Effects of YM905, a Novel Muscarinic M3-Receptor Antagonist, on Experimental Models of Bowel Dysfunction in Vivo

Jpn. J. Pharmacol. 86, 281 – 288 (2001)

Effects of YM905, a Novel Muscarinic M3-Receptor Antagonist, on Experimental Models of Bowel Dysfunction In Vivo

Seiji Kobayashi*, Ken Ikeda, Mami Suzuki, Toshimitsu Yamada and Keiji Miyata

Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan

Received January 24, 2001 Accepted April 2, 2001

ABSTRACT—We investigated the effects of YM905 [(+)-(1S,3'R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate monosuccinate], a new orally active muscarinic M3-receptor antagonist, on bowel dysfunction in vivo using experimental models that reproduce the symptoms present in irritable bowel syndrome (IBS). YM905 potently inhibited restraint stress-induced fecal pellet output in fed rats (ED50: 4.0 mg/kg) and diarrhea in fasted rats (ED50: 1.7 mg/kg), with similar potencies to the inhibition of bethanechol-, neostigmine- and nicotine-induced fecal pellet output in rats (ED50: 3.3, 7.9 and 4.5 mg/kg, respectively). YM905 also inhibited 5-hydroxytryptamine (5-HT)-, prostaglandin E2- and castor oil-induced secretory diarrhea in mice (ED50: 5.5, 14 and 6.3 mg/kg, respectively), but showed no significant effect on cholera toxin-induced intestinal secretion in mice. In addition, YM905 (3, 10 mg/kg) reversed morphine- decreased postprandial defecation in ferrets, a model of spastic constipation, whereas remosetron, a 5-HT3- receptor antagonist, was not effective. The mode of YM905 action was similar to that of darifenacin, a selective M3-receptor antagonist, with equivalent potencies. By contrast, propantheline, an antimuscarinic drug that has been used for IBS, was much less potent. These results show that YM905 ameliorates a wide spec- trum of bowel dysfunctions through the blockade of M3 receptors, suggesting its therapeutic potential for treating IBS.

Keywords: YM905, Muscarinic M3 receptor, Irritable bowel syndrome, Restraint stress, Defecation

Irritable bowel syndrome (IBS) is a common functional ileum (10) and colon (11) under physiological conditions. disorder characterized by symptoms of altered bowel habits, M3-receptor antagonists that display fewer effects on M2 such as frequent stool, diarrhea and/or spastic constipation receptors would therefore have some therapeutic utility (1, 2), probably resulting from a dysfunction of the auto- because they scarcely cause M2-associated side effects. nomic nerve system (3, 4). In the current therapy, anti- YM905 [(+)-(1S,3'R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4- cholinergic agents have become the standard class of drugs tetrahydroisoquinoline-2-carboxylate monosuccinate, Fig. 1] for the control of IBS (5), since they suppress the cholinergic is a novel muscarinic M3-receptor antagonist that shows a neurotransmission that excitatory regulates gastrointestinal higher binding affinity for exogeneously expressed human motility by blockage of the muscarinic receptors. Muscarinic acetylcholine receptors have been catego- rized into five subtypes (M1 –M5) genetically and pharma- cologically, although the distribution and biological sig- nificance of the M4 and M5 subtypes are not yet well understood (6). Smooth muscle tissues heterogeneously express the M2 and M3 subtypes with a population of approximately 4:1 (7, 8), and M3 receptors are known to play a predominant role in contractile responses in intestinal smooth muscle tissues, including the small intestine (9),

*Corresponding author. FAX: +81-298-54-1616 E-mail: [email protected] Fig. 1. Chemical structure of YM905.

281 282 S. Kobayashi et al.

M3 receptors (Ki: 9.9 nM) than for M1 and M2 receptors (3 mg/kg), neostigmine (0.1 mg/kg) or nicotine (1 mg/kg). (Ki: 24 and 120 nM, respectively) as reported previously A preliminary experiment showed that each agent, at the (12). In the present study, we evaluated the therapeutic dosage described above, produces a submaximal increase potential of YM905 for the treatment of IBS by using experi- in fecal pellet output in fed rats, respectively. The animals mental animal models that reproduce bowel abnormalities were then placed in individual cages at room temperature, closely similar to those observed in IBS in vivo. YM905 and the number of fecal pellets excreted in the subsequent was examined for inhibitory activity on bowel dysfunction 2-h periods was counted. The test drug was given orally models caused by restraint stress in rats and on secretory 1 h before administration of bethanechol, neostigmine or diarrhea models in mice. YM905 was also tested on mor- nicotine. phine-suppressed postprandial defecation in ferrets, an experimental model of spastic constipation. The effects of 5-HT-, PGE2- and castor oil-induced diarrhea in mice YM905 were compared with those of darifenacin, a selec- According to the previously reported method (13), diar- tive M3-receptor antagonist, and propantheline, an antimus- rhea was induced by an injection of 5-hydroxytryptamine carinic agent that has been used clinically for the control (5-HT) (3 mg/kg, i.p.), prostaglandin E2 (PGE2) (0.3 mg/kg, of IBS. i.p.) or castor oil (0.3 ml/animal, p.o.). The animals were then maintained in individual cages and observed for 3 h at MATERIALS AND METHODS room temperature. The occurrence of diarrhea was defined as the evacuation of watery or unformed stools during the Animals observation period. The test drug was given 1 h before Male Sprague-Dawley rats (190 – 270 g) and male ICR administration of 5-HT, PGE2 or castor oil. mice (29 – 42 g) were purchased from Japan SLC (Shizu- oka). The animals were maintained in a 12-h light-dark Morphine-induced spastic constipation in ferrets cycle at room temperature (23°C) and were provided food The ferrets were fed at 10 a.m. on the day of the experi- and water ad libitum up to the time the experiment began, ment and treated subcutaneously with morphine (0.3 mg/kg) except when stated otherwise. Male ferrets (0.9 – 1.5 kg) 90 min later. The test drug was given orally 90 min after were purchased from Marshall Farms (North Rose, NY, the morphine treatment and the number of pellets excreted USA). The ferrets were housed under the same conditions during the 2-h period following drug administration was as described above and were fed once a day with free access counted. The ferrets were used in a randomized crossover to water. In the present study, all the animals were used for design, with a washout period of at least 7 days. the experiments with approval from the institute’s animal ethics committee at Yamanouchi Pharmaceutical Co., Ltd. Cholera toxin-induced intestinal fluid secretion in mice Mice fasted overnight were used in the experiment. Restraint stress-induced bowel dysfunction in rats Under ether anesthesia, the small intestine was closed by The effects of YM905, darifenacin and propantheline on ligating both the pyloric and ileocecal regions, and 100 mg restraint stress-induced bowel dysfunction models in rats of cholera toxin in a 0.2 ml saline solution was then injected were examined according to the method of Miyata et al. into the ligated intestinal loop. The loop was isolated and (13). In the first series, a restraint stress-induced fecal pellet weighed 3 h after cholera toxin injection. The net fluid output model was produced using rats with no restrictions secretion was defined as the difference in the weights of on food intake before testing. The rats were restrained in the loop determined before and after drying. The test drug individual restraint cages (KN-468, 53 ´ 47 ´ 200 mm; was injected into the loop immediately after cholera toxin Natsume Seisakusho, Tokyo) for 1 h at room temperature. injection. The number of fecal pellets excreted during restraint stress was counted. In the second series, a stress-induced diarrhea Data analyses model was produced using rats fasted overnight. The animals The value for the number of fecal pellets represents the were stressed for 3 h by the same procedure as described mean ± S.E.M. and the value for diarrhea the percentage of above, and the occurrence of diarrhea was defined as the incidences. A comparison of the values for fecal output evacuation of watery or unformed stools during restraint between the control and each treatment group was deter- stress. In each experiment, the test drug was given orally mined statistically using an analysis of variance (ANOVA), 1 h before exposure to restraint stress. followed by Dunnett’s multiple range test. The incidences of diarrhea (number of animals with diarrhea/total number Bethanechol-, neostigmine- and nicotine-induced fecal of animals tested) were compared by Pearson’s Chi-square pellet output in rats test or Fisher’s exact probability test. Probabilities of less Rats were subcutaneously administered bethanechol than 0.05 were considered significant. A 50% effective dose Effects of YM905 on Bowel Dysfunction 283

(ED50) value with a 95% confidence limit was estimated (Kanto Chemical, Tokyo); morphine hydrochloride (Takeda as a potency for the test drug by linear regression analysis. Chemical Industries, Osaka). The doses of the test drugs All the statistical analyses were performed using the SAS and reagents are expressed in terms of the free base. statistical software package (SAS Institute, Carry, NA, USA). RESULTS

Chemicals Effects of YM905 on stress-induced defecation and diar- YM905, darifenacin and ramosetron hydrochloride rhea (YM060) were synthesized at Yamanouchi Pharmaceutical Figure 2a shows the effects of YM905, darifenacin and Co., Ltd. (Ibaraki). Propantheline bromide was purchased propentheline on restraint stress-induced fecal pellet output from Sigma (St. Louis, MO, USA). The test drugs were in fed rats. The number of fecal pellets counted during the administered orally in 0.5% (w/v) methylcellulose solution observation period was negligible in unrestrained normal or intravenously in physiologic saline solution. The other rats. Restraint stress caused fecal pellet output with pellet reagents used were as follows: bethanechol, neostigmine counts of 6.0 ± 0.8 for the control group for YM905 and bromide, PGE2 and cholera toxin (Sigma); nicotine and darifenacin and 4.7 ± 0.6 for the control group for propan- 5-HT hydrochloride (Nacalai Tesque, Kyoto); castor oil theline (n = 10). YM905 at oral doses of 1 – 30 mg/kg

Fig. 2. Effects of YM905, darifenacin and propantheline on restraint stress (a)-, bethanechol (b)-, neostigmine (c)- and nicotine (d)-induced increases in fecal pellet output in rats. The drugs were administered orally 1 h before restraint stress or injection of bethanechol (3 mg/kg, s.c.), neostigmine (0.1 mg/kg, s.c.) or nicotine (1 mg/kg, s.c.). Each bar represents the mean ± S.E.M. for 9 – 10 animals. *P<0.05, **P<0.01, ***P<0.001 vs control group (Dunnett’s multiple range test). 284 S. Kobayashi et al. dose-dependently decreased the number of pellets excreted 1.0, 7.3 ± 0.6 and 4.2 ± 0.6 for the control group for YM177 with an ED50 value (with 95% confidence interval) of 4.0 and darifenacin and 8.0 ± 0.9, 8.3 ± 0.7 and 4.7 ± 0.6 for (1.8 – 7.3) mg/kg, p.o. Darifenacin (0.3 – 10 mg/kg, p.o.) the control for propantheline, respectively (n = 10). Oral and propantheline (10 – 300 mg/kg, p.o.) also decreased administration of YM905 (1 – 30 mg/kg), darifenacin (0.3 – the fecal pellet count with ED50 values of 4.4 (2.4 – 14) 10 mg/kg) and propantheline (10 – 300 mg/kg) dose- and 41 (20 – 73) mg/kg, p.o., respectively. In another ex- dependently suppressed bethanechol-induced fecal pellet periment using rats fasted overnight, restraint stress caused output, with ED50 values of 3.3 (2.1 – 4.8), 2.7 (2.0 – 3.9) diarrhea with an incidence of 100% in the vehicle-control and 67 (48 – 97) mg/kg, p.o., respectively (Fig. 2b). Oral group. As shown in Fig. 3a, YM905 (0.3 – 10 mg/kg, p.o.), YM905, darifenacin and propantheline also caused a de- darifenacin (0.1 – 3mg/kg, p.o.) and propantheline (10 – crease in neostigmine- and nicotine-induced fecal pellet 300 mg/kg, p.o.) dose-dependently decreased the incidences output dose-dependently. Their ED50 values were 7.9 of diarrhea with ED50 values of 1.7 (0.6 – 4.8), 0.9 (0.3 – (3.8 – 22), 4.4 (2.1 – 19) and 56 (40 – 79) mg/kg, p.o. for 6.0) and 64 (29 – 160) mg/kg, p.o., respectively. neostigmine-induced defecation and 4.5 (2.0 – 8.7), 1.7 (0.7 – 4.4) and 97 (52 – 270) mg/kg, p.o. for nicotine- Effects of YM905 on bethanechol-, neostigmine- and nico- induced defecation, respectively (Fig. 2: c and d). In the tine-induced fecal pellet output preliminary experiment, the potencies of YM905 and Subcutaneous administration of bethanechol (3 mg/kg), darifenacin on bethanechol-induced fecal pellet output neostigmine (0.1 mg/kg) or nicotine (1 mg/kg) induced were estimated for the intravenous route as ED50 values of fecal pellet output in fed rats, with pellet counts of 9.2 ± 2.3 (1.6 – 3.4) and 0.2 (0.1 – 0.3) mg/kg, respectively (data

Fig. 3. Effects of YM905 (square), darifenacin (circle) and propantheline (triangle) on restraint stress (a)-induced diarrhea in fasted rats and 5-HT (b)-, PGE2 (c)- and castor oil (d)-induced diarrhea in mice. The drugs were administered orally 1 h before restraint stress or injection of 5-HT (3 mg/kg, i.p.), PGE2 (0.3 mg/kg, i.p.) or castor oil (0.3 ml/animal, p.o.). Each point represents the % inhibition of diarrhea in 7 – 10 animals. *P<0.05, **P<0.01, ***P<0.001 vs control (? 2 test). Effects of YM905 on Bowel Dysfunction 285 not shown). stimulated fecal pellet output in ferrets. In the morphine- treated control group, about half of the ferrets defecated Effects of YM905 on 5-HT-, PGE2- and castor oil-induced only once during the 2-h periods. Oral doses of YM905 (3, diarrhea 10 mg/kg) significantly increased the morphine-decreased The effects of YM905, darifenacin and propantheline on number of fecal pellets dose-dependently, and the number of 5-HT-, PGE2- and castor oil-induced diarrhea in mice are pellets for the 10 mg/kg-treated group was approximately shown in Fig. 3, b – d. Administration of 5-HT (3 mg/kg, equal to that for the group not treated with morphine. i.p.), PGE2 (0.3 mg/kg, i.p.) or castor oil (0.3 ml/animal, Darifenacin (10 mg/kg, p.o.) also significantly increased p.o.) caused diarrhea in vehicle-treated control mice with the morphine-decreased number of pellets. The selective 5- an incidence of 100%. YM905 (0.3 – 10 mg/kg, p.o.), HT3-receptor antagonist ramosetron, on the other hand, had darifenacin (0.1 – 3mg/kg, p.o.) and propantheline (100 – no significant effect on morphine-induced constipation at 1000 mg/kg, p.o.) dose-dependently decreased the inci- doses up to 0.1 mg/kg, p.o. (Fig. 4). dence of diarrhea caused by 5-HT and PGE2, with ED50 values of 5.5 (2.7 – 11), 2.6 (1.2 – 8.0) and 330 (150 – Effect of YM905 on cholera toxin-induced intestinal fluid 1200) mg/kg, p.o. for 5-HT-induced diarrhea and 14 (2.6 – secretion in mice 330), 3.7 (0.9 – 20) and 180 (20 – 440) mg/kg, p.o. for Injection of cholera toxin significantly increased intestinal PGE2-induced diarrhea, respectively. In contrast, although fluid secretion in mice with net fluid volumes of 1.6 ± 0.1 YM905 and propantheline decreased the incidence of diar- and 3.1 ± 0.2 ml for the cholera toxin-untreated and treated rhea caused by castor oil dose-dependently, with ED50 groups, respectively (P<0.001, n = 10). YM905, at intra- values of 6.3 (2.6 – 17) and 290 (71 – 1200) mg/kg, p.o., duodenal doses of 3, 10 and 30 mg/kg, showed no signifi- respectively, darifenacin did not cause significant inhibi- cant effect on cholera toxin-induced increase in intestinal tion of castor oil-induced diarrhea at doses up to 30 mg fluid secretion with net fluid volumes of 2.9 ± 0.1, /kg, p.o. 3.0 ± 0.2 and 3.0 ± 0.2 ml, respectively (n = 10). Intra- duodenal darifenacin (30 mg/kg) and propantheline (100 mg Effect of YM905 on morphine-induced spastic constipation /kg) also did not affect the intestinal secretion with fluid Feeding stimulates colonic motility via the so-called volumes of 3.0 ± 0.1 (n = 10) and 3.7 ± 0.3 ml (n = 8), gastrocolonic response under nervous regulation (14), respectively. thereby resulting in the occurrence of defecation. In the present study, feeding caused almost all of the ferrets not DISCUSSION treated with morphine to defecate 1 or 2 pellets during the 2-h observation periods. Subcutaneously administration of Stress is known to be an important factor in causing IBS, morphine (0.3 mg/kg) significantly decreased feeding- since it significantly alters bowel functions (15). Several

Fig. 4. Effects of YM905 (left panel), ramosetron and darifenacin (right panel) on morphine-induced spastic constipation in ferrets. The drugs were administered orally 90 min after morphine injection (0.3 mg/kg, s.c.). The results are expressed as the number of pellets excreted during the 2-h period following drug administration. ††P<0.01, †††P<0.001 vs normal group; *P<0.05, **P<0.01, ***P<0.001 vs control group (Dunnett’s multiple range test). 286 S. Kobayashi et al. rodent models of bowel dysfunction caused by restraint tract, animal models for spastic constipation have been stress have been investigated for pharmacological analysis poorly developed. In the present study, the effect of YM905 of a stress-related bowel disorder like IBS (13, 16, 17). on spastic constipation was tested using the morphine- Previous studies demonstrated that restraint stress results induced constipation model in ferrets, whose gastro- in an increase in fecal pellet output in fed rats, as well as in intestinal motility pattern resembles that in humans (24). diarrhea in food-deprived rats, which are equally mediated Since morphine, a m-opioid receptor agonist, causes consti- through the endogenous activation of the 5-HT3 receptor pation in vivo by potentiating intermittent tonic contraction (13). 5-HT3 receptors are prevalent in enteric neurons that and reducing propulsive waves of the colon (25, 26), this are responsible for a variety of gastrointestinal functions model can reproduce the bowel behavior similar to that (18), and their activation is thought to enhance cholinergic shown in IBS patients with spastic constipation. Our results transmission via the release of acetylcholine from parasym- showed that YM905 reverses morphine-induced constipa- pathetic nerve terminals (13), thus resulting in an increase tion with a similar potency to those shown in improving in gastrointestinal motility and fluid secretion. In the defecation and diarrhea. The exact mechanism explaining present study, oral YM905 potently suppressed both fecal why YM905 blocked the action of morphine remains output and diarrhea caused by restraint stress within an unknown, since, at least from the in vitro experiments, equal dose range. YM905 also suppressed cholinergic morphine is generally known as an inhibitor of acetyl- enhancement of fecal output responses following injection choline release from cholinergic nerve terminals in the of bethanechol (a muscarinic agonist), neostigmine (an myenteric plexus (27). In the enteric nervous system, how- acetylcholine esterase inhibitor) and nicotine (an agent that ever, m-opioid receptors are located in not only cholinergic releases acetylcholine from nerve endings), with similar neurons, but also adrenergic neurons that negatively regu- potencies to those in restraint stress models. In addition, late cholinergic tone (28). Moreover, it has been reported YM905 does not have a significant affinity for any 5-HT- that stimulation of the m-opioid receptor enhances intesti- receptor subtypes (K. Ikeda et al., unpublished data). The nal contraction by preventing the adrenergic inhibition of inhibitory effects of YM905 in restraint stress models are acetylcholine release from cholinergic neurons (28, 29), therefore caused by the antimuscarinic action. providing the putative mechanism whereby morphine in- The antidiarrheal activity of YM905 was also evaluated duces intestinal spasm. This notion is supported by the using exogenous 5-HT-, PGE2- and castor oil-induced diar- previous report showing that morphine-caused colonic rhea in mice, which are well-known models for secretory hypermotility was abolished by pretreatment with atropine diarrhea (19 – 21). Although 5-HT, PGE2 and castor oil in conscious dogs (30). Taken together, YM905 improved stimulate diarrhea through distinct mechanisms of action, constipation possibly by inhibiting the cholinergically- these diarrhea models are suitable for evaluating the anti- enhanced intestinal segmental spasms. It has been reported diarrheal activity of drugs, since these models are reported that ramosetron, a selective 5-HT3-receptor antagonist, to be sensitive to clinically efficacious antidiarrheal drugs suppresses restraint stress-induced bowel dysfunction and such as loperamide (22) and trimebutine (23). In the present 5-HT-induced diarrhea (13, 22), and this class of agent study, the antidiarrheal activities of YM905 were similarly holds therapeutic promise for gastrointestinal disorders, observed in these models, and the potencies were almost including IBS (18). In the present study, ramosetron had equivalent to those in both of cholinergically-enhanced no effects on ferret constipation, even at doses sufficient and stress-induced bowel dysfunction models. This YM905 to protect against stress-induced bowel dysfunction. These activity pattern resembles that of atropine, a non-selective findings suggest that YM905 may be efficacious for spastic muscarinic antagonist, as reported previously (13). How- constipation in IBS that is resistant to the 5-HT3-receptor ever, YM905 actions are distinguishable from those of tri- antagonist. mebutine since trimebutine is not efficacious for inhibiting In the present study, the effects of YM905 were com- restraint stress-induced fecal pellets output (23). In addi- pared with those of darifenacin, a selective M3-receptor tion, YM905 showed no effect on cholera toxin-induced antagonist, to validate the role of M3 receptors in stress- intestinal fluid secretion in vivo, which is mediated via related bowel dysfunction, because YM905 selectivity for the activation of the cyclic AMP pathway, suggesting that muscarinic receptor subtypes seems not to be sufficient to YM905 may have no other antisecretory effect beyond its distinguish the inhibitory action on smooth muscle M3 antimuscarinic action. The antidiarrheal actions of YM905 receptors from that on ganglionic M1 receptors facilitating therefore appear to be predominantly dependent on its vagal neurotransmission. Darifenacin displays a more than inhibitory effects on cholinergic-mediated enteric propul- tenfold selectivity for M3 receptors as compared not only sion of watery stool, rather than its antisecretory action. with M2 but also with M1 receptors, as a result of which Although a large proportion of IBS patients suffer consti- the compound has been extensively used as a tool for phar- pation derived from spasms of the lower gastrointestinal macologically characterizing the smooth muscle M3 recep- Effects of YM905 on Bowel Dysfunction 287 tors (6, 7, 31). In the present study, darifenacin suppressed suppressed morphine-induced spastic constipation. 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