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The effect of the selective 5-HT3 receptor agonist on ferret gut motility
Life Sciences 71 (2002) 1313 – 1319 www.elsevier.com/locate/lifescie
The effect of the selective 5-HT3 receptor agonist on ferret gut motility Yukinori Nagakura *, Tetsuo Kiso, Keiji Miyata, Hiroyuki Ito, Kiyoshi Iwaoka, Tokio Yamaguchi Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan Received 9 October 2001; accepted 7 March 2002
Abstract The effect of the selective 5-hydroxytryptamine (5-HT)3 receptor agonist YM-31636 (2-(1H-imidazol-4ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate) on gut motility of fed ferrets was investigated. YM-31636 (0.1 mg/kg p.o.) induced a giant migrating contraction (GMC)-like, high-amplitude, ungrouped colonic contraction although it did not change the basal colonic motility pattern. This GMC-like contraction was always accompanied by defecation. Both GMC-like contraction and defecation were inhibited with the selective 5-HT3 receptor antagonist ramosetron. YM-31636 affected gastric, duodenal and ileal motility pattern only slightly. These results suggest that 5-HT3 receptor agonists such as YM-31636 are useful in treating constipation since they facilitate GMC-like contractions and defecation without undesired changes in gut motility pattern. D 2002 Elsevier Science Inc. All rights reserved. Keywords: 5-HT3 receptor agonist; YM-31636; Gut motility; Giant migrating contraction; Constipation
Introduction The 5-HT3 receptor is deeply involved in the regulation of colonic motor activity in animals and humans. Recent reports indicate that 5-HT3 receptor agonists stimulate colonic propulsive activity [1,2] although 5-HT3 receptor antagonists inhibit it [3,4]. YM-31636 (2-(1H-imidazol-4-ylmethyl)-
*
Corresponding author. Tel.: +81-298-52-5111; fax: +81-298-52-2965. E-mail address: [email protected] (Y. Nagakura). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 8 4 1 - 6
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8H-indeno[1,2-d]thiazole monofumarate) is a newly synthesized, potent and selective 5-HT3 receptor agonist. This compound has almost full agonistic activity in inducing contraction (EC50: 0.12 AM) and partial agonistic activity in increasing short-circuit current (EC50: 0.16 AM) in isolated guinea-pig distal colon. Only ramosetron (a selective 5-HT3 receptor antagonist) but not methysergide (a 5-HT1 and 5-HT2 receptor antagonist) and SB204070 (a selective 5-HT4 receptor antagonist) inhibited its contractile effect in the guinea-pig colon even though the 5-HT-induced contraction in this preparation is mediated through 5-HT3, 5-HT4 and 5-HT1-like receptors, suggesting that the effect of YM-31636 on the colon is mediated exclusively through the 5-HT3 receptor. This is also supported by the fact that YM-31636 has negligible binding affinities for several receptors tested (5-HT1, 5-HT2, adrenalin a1, a2, h, dopamine D1, D2, histamine H1, muscarinic M2, opioid A and benzodiazepine receptors) [5]. Ferrets, like humans, are intermittent feeders. Ferret gut exhibits similarities to human gut both anatomically and physiologically [6]. Indeed, measurements of gut motility in conscious ferrets revealed that their spontaneous gut motility pattern is very similar to that reported for humans [7], and that the role of 5-HT3 receptor system in the regulation of ferret gut motility is similar to that in humans [8]. Consequently, the ferret is a suitable model animal to estimate the effect of 5-HT3 receptor agonists and antagonists on human gut functions. In ferrets, YM-31636 facilitates defecation with greater efficacy and earlier onset of action compared to the currently used laxative sodium picosulfate [9]. These results suggested 5-HT3 receptor agonists such as YM-31636 could treat patients with constipation. The colonic motility pattern correlated with the induction of defecation is known as the giant migrating contraction (GMC). GMCs occur in the colon, causing mass movement of colonic contents and consequently defecation [10]. In constipated patients, the most important motility abnormality seems to be a decrease in the frequency, duration and amplitude of GMCs. Therefore, one promising approach to treat constipation is to design drugs that can stimulate GMCs in order to produce mass movement and consequently defecation [11]. In the previous report [9], the dosedependency and time course for YM-31636-induced defecation were precisely investigated. According to the report, YM-31636 dose-dependently (0.01–3 mg/kg po) increases defecation frequency with a minimum effective dose of 0.03 mg/kg p.o. At the minimum effective dose, frequency of defecation (counts/animal in 1 hour) was less than 1 (indicating that defecation occurred only in some population of animals treated) although it reached over 1 at 0.1 mg/kg p.o. (indicating that defecation occurred in almost all animals treated). With respect to the time course, the defecation-evoking effect peaked within 1h, but did not last over 2h. However, it has never been clarified how this compound affects gastrointestinal motility. In this report, we directly measured the gut motility in ferrets and examined effects on the motility from gastric antrum to colon to determine whether YM-31636 stimulates GMCs, or induces undesired changes in gut motility. Because the goal of this study was to investigate the gut motility at a condition that YM-31636 evokes defecation, the dose of 0.1 mg/kg p.o., which had been shown to certainly induce defecation [9], was employed.
Methods All experiments were performed in compliance with the regulations of the Animal Ethical Committee of Yamanouchi Pharmaceutical Co., Ltd.
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Materials YM-31636 (2 - (1H - imidazol - 4 - ylmethyl) - 8H - indeno[1,2 - d]thiazole monofumarate) and ramosetron hydrochloride were prepared by Yamanouchi Pharmaceutical Co. Ltd. (Tsukuba, Japan). Preparation of animals Experiments were performed according to previously reported methods [7]. Four male ferrets (Marshall Farms; North Rose, NY) weighing 1.1–1.3 kg were used. They were fed a standard laboratory diet for carnivores and allowed free access to water. The animals were not fed for 18 h before surgery but were allowed free access to water. Under halothane anesthesia, force transducers (F-08IS, Star Medical; Tokyo, Japan), 8 mm wide and 5 mm long, were sutured to the gastric antrum (3 cm proximal to the pyloric ring), duodenum (3 cm distal to the pyloric ring), ileum (20 cm distal to the pyloric ring) and colon (6 cm proximal to the anus) to measure circular muscle contractions. Wires leading from the transducers were brought subcutaneously to a skin incision made between the scapulae and were protected with a jacket (PJ-F3, Star Medical). The animals were allowed to recover for at least 7 days after surgery before beginning the experiments. Recording of gut motility Ferrets were not fed for 18 h before all experiments but were allowed free access to water. During the experiments, each animal was placed in an observation box (40 cm wide 40 cm long 40 cm high) and was allowed to roam freely. The free ends of the force transducers were connected to a gut motility measuring system (ESC-820A, Star Medical) via a connecting cable suspended from the ceiling. The analog signals of gut motor activity, stored in the measuring system, were digitized at a sampling frequency of 10 Hz by an analog-to-digital converter (ESC1012, Star Medical) and analyzed by software (ESC-820C, Star Medical). Gut motility was quantified by determining the motility index, the value calculated by the computer. The motility index was determined by integrating the area under the contractile wave, i.e., the product of the amplitude (voltage) and the time (min) during the 1-h period after drug or vehicle administration. Evaluating the effect of the drug Evaluation of drug effects was conducted using the paired-control method, alternating the vehicle, YM-31636 and ramosetron plus YM31636 treatments in each ferret following a 3 day or longer washout period. For each ferret, the measurement of gut motility started while they were unfed. Animals were then fed carnivore food (Boiled Chicken Meal, 20 g/kg body weight, Excel Foods Lab. Ltd.; Tokyo, Japan). YM-31636 was suspended in 0.5 % methylcellose and administered orally in a volume of 2 ml/kg about 90 min after feeding. Ramosetron was dissolved in saline and administered subcutaneously in a volume of 1 ml/kg 10 min before YM-31636 administration. All drug doses are in terms of the free base. The motility index for the 1-h period after YM-31636 or vehicle administration was determined.
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Statistical analysis All results are presented as the mean F S.E.M. Statistical analysis of data was performed using the paired Student’s t-test. Values of P < 0.05 were considered significant.
Results Basal gut motility A basal motility pattern, consistent with patterns previously reported [7], was observed in each ferret. Phase III contractions of the migrating motor complex occurred cyclically, beginning in the gastric antrum and migrating distally to the duodenum and small intestine but not to the colon. This pattern was disrupted by feeding. Non-cyclical contractions continued in the gastric antrum, duodenum and ileum. The contractile activity of the colon was independent of activity observed at other recording sites. Colonic contractions consisted of relatively low-amplitude, sporadic contractions occurring irregularly in both fed and unfed animals. Defecation was always preceded by a colon-specific contraction which was characterized by an ungrouped and high-amplitude wave complex. Effect of YM-31636 on gut motility in fed animals Vehicle (0.5% methylcellulose 2 ml/kg p.o.) treatment neither affected gut motility pattern nor induced defecation within 120 min after administration (data not shown). Representative data showing
Fig. 1. A representative tracing showing the effect of YM-31636 on fed ferret gut motility. YM-31636 was administered at the point indicated. In this recording, a defecation before the drug administration (a spontaneous defecation) in addition to that after the administration (a drug-induced defecation) occurred. Note that there were also the cases in which only the drug-induced defecation occurred.
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Table 1 The motility index in fed animals after vehicle or drug administration Drugs
Gastric antrum
Duodenum
Ileum
Colon
Vehicle (2 ml/kg p.o.) 137 F 16 (P = 0.06) 102 F 5 (P = 0.95) 118 F 29 (P = 0.45) 69 F 11 (P = 0.34) YM-31636 (0.1 mg/kg p.o.) 84 F 16 104 F 24 150 F 16 104 F 28 Ramosetron (0.1 mg/kg s.c.) 147 F 18 (P = 0.16) 149 F 29 (P = 0.20) 108 F 37 (P = 0.37) 61 F 3 (P = 0.26) + YM-31636 (0.1 mg /kg p.o.) The motility index (g min) for the 1-h period after vehicle or drug administration was determined, and values are presented as the mean F S.E.M. of results from 4 animals. P values mean results from Student’s t-test (vs. YM-31636 treatment).
the effect of YM-31636 on fed ferret gut motility is presented in Fig. 1. At around 60 min (60.1 F 11.5 min.) after YM-31636 administration, YM-31636 evoked a single high-amplitude colonic contraction in all animals, preceding defecation. This colonic contraction induced by YM-31636 was similar in amplitude, duration and shape, to the contraction preceding spontaneous defecation (Fig. 1). However, YM-31636 had no effect on the basal colonic motility pattern. Although YM-31636 slightly reduced the amplitude of gastric antral contraction immediately after administration, the reduction of the antral motility index was not statistically significant (Table 1). YM-31636 did not affect duodenal or ileal motility patterns. As reported by Ito et al. [9], there were slight changes in stool consistency and no fluid stools were observed after the administration of YM-31636. Additionally, YM-31636 induced neither emesis nor any behavioral changes in animals. Effect of ramosetron on YM-31636-induced response Pretreatment with ramosetron 0.1 mg/kg s.c. inhibited YM-31636-induced colonic contraction and defecation. As previously reported [8], ramosetron by itself did not affect motility patterns at any measured site in fed animals. Additionally, ramosetron by itself did not induce any behavioral changes in animals.
Discussion GMC is a colonic motility pattern which migrates along the colon. It is responsible for movement of contents through the colon and for induction of defecation [10]. Since severe constipation is associated with a decreased frequency, duration and amplitude of GMCs, one promising approach to treat constipation is to design drugs that induce GMCs to produce mass movement and consequently defecation of intestinal contents [11]. Results of the present study showed that the selective 5-HT3 receptor agonist YM-31636 induces a colonic motility pattern specifically related to the induction of defecation. The length of the ferret colon is rather short (about 10 cm), making the distal migration of the high-amplitude contraction difficult to confirm in the present study. To confirm the distal migration and analyze details (propagation velocity etc.), further study with miniature but elaborate strain gages is needed. Nevertheless, because defecation was immediately preceded by a YM-31636-induced contraction without exception, it is likely that the YM-31636-induced colonic motility pattern is a GMC which produces a mass movement and
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consequently defecation. These results agree with previous findings on the role of 5-HT3 receptors in the regulation of colonic motor activity. It has been reported that selective 5-HT3 receptor antagonists evoke constipation in humans [12] and decrease of colonic transit in animals [2] and humans [3,4]. In contrast, the 5-HT3 receptor agonist m-chlorophenylbiguanide induces a single, high-amplitude colonic GMC-like contraction in dogs [13]. The present and previous findings suggest that the 5-HT3 receptor regulates, at least in part, the induction of GMC in the colon and defecation. YM-31636 did not affect the basal colonic motility pattern of ferrets, except for inducing a GMC-like contraction. In addition, it had a slight effect on gastric, duodenal and ileal motility patterns. Although it has been reported that intravenous injection of the selective 5-HT3 receptor agonist m-chlorophenylbiguanide reduces the amplitude of gastric contractions in fed dogs [14], no such inhibitory effect on gastric motility resulting from YM-31636 treatment was observed in the present study, possibly due to differences in the route of administration. Because unnecessary changes in the gut motility pattern may lead to side effects such as abdominal pain and diarrhea, the specific induction of a GMC-like contraction without any change to the basal gut motility pattern is a potential merit in treating constipation. Laxatives are widely used to treat constipation. However, because they act in the lumen of the colon, the onset of their action usually occurs many hours after their administration. It has been reported that YM-31636-induced defecation occurs within 1 hour after oral administration in most of animals treated, whereas sodium picosulfate, a widely used laxative, needs several hours to induce defecation [7]. These results demonstrate that YM-31636 has an earlier onset of action compared to existing laxatives, indicating that YM-31636 does not have the limitations of existing laxatives. Taken together, 5-HT3 receptor agonists including YM-31636 promise to become useful agents in treating the constipation. Indeed, the 5-HT3 receptor agonist MKC-733 has recently been found to decrease colonic transit time in humans and to be effective in treating patients with constipation [15]. The site of 5-HT3 receptors involved in the action of YM-31636 remains to be clarified. Because it has been well known that 5-HT3 receptors in the gastrointestinal neuronal plexus are deeply involved in the regulation of gastrointestinal motor functions, it is possible that they are responsible for the effect of this compound. However, the possibility of the involvement of other 5-HT3 receptors such as those in the brain cannot be excluded. Investigations of pharmacokinetics including brain concentration of this drug in ferrets might be a way to elucidate this question although it remains to be as a future work.
Conclusion The selective 5-HT3 receptor agonist YM-31636 evokes a GMC-like colonic contraction which induces defecation, while only slightly affecting the basal gut motility pattern. This result suggests that 5-HT3 receptor agonists including YM-31636 will prove very useful in treating constipation because of their high efficacy with few accompanying side effects.
Acknowledgements I thank Mr. Steven E. Johnson for editing this manuscript.
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References [1] Yano T, Yamazaki S, Tobe A. Effects of MKC-733, a new prokinetic agent, on the gastrointestinal tract. Digestion 1998;59:Suppl. 3 (ExhB5335). [2] Jin JG, Foxx OAE, Grider JR. Propulsion in guinea pig colon induced by 5-hydroxytrypatmine (HT) via 5-HT4 and 5-HT3 receptors. Journal of Pharmacology and Experimental Therapeutics 1999;288:93 – 7. [3] Gore S, Gilmore IT, Haigh CG, Brownless SM. Colonic transit in man is slowed by ondansetron (GR38023F), a selective 5-hydroxytryptamine receptor (type 3) antagonist. Alimentary Pharmacology and Therapeutics 1990;4:139 – 44. [4] Houghton LA, Foster J, Whorwell PJ, McDonald JN. Effect of alosetron, a new selective 5-HT3 antagonist, on gastrointestinal (GI) transit in normal healthy volunteers. Gastroenterology 1995;108:Suppl. A619. [5] Ito H, Kiso T, Miyata K, Kamato T, Yuki H, Akuzawa S, Nagakura Y, Yamano M, Suzuki M, Naitoh Y, Sakai H, Iwaoka K, Yamaguchi T. Pharmacological profile of YM-31636, a novel 5-HT3 receptor agonist, in vitro. European Journal of Pharmacology 2000;409:195 – 201. [6] Mackay TW, Andrews PLR. A comparative study of the vagal innervation of the stomach in man and the ferret. Journal of Anatomy 1983;136:449 – 81. [7] Nagakura Y, Sohda K, Ito H, Miyata K. Gastrointestinal motor activity under the conscious condition in ferrets. European Journal of Pharmacology 1997;321:53 – 7. [8] Nagakura Y, Kiso T, Ito H, Miyata K, Yamaguchi T. The role of 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in the regulation of gut motility in the ferret. Life Science 2000;66:PL331 – 8. [9] Ito H, Kiso T, Yuki H, Naitoh Y, Miyata K, Iwaoka K, Yamaguchi T. Investigation of the effects of YM-31636, a novel 5HT3 receptor agonist, on defecation in normal and constipated ferrets. European Journal of Pharmacology 2001;424:151 – 7. [10] Sarna SK. Physiology and pathophysiology of colonic motor activity. Digestive Diseases and Sciences 1991;36:827 – 62. [11] Briejer MR, Schuurkes JAJ, Sarna SK. Idiopathic constipation: too few stools and too little knowledge. Trends in Pharmacological Sciences 1999;20:1 – 3. [12] Perez EA. Comparative efficacy of oral and intravenous granisetron for the prevention of acute chemotherapy-induced emesis. Clinica Terapeutica 1996;18:578 – 90. [13] Nagakura Y, Kamato T, Nishida A, Ito H, Yamano M, Miyata K. Characterization of 5-hydroxytryptamine (5-HT) receptor subtypes influencing colonic motility in conscious dogs. Naunyn-Schmiedeberg’s Archives0 of Pharmacology 1996; 353:489 – 98. [14] Nagakura Y, Ito H, Kamato T, Nishida A, Miyata K. Effect of a selective 5-HT3 receptor agonist on gastric motility in fasted and fed dogs. European Journal of Pharmacology 1997;327:189 – 93. [15] Fujita T, Kumagai Y, Yokota S, Sawada M, Saigenji K, Muramatu M, Majima M. The effects of a novel 5-HT3 agonist on symptoms of subjects with constipation and their intestinal motilities. Digestion 1998;59:Suppl. 3 (ExhB5359).
The effect of the selective 5-HT3 receptor agonist on ferret gut motility Yukinori Nagakura *, Tetsuo Kiso, Keiji Miyata, Hiroyuki Ito, Kiyoshi Iwaoka, Tokio Yamaguchi Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan Received 9 October 2001; accepted 7 March 2002
Abstract The effect of the selective 5-hydroxytryptamine (5-HT)3 receptor agonist YM-31636 (2-(1H-imidazol-4ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate) on gut motility of fed ferrets was investigated. YM-31636 (0.1 mg/kg p.o.) induced a giant migrating contraction (GMC)-like, high-amplitude, ungrouped colonic contraction although it did not change the basal colonic motility pattern. This GMC-like contraction was always accompanied by defecation. Both GMC-like contraction and defecation were inhibited with the selective 5-HT3 receptor antagonist ramosetron. YM-31636 affected gastric, duodenal and ileal motility pattern only slightly. These results suggest that 5-HT3 receptor agonists such as YM-31636 are useful in treating constipation since they facilitate GMC-like contractions and defecation without undesired changes in gut motility pattern. D 2002 Elsevier Science Inc. All rights reserved. Keywords: 5-HT3 receptor agonist; YM-31636; Gut motility; Giant migrating contraction; Constipation
Introduction The 5-HT3 receptor is deeply involved in the regulation of colonic motor activity in animals and humans. Recent reports indicate that 5-HT3 receptor agonists stimulate colonic propulsive activity [1,2] although 5-HT3 receptor antagonists inhibit it [3,4]. YM-31636 (2-(1H-imidazol-4-ylmethyl)-
*
Corresponding author. Tel.: +81-298-52-5111; fax: +81-298-52-2965. E-mail address: [email protected] (Y. Nagakura). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 8 4 1 - 6
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8H-indeno[1,2-d]thiazole monofumarate) is a newly synthesized, potent and selective 5-HT3 receptor agonist. This compound has almost full agonistic activity in inducing contraction (EC50: 0.12 AM) and partial agonistic activity in increasing short-circuit current (EC50: 0.16 AM) in isolated guinea-pig distal colon. Only ramosetron (a selective 5-HT3 receptor antagonist) but not methysergide (a 5-HT1 and 5-HT2 receptor antagonist) and SB204070 (a selective 5-HT4 receptor antagonist) inhibited its contractile effect in the guinea-pig colon even though the 5-HT-induced contraction in this preparation is mediated through 5-HT3, 5-HT4 and 5-HT1-like receptors, suggesting that the effect of YM-31636 on the colon is mediated exclusively through the 5-HT3 receptor. This is also supported by the fact that YM-31636 has negligible binding affinities for several receptors tested (5-HT1, 5-HT2, adrenalin a1, a2, h, dopamine D1, D2, histamine H1, muscarinic M2, opioid A and benzodiazepine receptors) [5]. Ferrets, like humans, are intermittent feeders. Ferret gut exhibits similarities to human gut both anatomically and physiologically [6]. Indeed, measurements of gut motility in conscious ferrets revealed that their spontaneous gut motility pattern is very similar to that reported for humans [7], and that the role of 5-HT3 receptor system in the regulation of ferret gut motility is similar to that in humans [8]. Consequently, the ferret is a suitable model animal to estimate the effect of 5-HT3 receptor agonists and antagonists on human gut functions. In ferrets, YM-31636 facilitates defecation with greater efficacy and earlier onset of action compared to the currently used laxative sodium picosulfate [9]. These results suggested 5-HT3 receptor agonists such as YM-31636 could treat patients with constipation. The colonic motility pattern correlated with the induction of defecation is known as the giant migrating contraction (GMC). GMCs occur in the colon, causing mass movement of colonic contents and consequently defecation [10]. In constipated patients, the most important motility abnormality seems to be a decrease in the frequency, duration and amplitude of GMCs. Therefore, one promising approach to treat constipation is to design drugs that can stimulate GMCs in order to produce mass movement and consequently defecation [11]. In the previous report [9], the dosedependency and time course for YM-31636-induced defecation were precisely investigated. According to the report, YM-31636 dose-dependently (0.01–3 mg/kg po) increases defecation frequency with a minimum effective dose of 0.03 mg/kg p.o. At the minimum effective dose, frequency of defecation (counts/animal in 1 hour) was less than 1 (indicating that defecation occurred only in some population of animals treated) although it reached over 1 at 0.1 mg/kg p.o. (indicating that defecation occurred in almost all animals treated). With respect to the time course, the defecation-evoking effect peaked within 1h, but did not last over 2h. However, it has never been clarified how this compound affects gastrointestinal motility. In this report, we directly measured the gut motility in ferrets and examined effects on the motility from gastric antrum to colon to determine whether YM-31636 stimulates GMCs, or induces undesired changes in gut motility. Because the goal of this study was to investigate the gut motility at a condition that YM-31636 evokes defecation, the dose of 0.1 mg/kg p.o., which had been shown to certainly induce defecation [9], was employed.
Methods All experiments were performed in compliance with the regulations of the Animal Ethical Committee of Yamanouchi Pharmaceutical Co., Ltd.
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Materials YM-31636 (2 - (1H - imidazol - 4 - ylmethyl) - 8H - indeno[1,2 - d]thiazole monofumarate) and ramosetron hydrochloride were prepared by Yamanouchi Pharmaceutical Co. Ltd. (Tsukuba, Japan). Preparation of animals Experiments were performed according to previously reported methods [7]. Four male ferrets (Marshall Farms; North Rose, NY) weighing 1.1–1.3 kg were used. They were fed a standard laboratory diet for carnivores and allowed free access to water. The animals were not fed for 18 h before surgery but were allowed free access to water. Under halothane anesthesia, force transducers (F-08IS, Star Medical; Tokyo, Japan), 8 mm wide and 5 mm long, were sutured to the gastric antrum (3 cm proximal to the pyloric ring), duodenum (3 cm distal to the pyloric ring), ileum (20 cm distal to the pyloric ring) and colon (6 cm proximal to the anus) to measure circular muscle contractions. Wires leading from the transducers were brought subcutaneously to a skin incision made between the scapulae and were protected with a jacket (PJ-F3, Star Medical). The animals were allowed to recover for at least 7 days after surgery before beginning the experiments. Recording of gut motility Ferrets were not fed for 18 h before all experiments but were allowed free access to water. During the experiments, each animal was placed in an observation box (40 cm wide 40 cm long 40 cm high) and was allowed to roam freely. The free ends of the force transducers were connected to a gut motility measuring system (ESC-820A, Star Medical) via a connecting cable suspended from the ceiling. The analog signals of gut motor activity, stored in the measuring system, were digitized at a sampling frequency of 10 Hz by an analog-to-digital converter (ESC1012, Star Medical) and analyzed by software (ESC-820C, Star Medical). Gut motility was quantified by determining the motility index, the value calculated by the computer. The motility index was determined by integrating the area under the contractile wave, i.e., the product of the amplitude (voltage) and the time (min) during the 1-h period after drug or vehicle administration. Evaluating the effect of the drug Evaluation of drug effects was conducted using the paired-control method, alternating the vehicle, YM-31636 and ramosetron plus YM31636 treatments in each ferret following a 3 day or longer washout period. For each ferret, the measurement of gut motility started while they were unfed. Animals were then fed carnivore food (Boiled Chicken Meal, 20 g/kg body weight, Excel Foods Lab. Ltd.; Tokyo, Japan). YM-31636 was suspended in 0.5 % methylcellose and administered orally in a volume of 2 ml/kg about 90 min after feeding. Ramosetron was dissolved in saline and administered subcutaneously in a volume of 1 ml/kg 10 min before YM-31636 administration. All drug doses are in terms of the free base. The motility index for the 1-h period after YM-31636 or vehicle administration was determined.
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Statistical analysis All results are presented as the mean F S.E.M. Statistical analysis of data was performed using the paired Student’s t-test. Values of P < 0.05 were considered significant.
Results Basal gut motility A basal motility pattern, consistent with patterns previously reported [7], was observed in each ferret. Phase III contractions of the migrating motor complex occurred cyclically, beginning in the gastric antrum and migrating distally to the duodenum and small intestine but not to the colon. This pattern was disrupted by feeding. Non-cyclical contractions continued in the gastric antrum, duodenum and ileum. The contractile activity of the colon was independent of activity observed at other recording sites. Colonic contractions consisted of relatively low-amplitude, sporadic contractions occurring irregularly in both fed and unfed animals. Defecation was always preceded by a colon-specific contraction which was characterized by an ungrouped and high-amplitude wave complex. Effect of YM-31636 on gut motility in fed animals Vehicle (0.5% methylcellulose 2 ml/kg p.o.) treatment neither affected gut motility pattern nor induced defecation within 120 min after administration (data not shown). Representative data showing
Fig. 1. A representative tracing showing the effect of YM-31636 on fed ferret gut motility. YM-31636 was administered at the point indicated. In this recording, a defecation before the drug administration (a spontaneous defecation) in addition to that after the administration (a drug-induced defecation) occurred. Note that there were also the cases in which only the drug-induced defecation occurred.
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Table 1 The motility index in fed animals after vehicle or drug administration Drugs
Gastric antrum
Duodenum
Ileum
Colon
Vehicle (2 ml/kg p.o.) 137 F 16 (P = 0.06) 102 F 5 (P = 0.95) 118 F 29 (P = 0.45) 69 F 11 (P = 0.34) YM-31636 (0.1 mg/kg p.o.) 84 F 16 104 F 24 150 F 16 104 F 28 Ramosetron (0.1 mg/kg s.c.) 147 F 18 (P = 0.16) 149 F 29 (P = 0.20) 108 F 37 (P = 0.37) 61 F 3 (P = 0.26) + YM-31636 (0.1 mg /kg p.o.) The motility index (g min) for the 1-h period after vehicle or drug administration was determined, and values are presented as the mean F S.E.M. of results from 4 animals. P values mean results from Student’s t-test (vs. YM-31636 treatment).
the effect of YM-31636 on fed ferret gut motility is presented in Fig. 1. At around 60 min (60.1 F 11.5 min.) after YM-31636 administration, YM-31636 evoked a single high-amplitude colonic contraction in all animals, preceding defecation. This colonic contraction induced by YM-31636 was similar in amplitude, duration and shape, to the contraction preceding spontaneous defecation (Fig. 1). However, YM-31636 had no effect on the basal colonic motility pattern. Although YM-31636 slightly reduced the amplitude of gastric antral contraction immediately after administration, the reduction of the antral motility index was not statistically significant (Table 1). YM-31636 did not affect duodenal or ileal motility patterns. As reported by Ito et al. [9], there were slight changes in stool consistency and no fluid stools were observed after the administration of YM-31636. Additionally, YM-31636 induced neither emesis nor any behavioral changes in animals. Effect of ramosetron on YM-31636-induced response Pretreatment with ramosetron 0.1 mg/kg s.c. inhibited YM-31636-induced colonic contraction and defecation. As previously reported [8], ramosetron by itself did not affect motility patterns at any measured site in fed animals. Additionally, ramosetron by itself did not induce any behavioral changes in animals.
Discussion GMC is a colonic motility pattern which migrates along the colon. It is responsible for movement of contents through the colon and for induction of defecation [10]. Since severe constipation is associated with a decreased frequency, duration and amplitude of GMCs, one promising approach to treat constipation is to design drugs that induce GMCs to produce mass movement and consequently defecation of intestinal contents [11]. Results of the present study showed that the selective 5-HT3 receptor agonist YM-31636 induces a colonic motility pattern specifically related to the induction of defecation. The length of the ferret colon is rather short (about 10 cm), making the distal migration of the high-amplitude contraction difficult to confirm in the present study. To confirm the distal migration and analyze details (propagation velocity etc.), further study with miniature but elaborate strain gages is needed. Nevertheless, because defecation was immediately preceded by a YM-31636-induced contraction without exception, it is likely that the YM-31636-induced colonic motility pattern is a GMC which produces a mass movement and
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consequently defecation. These results agree with previous findings on the role of 5-HT3 receptors in the regulation of colonic motor activity. It has been reported that selective 5-HT3 receptor antagonists evoke constipation in humans [12] and decrease of colonic transit in animals [2] and humans [3,4]. In contrast, the 5-HT3 receptor agonist m-chlorophenylbiguanide induces a single, high-amplitude colonic GMC-like contraction in dogs [13]. The present and previous findings suggest that the 5-HT3 receptor regulates, at least in part, the induction of GMC in the colon and defecation. YM-31636 did not affect the basal colonic motility pattern of ferrets, except for inducing a GMC-like contraction. In addition, it had a slight effect on gastric, duodenal and ileal motility patterns. Although it has been reported that intravenous injection of the selective 5-HT3 receptor agonist m-chlorophenylbiguanide reduces the amplitude of gastric contractions in fed dogs [14], no such inhibitory effect on gastric motility resulting from YM-31636 treatment was observed in the present study, possibly due to differences in the route of administration. Because unnecessary changes in the gut motility pattern may lead to side effects such as abdominal pain and diarrhea, the specific induction of a GMC-like contraction without any change to the basal gut motility pattern is a potential merit in treating constipation. Laxatives are widely used to treat constipation. However, because they act in the lumen of the colon, the onset of their action usually occurs many hours after their administration. It has been reported that YM-31636-induced defecation occurs within 1 hour after oral administration in most of animals treated, whereas sodium picosulfate, a widely used laxative, needs several hours to induce defecation [7]. These results demonstrate that YM-31636 has an earlier onset of action compared to existing laxatives, indicating that YM-31636 does not have the limitations of existing laxatives. Taken together, 5-HT3 receptor agonists including YM-31636 promise to become useful agents in treating the constipation. Indeed, the 5-HT3 receptor agonist MKC-733 has recently been found to decrease colonic transit time in humans and to be effective in treating patients with constipation [15]. The site of 5-HT3 receptors involved in the action of YM-31636 remains to be clarified. Because it has been well known that 5-HT3 receptors in the gastrointestinal neuronal plexus are deeply involved in the regulation of gastrointestinal motor functions, it is possible that they are responsible for the effect of this compound. However, the possibility of the involvement of other 5-HT3 receptors such as those in the brain cannot be excluded. Investigations of pharmacokinetics including brain concentration of this drug in ferrets might be a way to elucidate this question although it remains to be as a future work.
Conclusion The selective 5-HT3 receptor agonist YM-31636 evokes a GMC-like colonic contraction which induces defecation, while only slightly affecting the basal gut motility pattern. This result suggests that 5-HT3 receptor agonists including YM-31636 will prove very useful in treating constipation because of their high efficacy with few accompanying side effects.
Acknowledgements I thank Mr. Steven E. Johnson for editing this manuscript.
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