not be predictive of the clinical setting. We would suggest, therefore, that future studies should concentrate on studying the pathogenesis of antral ulceration induced by NSAIDs. In particular, we believe that future research should assess whether NSAIDs do indeed reduce blood flow in the gastric antrum and define the mechanism(s) involved. Identification of these processes should significantly advance our understanding of antral ulceration and may suggest novel approaches in the design of cytoprotective agents. Recent work has established that two distinct forms of the enzyme cyclooxygenase (COX) can catalyse the metabolism of arachidonic acid and initiate prostaglandin synthesis. It is hypothesised (De Witt et al., 1993) that the analgesic/anti-inflammatory effect of current NSAIDs is achieved through inhibition of COX 2, whereas their side effects (such as antral ulceration) result as a consequence of inhibition of gastric COX 1. The recently described selective inhibitor of COX 2, NS398, has been shown to be analgesic and anti-inflammatory without causing gastric ulceration (Futaki et al., 1993; Masferrer et al., 1994). If key mechanisms (such as alterations in blood flow) of NSAID-induced antral damage can be identified, then it would be hypothesised that selective inhibitors of COX 1 would be ulcerogenic and reduce antral blood flow, whereas inhibitors of COX 2 would not share either of these properties. If this hypothesis can be substantiated, then inhibitors of COX 2 may be the next generation of "gastric safe" NSAIDs.

Neutrophil infiltration does not contribute to the ulcerogenic effects of indomethacin in the rat gastric antrum.

We have previously suggested (Trevethick et al., Gut 34, 156-160) that indomethacin-induced ulceration of the rat gastric antrum may be a neutrophil-dependent process. Accordingly, in this study we have used an anti-neutrophil serum (ANS) to investigate the effects of neutrophil depletion on this pathology. In animals pretreated with the ANS to induce a nearly total neutropaenia, indomethacin-induced increases in blood neutrophilia and cell infiltration into the gastric antrum (assessed as LTB4 release ex vivo) were eliminated. In marked contrast, however, ANS pretreatment affected neither the area of mucosa damaged nor the microscopic characteristics or distribution of the lesions. These results suggest that, in contrast to the published reports examining indomethacin-induced ulceration of the gastric fundus, neutrophil infiltration is not involved in the pathogenesis of indomethacin-induced ulceration of the rat gastric antrum.

Leukotrienes do not contribute to the pathogenesis of indomethacin-induced ulceration of the gastric antrum in the re-fed rat.

The potential involvement of leukotrienes in the pathogenesis of indomethacin-induced ulceration of the rat gastric antrum has been studied. Pretreatment with the leukotriene biosynthesis inhibitor, MK886 (30 mg/kg p.o.), inhibited the increases in blood and antral leukotriene B4 release ex vivo associated with the evolution of antral ulceration. Despite this, however, there was no significant reduction in either the area of antral ulceration, or in the associated blood neutrophilia and neutrophil infiltration into the gastric antrum. Similarly, pretreatment with the leukotriene B4 antagonist, SC41930 (50 mg/kg p.o.) or the peptidyl leukotriene antagonist ICI198,615 (50 mg/kg p.o.) did not inhibit the area of antral ulceration induced by indomethacin. Thus, in contrast to published reports studying fundic ulceration, our results suggest that leukotrienes do not play a major role either in the pathogenesis of indomethacin-induced ulceration of the rat gastric antrum or neutrophil infiltration into the damaged antrum.

Gastric anti-secretory, mucosal protective, anti-pepsin and anti-Helicobacter properties of ranitidine bismuth citrate.

Ranitidine bismuth citrate is a novel compound formed from ranitidine and a bismuth citrate complex. In conscious dogs, ranitidine bismuth citrate had similar activity to ranitidine hydrochloride as an inhibitor of histamine-induced gastric acid secretion when oral doses containing equivalent amounts of ranitidine base (0.1 or 0.3 mg/kg) were compared. In the rat, ranitidine bismuth citrate (3-30 mg/kg p.o.) prevented gastric mucosal damage induced by ethanol (fundic damage) and indomethacin (antral damage). Ranitidine hydrochloride and tripotassium dicitrato bismuthate were also effective against indomethacin-induced damage, but were both significantly less potent than ranitidine bismuth citrate in this model. Ranitidine hydrochloride was inactive against ethanol-induced damage. In vitro, ranitidine bismuth citrate (1 mmol/L) inhibited human pepsin isoenzymes 1, 2, 3 and 5. Pepsin 1 was inhibited to a similar extent by ranitidine bismuth citrate, bismuth citrate and tripotassium dicitrato bismuthate at concentrations equivalent to 1 mmol/L bismuth, but ranitidine (1 mmol/L) was inactive. Ranitidine bismuth citrate was more potent than tripotassium dicitrato bismuthate as an inhibitor of pepsins 2, 3 and 5. Ranitidine bismuth citrate inhibited both Helicobacter pylori (effective concentration 4-32 micrograms bismuth/ml) and H. mustelae (1-4 micrograms bismuth/ml); similar results were obtained with tripotassium dicitrato bismuthate. Bismuth citrate was slightly less effective, and ranitidine hydrochloride was inactive (> 125 micrograms/ml). In ferrets naturally colonized with H. mustelae, oral treatment with ranitidine bismuth citrate, 12 or 24 mg/kg twice daily for 4 weeks, caused a dose related clearance of H. mustelae. Qualitatively similar results were obtained in a small study with tripotassium dicitrato bismuthate and bismuth citrate.

Do infiltrating neutrophils contribute to the pathogenesis of indomethacin induced ulceration of the rat gastric antrum?

The potential involvement of neutrophils in the pathogenesis of indomethacin induced ulceration of the gastric antrum in the re-fed rat was studied. Indomethacin was associated with a time dependent increase in the extent and severity of ulceration, blood neutrophilia, neutrophil infiltration into the gastric antrum, and calcium ionophore induced immunoreactive leukotriene B4 (LTB4) release from the antrum ex vivo. Neutrophil infiltration into the antrum was detectable 1 hour after dosing with indomethacin, at which time damage was apparent microscopically but not macroscopically. Thus, cell infiltration may contribute to the development, if not the initiation, of ulceration. Consistent with this suggestion, oral dexamethasone (5 mg/kg) significantly attenuated indomethacin induced ulceration, the associated neutrophil infiltration, and calcium ionophore induced immunoreactive leukotriene B4 release from the gastric antrum and whole blood ex vivo, although the blood neutrophilia was unaffected. These results suggest that indomethacin induced ulceration of the rat gastric antrum may have a dependence on neutrophil infiltration for its pathogenesis.

Early histological features of small intestinal injury induced by indomethacin.

The early histological features of indomethacin-induced jejunal injury in the rat are described in tissues preserved by perfusion-fixation with 10% formol-saline. After an oral dose of indomethacin (15 mg/kg, known to cause severe multifocal ulceration of the rat jejunum), groups of rats were anaesthetized with subsequent perfusion-fixation of the gastrointestinal tract at 1, 2, 3, 6 and 48 h after dosing. Using routine light microscopic techniques, we have observed a sequence of four distinct stages, in time, of small intestinal injury. The earliest histological features were shortening of the villi, epithelial stratification, basal lamina degeneration, eosinophil degranulation and infiltration of the epithelium prior to infiltration of the mucosa by neutrophils. We consider that these earliest changes, seen at 1, 2 and 3 h, represent a distinct histological entity termed Type 1 change or villous 'tufting'. Type 2 change includes all of the features of Type 1 change plus the subsequent infiltration of the mucosa by neutrophils at 2, 3 and 6 h. Type 3 change includes necrosis of the upper-third of the villi and was mainly seen at 3 and 6 h. Type 4 change describes extreme injury to more than one-third of the mucosa with severe, acute inflammation and perforation of the bowel wall by 48 h. Although a small number of neutrophils had appeared to infiltrate the mucosa as early as 2 h after dosing, they were only significantly increased at 3, 6 and 48 h. Possible pathogenic mechanisms involved in shortening of villi as a result of smooth muscle contraction and the role of mucosal eosinophils in NSAID-induced jejunal injury in the rat are discussed.

The effects of the stable thromboxane A2-mimetic, U46619, on gastric mucosal damage and gastric non-parietal secretion in the rat.

The effects of the thromboxane A2-mimetic, U46619, and the thromboxane receptor antagonist, AH23848, on ethanol-induced gastric mucosal damage and gastric non-parietal secretion have been examined in the rat. Oral dosing with U46619 or AH23848 produced a dose-related inhibition of ethanol-induced gastric mucosal damage in the conscious rat, and these effects were partially blocked by indomethacin treatment. Intragastric application of U46619 or AH23848 to the stomach of the anaesthetized rat stimulated the gastric secretion of a juice which consisted principally of Na+ and Cl- ions. These secretagogue effects of both compounds were blocked by indomethacin treatment. These results show that U46619 and AH23848 induce secretory and protective effects in the stomach of the rat, although these responses probably do not involve thromboxane receptors and are mediated, at least in part, by endogenous prostaglandins. The results are discussed in relation to the role of endogenous thromboxane A2 in gastric mucosal protection, and of the possible protective function of non-parietal secretion in the stomach.