Introduction

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The prokinetic effect of 5-hydroxytryptamine-4 (5-HT₄) receptor agonists is well documented in a number of experimental models (for a review, see De Ponti and Malagelada, 1998): in vivo, they promote gastric emptying and reduce colonic transit time. In vitro studies have proposed enhanced acetylcholine release via activation of neuronal 5-HT₄ receptors as the main mechanism responsible for the motor-stimulating effect. However, pharmacological evidence for a functional role of 5-HT₄ receptors in the modulation of small bowel motility in vivo is at present still fragmentary, because most of the available data were obtained in vitro and some early in vivo studies with this class of prokinetics did not verify the effect of selective 5-HT₄ receptor antagonists (Scheman and Ehrlein, 1986; Summers and Flatt, 1988; Gullikson et al., 1993; Orihata and Sarna, 1994; Edelbroek et al., 1995), some of which were not available at that time or unsuitable for in vivo use because of their short half-life (Hegde and Eglen, 1996).

One of the most widely studied prokinetics, cisapride, is known to stimulate propulsive motility in the canine small bowel (Scheman and Ehrlein, 1986; Summers and Flatt, 1988) and to increase the number of propagated spike bursts in the human small bowel (Coremans et al., 1988). Cisapride, however, is a mixed 5-HT₄ receptor partial agonist and a 5-HT₃ receptor antagonist, a property that may be responsible for some differences observed in its prokinetic activity at different gut levels (for a review, see De Ponti and Malagelada, 1998).

Cisapride is generally safe and well tolerated, according to a large postmarketing surveillance study (Wager et al., 1997). However, pharmacological evidence for the presence of 5-HT₄ receptors in other organs, such as urinary bladder (Tonini et al., 1994), atrium (Kaumann et al., 1996), and adrenocortical tissue (Lefebvre et al., 1998) suggests some caution at least in some patient populations. In particular, 5-HT₄ receptors have been detected in the porcine and human atrium, where they mediate tachycardia and are hypothesized to trigger arrhythmias (Kaumann and Sanders, 1994; Hegde and Eglen, 1996; Kaumann et al., 1996). Cardiac adverse effects indeed have been reported with cisapride (Wysowski and Bacsanyi, 1996), a compound also possessing class III antiarrhythmic properties (Puisieux et al., 1996; Mohammad et al., 1997; Rampe et al., 1997; Drolet et al., 1998). These can lead to a prolongation of the QT interval, especially when cisapride is used at high doses or in case of pharmacokinetic interactions resulting in higher than expected plasma levels (Bedford and Rowbotham, 1996).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Molecular structures of ML 10302, SR 59768, and cisapride.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Dose-response relationship for stimulation of spike activity (expressed as  spikes/15 min) in the canine duodenum and jejunum by SR 59768 (), ML 10302 (), and cisapride (). Values are means ± S.E. (numbers indicate the value of n for each dose).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Computer plot showing the stimulatory effect of ML10302 (100 nmol/kg i.v.) (administered at arrow) on small bowel spike activity in one representative fasting dog (E1-E2, duodenum; E5-E6, jejunum). On the y-axis: number of spikes/30 s. Note simultaneous stimulation of spike activity at all recording sites.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Computer plot of small bowel spike activity recorded before and after GR125487 (20 nmol/kg i.v.) (administered at arrow) in the same dog as in Fig. 3 (E1-E2, duodenum; E5-E6, jejunum). On the y-axis: number of spikes/30 s. Note lack of effect of GR125487.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Computer plot of small bowel spike activity recorded in the same dog as in Figs. 3 and 4. Previous administration of GR125487 (20 nmol/kg i.v., left arrow) suppresses the stimulatory effect of ML10302 (100 nmol/kg i.v., right arrow) (E1-E2, duodenum; E5-E6, jejunum). On the y-axis: number of spikes/30 s.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Antagonism of the stimulatory effect of SR59768 (100 nmol/kg) and ML10302 (100 nmol/kg) (expressed as  spikes/15 min) by atropine (300 nmol/kg) or GR125487 (20 nmol/kg) in the canine duodenum and jejunum. Solid bars, agonist alone; open bars, agonist preceded by atropine; hatched bars, agonist preceded by GR125487. Values are means ± S.E. (n = 4); *p < .05; **p < .01 versus agonist alone.

The QT interval is an electrocardiographic measurement of the period between the beginning of ventricular activation and the completion of ventricular recovery. Drugs possessing class III antiarrhythmic properties delay ventricular repolarization, thus leading to QT-interval prolongation. Because the heart rate is a major determinant of the duration of the QT interval, measurements of this parameter usually are rate-corrected (rate-corrected QT or QT_c). From a clinical point of view, a prolonged QT interval is associated with the possible occurrence of torsades de pointes, a polymorphous ventricular arrhythmia that may cause syncope and degenerate into ventricular fibrillation (Wysowski and Bacsanyi, 1996).

The specific aims of the present study were 1) to compare the prokinetic action of two selective 5HT₄ receptor agonists with low affinity for 5-HT₃ receptors ML10302 and SR59768 (Langlois et al., 1994; Yang et al., 1997) in the canine duodenum and jejunum in vivo, using cisapride as a reference compound; 2) to determine the contribution of 5-HT₄ and muscarinic receptors in mediating these effects, resorting to GR125487, a selective 5-HT₄ receptor antagonist with a long duration of action in vivo (Gale et al., 1994a,b); and 3) to study the effects of the three 5-HT₄ receptor agonists on heart rate and QT interval, to assess whether or not the cardiac action observed with cisapride is a class effect, shared by other 5-HT₄ receptor agonists.

	Materials and Methods

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Experimental Model. Experiments were carried out on five Beagle dogs (all females, 8-14 kg b.wt.), purchased from an authorized local breeder, and housed in single, air-conditioned boxes. All experiments were performed according to European Union Directive 86/609 on the care and use of experimental animals. The animals were fed with dog food in pellets (Mil; Stefano Morini, S. Polo d'Enza, Italy). Water was available ad libitum.

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Experimental Procedure. Experiments were performed on conscious dogs after allowing at least 15 days for recovery after surgery. Before each experimental session, the dogs were fasted for at least 18 h, while water was available ad libitum.

Pharmacological Agents. The following drugs were used (Fig. 1): cisapride, 2-piperidinoethyl 4-amino-5-chloro-2-methoxybenzoate (ML10302) and 2-[(3S)-3-hydroxypiperidino]ethyl 4-amino-5-chloro-2-methoxybenzoate (SR59768) (all synthesized at Sanofi Midy Research Center, Milan, Italy); atropine sulfate (Sigma Chemical Co., St. Louis, MO); and 1-[2-[(methylsulphonyl)amino]ethyl]-4-piperidinyl-methyl 5-fluoro-2-methoxy-1H-indole-3-carboxylate (GR125487, sulfamate salt; Glaxo, Stevenage, Herthfordshire, England). Atropine sulfate and GR125487 were dissolved in 154 mM NaCl. Stock solutions of cisapride were prepared in acetic acid; for SR59768 and ML10302, 1 N HCl was used. The solvents previously had been found to have no effect on the parameters to be studied at their final concentration.

Experimental Design. Three sets of experiments were carried out, with each dog serving as its own control. In each experimental session, only one dose of 5-HT₄ receptor agonist was tested, allowing an interval of at least 2 days between two experimental sessions with the same dog. In the first set of experiments, dose-response studies were performed for each agonist, testing the following doses by the i.v. route: 3, 10, 30, 100, and 300 nmol/kg for SR59768 and ML10302; and 30, 100, 300, 1000, and 3000 nmol/kg for cisapride.

Data Analysis. Intestinal spike activity was evaluated both visually and with the aid of a previously validated computer system, which allows quantitative analysis of intestinal spike activity (De Ponti et al., 1993). Briefly, the number of spikes per unit of time (30 s or 15 min in the present experiments) was calculated automatically by original software that identifies spikes by means of a discriminant function. The variation in spike activity induced by the agonists was assessed by calculating the difference () between the numbers of spikes recorded during 15-min control periods and 15-min periods immediately after drug administration (this time was sufficient to reach the peak effect; see Results). As controls, we used 15-min drug-free periods taken at the same point of the MMC cycle in the same dog (i.e., the control period started 15 min after the end of an activity front; this is usually a quiescent period, termed phase I). The different phases of the MMC were defined according to criteria previously established in our laboratory (De Ponti et al., 1989). Briefly, phase III (activity front) was defined as the period of maximal activity (more than 80% of slow waves with superimposed spikes) lasting more than 3 min and migrating over at least three electrode sites. The MMC period was defined as the time interval between the end of two consecutive activity fronts at the same electrode.

	Results

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Intestinal Effects of 5-HT₄ Receptor Agonists. Both ML10302 and SR59768 dose-dependently stimulated spike activity in the duodenum and the jejunum (Fig. 2). The dose of 100 nmol/kg was considered maximal for both agonists and was used after administration of the antagonists in the third set of experiments. ED₅₀ values are shown in Table 1. The stimulatory effect of both compounds started immediately after their administration, appeared simultaneously at all recording sites, and lasted at least 30 min after the dose of 100 nmol/kg (Fig. 3).

Effect of 5-HT₄ Receptor Agonists on Heart Rate and QT_c. None of the doses of ML10302 or SR59768 had any effect on heart rate or QT_c (Tables 4 and 5). Likewise, cisapride had no significant effect on heart rate or QT_c up to the dose of 1000 nmol/kg, whereas the highest dose (3000 nmol/kg) induced statistically significant tachycardia and significantly lengthened the QT_c interval (Tables 4 and 5). This effect, however, had a short duration (~10 min).

Intestinal Effects of Antagonists Per Se. Atropine (300 nmol/kg i.v.) or GR125487 (20 nmol/kg i.v.), administered immediately after the end of phase III (i.e., during the quiescent period), did not affect the number of spikes recorded in the subsequent 30-min period with respect to control (drug-free) periods (Table 6 and Fig. 4). Atropine delayed the appearance of the next activity front (see also De Ponti et al., 1993), whereas GR125487 had no effect on MMC cycling (Fig. 4).

Intestinal Effects of Combined Agonists and Antagonists. The stimulatory effect of maximal doses (100 nmol/kg i.v.) of ML10302 or SR59768 was reduced significantly both in duodenum and jejunum by pretreatment with atropine (300 nmol/kg i.v.) or GR125487 (20 nmol/kg i.v.) (Figs. 5 and 6).

	Discussion

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The results of the present study can be summarized as follows: 1) ML10302 and SR59768 are equipotent intestinal prokinetics acting via 5-HT₄ and muscarinic receptors in the dog in vivo; 2) the prokinetic effect is similar at the two intestinal levels studied (duodenum and jejunum); and 3) maximal intestinal prokinesia by ML10302 and SR59768 is achieved with no significant effects on heart rate or QT_c.

As regards the dose-response curves of ML10302 and SR59768, they tended to be bell-shaped, especially in the duodenum. This may depend on the fact that 5-HT₄ receptors can act at different sites to modulate gastrointestinal motility, and the net effect may depend on a balance between stimulatory and inhibitory effects (De Ponti and Malagelada, 1998). Apart from the well known facilitatory effects of 5-HT₄ receptor stimulation on acetylcholine release (and this study confirmed the involvement of muscarinic pathways in mediating the in vivo effect of ML10302 and SR59768), 5-HT₄ receptors also have been hypothesized, at least in some models, on inhibitory neural pathways (Graf and Sarna, 1996; De Ponti and Malagelada, 1998) and, perhaps more importantly, on smooth muscle cells, where they mediate relaxation via an increase in cyclic AMP (Kuemmerle et al., 1995).

Although a formal evaluation of the potency of ML10302 and SR59768 with respect to that of cisapride cannot be made, because no ED₅₀ was calculated for cisapride, it should be noted that dose-response curves for cisapride were definitely to the right and that the MI obtained with 100 nmol/kg ML10302 or SR59768 was similar to that obtained with 1000 nmol/kg cisapride. Interestingly, the dose-response curve for cisapride in the duodenum appeared biphasic, with a steep slope in the 1000 to 3000 dose range. This may be due to the recruitment of additional mechanisms of action, unrelated to 5-HT₄ receptor activation, such as influx of extracellular calcium (Hasler and Washabau, 1997), interaction with muscarinic receptors (Hasler et al., 1991), or blockade of K⁺ channels (Mohammad et al., 1997; see below). Indeed, the stimulatory effect of cisapride (1000 nmol/kg i.v.) on small bowel spike activity is only partially antagonized by GR125487 (unpublished observation).

The lack of effect of GR125487 on fasting small bowel motility deserves some comment, because it suggests that 5-HT₄ receptors are not involved in the control of the MMC pattern, at least during the quiescent phase. This is in line with similar findings by Haga et al. (1997), who gave GR125487 during phase I to conscious dogs, and by Clayton and Gale (1996), who found no effect of GR125487 on intestinal transit in conscious rats. Graf and Sarna (1996) reported a reduced amplitude and duration of phase III activity after 5-HT₄ receptor blockade in conscious dogs; hence, they hypothesized the existence of 5-HT₄ receptors on inhibitory neurons. If we accept a pharmacodynamic half-life of approximately 145 min for GR125487 (Gale et al., 1994a), i.e., much longer than the MMC cycle period in our dogs, our results do not support an involvement of 5-HT₄ receptors during phase III either.

An interesting aspect of this study is that, unlike cisapride, ML10302 and SR59768 show a clear dissociation between intestinal prokinetic and cardiac effects: doses inducing maximal intestinal stimulation had no effect on QT_c or heart rate, a finding that raises questions on the functional relevance of cardiac 5-HT₄ receptors. In human atrium, it has been suggested that the small 5-HT₄ receptor population may, in part, explain why the positive inotropic effects of 5-hydroxytryptamine are smaller than those of catecholamines (Kaumann et al., 1996).

While this study was in progress, several reports (Puisieux et al., 1996; Carlsson et al., 1997; Kii and Ito, 1997; Mohammad et al., 1997; Rampe et al., 1997; Drolet et al., 1998) have been published on the class III antiarrhythmic properties of cisapride at therapeutic concentrations (approximately 0.1 µM). If we accept a value of 1 liter/kg for the volume of distribution of cisapride in the dog (Michiels et al., 1987), then our theoretical total plasma concentration after administration of the highest dose would be 3 µM. With a protein binding of 95% (Michiels et al., 1987), the free cisapride plasma concentration would be 150 nM, i.e., well above the concentration displaying class III antiarrhythmic properties (Puisieux et al., 1996; Carlsson et al., 1997; Mohammad et al., 1997; Rampe et al., 1997; Drolet et al., 1998). The K⁺-channel-blocking properties of cisapride thus explain its effects on the QT_c. On the other hand, the structural requirements for class III antiarrhythmic properties seem to be absent in the two esters of 4-amino-5-chloro-2-methoxybenzoic acid, as already reported for mosapride (Carlsson et al., 1997).

The other effect of the highest dose of cisapride was tachycardia, an effect not observed even with supramaximal prokinetic doses of ML10302 or SR59768 (300 nmol/kg). A possible explanation for the lack of chronotropic effect of ML10302 is a difference between cardiac and neuronal 5-HT₄ receptors, as suggested by a recent study carried out in human atrium (Blondel et al., 1997), in which ML10302 displayed poor agonistic effect. Further studies are needed to clarify this issue.

An alternative explanation for the lack of cardiac effect of the two esters may be that 5-HT₄ receptors are not involved in mediating tachycardia in dogs, because 5-HT-induced tachycardia is known to be species-dependent (Saxena and Villalón, 1991). Indeed, 5-HT₄ receptor-mediated tachycardia has been characterized in the pig (in vivo and in vitro) as well as in human tissues, whereas the dog has received relatively little attention. Thus, tachycardia induced by high-dose cisapride tentatively could be ascribed to hypotension (Onat et al., 1994).

As regards the choice of the experimental model, we were concerned that our canine model would not be adequate to discriminate between intestinal and cardiac effects and considered the pig as an alternative. However, the porcine small bowel does not seem to respond adequately to 5-HT₄ receptor agonists (Wechsung and Houvenaghel, 1998), which, on the contrary, induce similar motor patterns in dogs and humans (Schemann and Ehrlein, 1986; Coremans et al., 1988). In addition, the dog is a suitable model with which to study QT_c variations (which depend on K⁺-channel blockade), and we performed ancillary (unpublished) experiments to compare the cardiac effects of 300 nmol/kg i.v. ML10302 and 3000 nmol/kg i.v. cisapride in three pigs. Again, cisapride prolonged the QT_c (QT_c = 62 ± 9 ms · s^1/2; p < .05) and induced tachycardia (heart rate = 45 ± 10 beats/min; p < .05), whereas ML10302 had no effect on QT_c (QT_c = 12 ± 19 ms · s^1/2) or heart rate (heart rate = 5 ± 3 beats/min).

In conclusion, this study has shown that ML10302 and SR59768 are equipotent prokinetics in the canine duodenum and jejunum in vivo. Their intestinal effects are mediated by the activation of 5-HT₄ receptors and muscarinic pathways and are not accompanied by effects on QT_c or heart rate.