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Inhibitory Effects of Ramosetron, a Potent and Selective 5-HT3 J Pharmacol Sci 106, 264 – 270 (2008)2 Journal of Pharmacological Sciences ©2008 The Japanese Pharmacological Society Full Paper 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), loperam- ide (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
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