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Inhibitory Effects of Ramosetron, a Potent and Selective 5-HT3–Receptor Antagonist, on Conditioned Fear Stress–Induced Abnormal Defecation and Normal Defecation in Rats: Comparative Studies With Antidiarrheal and Spasmolytic Agents

Takuya Hirata1,*, Toshiyuki Funatsu1, Yoshihiro Keto1, Shinobu Akuzawa1, Masao Sasamata1, and Keiji Miyata1 1Applied Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma, Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan

Received Novemer 2, 2007; Accepted December 13, 2007

Abstract. We examined the effect of ramosetron, a potent serotonin (5-HT)3-receptor antagonist for irritable bowel syndrome with diarrhea, on conditioned fear stress (CFS)-induced defecation and normal (non-stressed) defecation in rats and compared ramosetron with the antidiarrheal agent loperamide and the spasmolytic agents trimebutine and tiquizium. Ramosetron, loperamide, trimebutine, and tiquizium significantly inhibited CFS-induced defecation in a dose-dependent manner with ED50 (95% confidence limit) values of 0.019 (0.01 – 0.028), 9.4 (4.0 – 22), 850 (520 – 2,400), and 300 (190 – 450) mg/kg, respectively. A significant effect of ramosetron on CFS-induced defecation appeared at 10 min after dosing and was sustained for 8 h. In contrast, loperamide, trimebutine, and tiquizium significantly inhibited CFS-induced defecation between 1 – 8, 1 – 4, and 1–8h after administration, respectively. High doses of ramosetron did not affect normal defecation, whereas loperamide, trimebutine, and tiquizium significantly inhibited this process. In conclusion, ramosetron has potent, rapid-onset, and long-lasting inhibitory effects on CFS-induced defecation in rats, but does not influence normal defecation. The present findings indicate that ramosetron will be a useful therapeutic agent for irritable bowel syndrome with diarrhea, showing greater efficacy and safety than other antidiarrheal and spasmolytic agents.

Keywords: ramosetron, antidiarrheal agent, spasmolytic, conditioned fear stress, normal defecation

Introduction (IBS-D), IBS with constipation, mixed IBS, and unsub- typed IBS. In clinical settings, antidiarrheal agents, spas- Irritable bowel syndrome (IBS) is a functional disease molytic agents, synthetic polymers, and lactobacillus with persistent gastrointestinal symptoms, mainly preparations are widely used to treat IBS-D, but the abdominal pain/discomfort and abnormal defecation, effectiveness of these drugs remains unclear (3). not accompanied by organic disease (1). Although the In contrast, several serotonin (5-HT)3-receptor precise pathophysiology of IBS is still not fully antagonists have recently been developed as therapeutic understood, factors thought to play a role in this disease agents for IBS-D, and their effectiveness has now been include altered gastrointestinal motility, increased established (4, 5). It has been reported that 5-HT3 sensitivity of the colon, and psychosocial factors (1). receptors are widely distributed within the gastro- According to the Rome III criteria presented in 2006 (2), intestinal tract, and activation of these receptors by IBS is classified into four subtypes: IBS with diarrhea endogenous 5-HT results in intestinal secretion and peristaltic activity (6). The clinical study of Bearcroft *Corresponding author. [email protected] et al. (7) showed that the postprandial plasma concentra- Published online in J-STAGE tion of 5-HT in IBS patients was higher than that in doi: 10.1254/jphs.FP0071943 healthy volunteers. Furthermore, 5-HT3–receptor antago-

264 Effects of Ramosetron on Defecation 265 nists have been reported to inhibit stress-induced to the electric shock. About 24 h after conditioning, rats abnormal defecation in animals (8, 9), suggesting the were returned to the electric shock chamber, and the involvement of 5-HT3 receptors in the pathogenesis of warning beep and illumination, but without the electric IBS. shock, were applied over 30 min to evoke CFS. Feces Ramosetron (YM060), a potent and selective 5-HT3– were collected after the 30-min session of CFS to receptor antagonist (10, 11), has been launched in some determine total stool weight. Experiments examining the Asian countries as a medication for gastrointestinal inhibitory potencies of test drugs involved the following symptoms caused by antitumor agents. Ramosetron is treatment groups (nine rats per treatment): normal (non- also known to potently inhibit stress-induced abnormal stressed), 0.5% (w/v) methylcellulose (MC) solution, defecation in animals (12, 13) and is currently under ramosetron (0.01, 0.03, 0.1 mg/kg), loperamide (3, 10, development for use in patients suffering from IBS-D. 30 mg/kg), trimebutine (100, 300, 1000 mg/kg), and No study, however, has directly analyzed the inhibitory tiquizium (100, 300, 1,000 mg/kg). The test compounds effects of ramosetron on stress-induced abnormal were administered orally 1 h before initiation of CFS. defecation, in comparison with existing drugs prescribed This experiment was performed three times separately, for IBS-D. and each experiment was conducted with all treatment In the present study, therefore, we examined the effect groups including three rats per group, which were of ramosetron on stress-induced abnormal defecation randomly assigned so that mean body weight in each using a rat model of conditioned fear stress (CFS), which group was approximately balanced. In the second series is known to be psychological stress model useful for the of experiments, evaluating time course changes in the evaluation of anxiolytic, antidepressant, and anti-IBS inhibitory effects of test drugs, the following compounds agents (12, 14), and compared ramosetron with the were administered orally 10 min, 1, 4, 8, and 12 h before antidiarrheal and spasmolytic agents such as loperamide, initiation of CFS (10 rats per treatment): ramosetron trimebutine (opioid receptor agonist), and tiquizium (0.1 mg/kg), loperamide (10 mg/kg), trimebutine (1,000 (muscarinic receptor antagonist). In addition, because mg/kg), and tiquizium (1,000 mg/kg). The dose levels certain antidiarrheal and spasmolytic agents have been were set to inhibit the CFS-induced defecation with the reported to simultaneously improve the symptoms of maximum inhibition of 60% – 70%. Control rat groups IBS and cause severe constipation (15, 16), we also (10 rats/group) treated with 0.5% (w/v) MC solution evaluated the influence of these drugs on normal (non- were used for all time points of each test compound. The stressed) defecation in rats. effect of trimebutine at 12 h after administration was not evaluated because the significant effect had already Materials and Methods disappeared at 8 h after dosing.

Animals Normal defecation in rats Male Sprague-Dawley rats (7 – 8-week-old; Clea Each rat without fasting was housed in an individual Japan, Inc., Tokyo) were used. Animals were housed in observation cage immediately after the oral administra- a temperature-controlled environment (22 ± 2°C) under tion of test compounds during 21:30 – 22:30. Feces a 12:12 h light/dark cycle and were given food and water were collected 12 and 24 h after administration to ad libitum. All experimental procedures were approved measure total stool weights. Five treatments (nine rats by the Animal Ethical Committee of Astellas Pharma, per treatment) were included in this experiment: 0.5% Inc. (Tokyo). MC solution, ramosetron (0.1, 0.3, 1 mg/kg), loperamide (3, 10, 30 mg/kg), trimebutine (100, 300, 1000 Conditioned fear stress-induced defecation in rats mg/kg), and tiquizium (100, 300, 1000 mg/kg). This This experiment was performed according to the experiment was performed three times separately, and method of Funatsu et al. (12). Using an electric-shock each experiment was conducted with all treatment machine (CB2000; O’Hara, Inc., Tokyo) with an groups including three rats per group, which were electrode grid-inlaid floor (17 × 17 × 39 cm), a maximal randomly assigned so that mean body weight in each 2 mA of electric current and illumination with three group was approximately balanced. 40 W electric bulbs were applied to rats for 5 s per min, with a total of 15 exposures in each session. Each Drugs application of electric shock and illumination was The drugs ramosetron hydrochloride (Astellas preceded by a 3-s warning beep. Animals in the normal Pharma, Inc.), loperamide hydrochloride, trimebutine (non-stressed) group were placed in the electric-shock maleate (Sigma-Aldrich Japan, Tokyo), and tiquizium machine under the same protocol, but were not exposed bromide (Permachem Asia, Ltd., Tokyo) were used in 266 T Hirata et al this study. Ramosetron was dissolved in distilled water control rats treated with 0.5% (w/v) MC (Fig. 1). (DW) and diluted with 0.5% (w/v) MC solution. Oral administration of ramosetron (0.01 – 0.1 mg/kg), Loperamide, trimebutine, and tiquizium were suspended loperamide (3 – 30 mg/kg), trimebutine (100 – 1000 in and diluted with 0.5% (w/v) MC solution. In this mg/kg), or tiquizium (100 – 1000 mg/kg) significantly study, all test compounds were used in their salt forms. inhibited CFS-induced defecation in a dose-dependent manner (Fig. 1), with ED50 (95% CLs) values of 0.019 Statistical analyses (0.011 – 0.028), 9.4 (4.0 – 22), 850 (520 – 2400), and All results were statistically analyzed using the 300 (190 – 450) mg/kg, respectively (Table 1). The Statistical Analysis System ver. 8.2 (SAS Institute Japan potencies of loperamide, trimebutine, and tiquizium Ltd., Tokyo). All data were rounded off to two signifi- relative to ramosetron (1) were 1/500, 1/44,000, and cant figures. The mean ± S.E.M. of the total weight of 1/15,000, respectively (Table 1). stools in each treatment group was calculated, and Student’s t-test and Dunnett’s multiple range test were Time course changes in the inhibitory effects on CFS- used to compare total stool weights between pairs of induced defecation in rats treatment groups and among multiple treatment groups, Ramosetron (0.1 mg/kg) administered orally 10 min respectively, with the significance level set at 5% before the initiation of CFS significantly inhibited (P<0.05). To determine the inhibitory effects of drugs CFS-induced defecation, and the effect was sustained at on defecation, an ED50 value and 95% confidence limits 1, 4, and 8 h after administration (Fig. 2A). The (CLs) for each test compound were estimated from the maximum effect of ramosetron was observed at 1 h total weights of stools, using linear regression analysis. after dosing (maximum inhibition rate: 70 ± 7.6%, Fig. 3). Loperamide (10 mg/kg) and tiquizium (1000 Results mg/kg) significantly inhibited CFS-induced defecation at 1, 4, and 8 h after administration (Fig. 2: B and D), CFS-induced defecation in rats and the maximum inhibition rates were 72 ± 5.4% at 4 h In normal rats without CFS treatment, the total weight after dosing and 67 ± 5.8% at 8 h after dosing, respec- of stools during 30 min was 0.53 ± 0.13 g, while CFS tively (Fig. 3). Trimebutine significantly inhibited CFS- for 30 min significantly increased it to 2.4 ± 0.25 g in induced defecation at 1 and 4 h after administration

Fig. 1. Inhibitory effects of ramosetron, loperamide, trimebutine, and tiquizium on CFS-induced defecation in rats. Each column with a vertical bar represents the mean ± S.E.M. for nine animals. ##P<0.01, compared to normal (without CFS) rats using Student’s t-test. *P<0.05 and **P<0.01, compared to control rats treated with 0.5% (w/v) MC solution, using Dunnett’s multiple range test. The dose levels of all test compounds refer to their salt forms. N: normal.

Table 1. Potencies of ramosetron, loperamide, trimebutine, and tiquizium on inhibition of CFS-induced defecation in rats Ramosetron Loperamide Trimebutine Tiquizium

ED50 [95% CL] mg/kg, p.o. 0.019 [0.011 – 0.028] 9.4 [4.0 – 22] 850 [520 – 2,400] 300 [190 – 450] (1) (1/500) (1/44,000) (1/15,000)

Values in parentheses represent the potencies relative to ramosetron. The ED50 values and 95% CLs of all test compounds refer to the salt forms. N = 9. Effects of Ramosetron on Defecation 267

Fig. 2. Inhibitory time profile of ramosetron (A), loperamide (B), trimebutine (C), and tiquizium (D) on CFS-induced defecation in rats. Each column with a vertical bar represents the mean ± S.E.M. for 10 rats. Black column: control rats treated with 0.5% (w/v) MC solution. Gray column: test drug-treated rats. White column: normal rats (without CFS). **P<0.01, compared to the respective control group using Student’s t-test. The dose levels of all test compounds refer to their salt forms. NT: not tested.

the 0 – 12, 12 – 24, and 0 – 24 h after administration of 0.5% (w/v) MC solution was 7.8 ± 0.56, 2.7 ± 0.24, and 10 ± 0.67 g respectively (Fig. 4). Oral administration of ramosetron (0.1 – 1 mg/kg) did not affect normal defecation in the 0 – 12, 12 – 24, and 0 – 24 h after administration, and the average stool weight of each measurement periods did not significantly differ from the values for control rats (Fig. 4). In contrast, loperamide (3 – 30 mg/kg), trimebutine (100 – 1,000 mg/kg), and tiquizium (100 – 1000 mg/kg) showed dose-dependent inhibitions of normal defecation for 0 – 12, 12 – 24, and 0 – 24 h after dosing (Fig. 4), and ED50 (95% CLs) values estimated from the total Fig. 3. Changes with time in the inhibition rates of ramosetron (0.1 mg/kg), loperamide (10 mg/kg), trimebutine (1,000 mg/kg), and stool weight for 0 – 24 h after administration were 9.5 tiquizium (1000 mg/kg) on CFS-induced defecation in rats. Each (7.5 – 12), >1000, and 550 (330 – 1500) mg/kg, respec- point with a vertical bar represents the mean ± S.E.M. for 10 rats. tively (Table 2).

Discussion (Fig. 2C), and the maximum effect was observed at 1 h after dosing (maximum inhibition rate: 58 ± 6.4%, This study has shown that ramosetron, a novel Fig. 3). therapeutic agent for IBS-D, inhibited CFS-induced defecation but did not affect normal (non-stressed) Normal defecation in rats defecation in rats, whereas the antidiarrheal agent loper- In control rats, the total weight of stools defecated in amide, and spasmolytic agents trimebutine and tiqui- 268 T Hirata et al

Fig. 4. Influences of ramosetron, loperamide, trimebutine, and tiquizium on normal defecation in rats. Each column with a vertical bar represents the mean ± S.E.M. of total stool weights in the interval of 0 – 12 and 12 – 24 h for nine animals. Significant differences from 0.5% (w/v) MC-treated rats of the respective time interval were analyzed by Dunnett’s multiple range test (*P<0.05 and **P<0.01, 0–12h; ##P<0.01, 12 – 24 h; $P<0.05 and $$P<0.01, 0 – 24 h). No significant difference was observed between rats treated with 0.5% (w/v) MC and ramosetron in all time intervals. The dose levels of all test compounds refer to their salt forms.

Table 2. Potencies of ramosetron, loperamide, trimebutine, and tiquizium on inhibition of normal defecation in rats Ramosetron Loperamide Trimebutine Tiquizium

ED50 [95% CL] mg/kg, p.o. >1 9.5 [7.5 – 12] >1000 550 [330 – 1,500]

The ED50 values and 95% CLs of all test compounds refer to the salt forms. N = 9. zium, inhibited both CFS-induced defecation and nor- administration of [14C]-ramosetron is approximately mal defecation. Furthermore, we found that a significant 15 min (17), we suggest that ramosetron becomes inhibitory effect of ramosetron on CFS-induced defeca- efficacious immediately after absorption into circulating tion appeared at 10 min after dosing and was sustained blood. In addition, a significant effect of ramosetron on for 8 h after administration. These results indicate that CFS-induced defecation was also observed 8 h after ramosetron has potent, rapid-onset, and long-lasting administration, although it has been shown that the inhibitory effects on CFS-induced defecation in rats, plasma elimination half-life of [14C]-ramosetron is about without any influence on normal defecation. 1 h (17). Previously, Ohta et al. (18) have suggested that The experimental CFS method used here is known to ramosetron may achieve long-lasting binding to 5-HT3 expose rats to psychological stress arising from re- receptors because ramosetron possesses the distinctive exposure to an environment where the animals pre- ability to maintain the active three-dimensional chemical viously received electric shocks, resulting in freezing conformation necessary for such binding. In fact, it has behavior and acceleration of defecation. The model is been reported that ramosetron dissociated slowly from widely used for the evaluation of anxiolytic, anti- 5-HT3 receptors (10, 19) and showed long-lasting 5- depressant, and anti-IBS agents (12, 14). In this model, HT3-receptor antagonism (11, 20). Taken together, these we found that all test compounds including ramosetron, data indicate that ramosetron produces a long-lasting loperamide, trimebutine, and tiquizium inhibited CFS- inhibitory effect on CFS-induced defecation because of induced defecation in a dose-dependent manner, with the persistent 5-HT3–receptor antagonism. potency (based on a comparison of ED50 values) decreas- Colonic giant migrating contractions propagate ing in the order ramosetron>> loperamide>> tiquizium> aborally over extended distances to evoke mass move- trimebutine. In addition, a significant inhibitory effect of ments of feces and are regulated by cholinergic path- ramosetron on CFS-induced defecation appeared 10 min ways (21). The enhancement of colonic motility by the after administration, whereas no other compound gastro-colic reflex, activated by gastric distension, and showed a significant effect at that time. Because the time contraction of the rectum with relaxation of the internal taken to reach maximum plasma concentration after oral anal sphincter by rectal reflex, caused by the ingression Effects of Ramosetron on Defecation 269 of feces into the rectum, are thought to be involved in and normal defecation in Mongolian gerbils. However, normal defecation. These reflexes are controlled by we also obtained the data showing that oral administra- parasympathetic nerves (22). On the other hand, acti- tion of ramosetron did not inhibit normal defecation in vation of the 5-HT3 receptor is known to be involved dogs, whereas tiquizium significantly inhibited it (24). in stress-induced abnormal defecation. In detail, Therefore, we considered that the discrepancy may be corticotropin-releasing factor secreted in response to attributable to species differences in the roles of 5-HT3 stress increases the release of endogenous 5-HT from receptor in normal defecation between rats, dogs, and 5-HT-containing nerves and/or enterochromaffin cells, Mongolian gerbils, although no exact cause is known so resulting in the activation of 5-HT3 receptors in the far. Taken together, it is suggested that 5-HT3 receptors enteric nervous system (23). Endogenous 5-HT stimu- are partly involved in normal colonic function, but 5- lates the release of various neurotransmitters, such as HT3–receptor antagonists do not possess the inhibitory acetylcholine, via the activation of 5-HT3 receptors, to effects on normal defecation in rats. induce the acceleration of colonic transit (12) and As with tiquizium, loperamide and trimebutine, which abnormal water transport (24), which in turn cause are known as opioid µ-receptor agonists (28, 29), abnormal defecation. Our present study showed that the suppressed both CFS-induced defecation and normal 5-HT3-receptor antagonist ramosetron did not affect defecation in rats. A previous report has shown that the normal defecation in rats at doses higher than were activation of opioid µ-receptors on the myenteric plexus needed to inhibit CFS-induced defecation, whereas the decreased the release of acetylcholine caused by electri- antimuscarinic agent tiquizium (25) inhibited both CFS- cal stimulation (30). Loperamide and trimebutine thus induced and normal defecation at the same dose. These inhibit intestinal motility in both stressed and non- results, and literature reports, indicate that muscarinic stressed animals by decreasing the release of endo- receptors mediate both normal defecation and stress- genous acetylcholine via the activation of opioid µ- induced abnormal defecation, whereas 5-HT3 receptors receptors. are involved in stress-induced abnormal defecation In conclusion, the present study has shown that but not in normal defecation. In contrast, it has been ramosetron inhibits stress-induced abnormal defecation previously reported that ramosetron significantly in rats better than existing antidiarrheal and spasmolytic inhibited normal colonic transit as well as wrap-restraint agents. The inhibitory effect of ramosetron appeared stress-stimulated defecation and colonic transit in rats immediately after dosing and lasted for many hours. (26). The literature showed that ramosetron at doses Furthermore, ramosetron at dose levels that inhibited of 3 µg/kg or more completely abolished the wrap stress-induced abnormal defecation did not affect nor- stress–stimulated colonic transit but inhibited the wrap mal defecation in rats, but antidiarrheal and spasmolytic stress–stimulated defecation to approximately 50% – agents inhibited both forms of defecation. These results 60% of control rats. In addition, ramosetron was shown indicate that ramosetron will be a useful therapeutic to inhibit normal colonic transit to just approximately agent for IBS-D, with greater efficacy and safety than 70% of control rats. These results indicate that the existing antidiarrheal and spasmolytic agents. inhibitory effect on colonic transit does not correlate with those on defecation and that ramosetron exerts References slight or negligible inhibitory effect on normal defecation in rats. Similarly, alosetron has been reported to 1 Grundy D, Al-Chaer ED, Aziz Q, Collins SM, Ke M, Tache Y, significantly prolong the migration myoelectric complex et al. Fundamentals of neurogastroenterology: basic science. (MMC) cycle length in rats (27). However, as described Gastroenterology. 2006;130:1391–1411. above, defecation is known to be regulated by not only 2 Longstreth GF, Thompson WG, Chey WD, Houghton LA, Mearin F, Spiller RC. Functional bowel disorders. 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