Effects of Novel TRPA1 Receptor Agonist ASP7663 in Models of Drug-Induced Constipation and Visceral Pain

European Journal of Pharmacology 723 (2014) 288–293

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European Journal of Pharmacology

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Neuropharmacology and analgesia Effects of novel TRPA1 receptor agonist ASP7663 in models of drug-induced constipation and visceral pain

Ryosuke Kojima a,n, Katsura Nozawa a, Hitoshi Doihara a, Yoshihiro Keto a, Hidetaka Kaku b, Toshihide Yokoyama a, Hiroyuki Itou a a Pharmacology Research Labs, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan b Chemistry Research Labs, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan article info abstract

Article history: Constipation is a major gastrointestinal motility disorder with clinical need for effective drugs. We Received 14 May 2013 previously reported that transient receptor potential ankyrin 1 (TRPA1) is highly expressed in Received in revised form enterochromaffin (EC) cells, which are 5-hydroxytryptamine (5-HT)-releasing cells, and might therefore 17 October 2013 be a novel target for constipation. Here, we examined the effects of ASP7663, a novel and selective TRPA1 Accepted 3 November 2013 agonist, in constipation models as well as an abdominal pain model. ASP7663 activated human, rat, and Available online 28 November 2013 mouse TRPA1 and released 5-HT from QGP-1 cells, and oral but not intravenous administration Keywords: of ASP7663 significantly improved the loperamide-induced delay in colonic transit in mice. While TRPA1 pretreatment with the TRPA1 antagonist HC-030031 and vagotomy both inhibited the ameliorating effect IBS of oral ASP7663 on the colonic transit, both orally and intravenously administered ASP7663 significantly Constipation model inhibited colorectal distension (CRD)-induced abdominal pain response in rats. Taken together, these Abdominal pain results demonstrate that ASP7663 exerts both anti-constipation and anti-abdominal pain actions, the former is likely triggered from the mucosal side of the gut wall via activation of vagus nerves while the latter is assumed to be provoked through systemic blood flow. We conclude that ASP7663 can be an effective anti-constipation drug with abdominal analgesic effect. & 2013 Elsevier B.V. All rights reserved.

1. Introduction focused on its role in diverse sensory processes including cold nociception, hearing and inflammatory pain (Bautista et al., 2006; Constipation is a common gastrointestinal disorder character- Kwan et al., 2006; Nagata et al., 2005). We previously reported ized by such symptoms as straining, hard stool, and infrequent that TPRA1 is extensively expressed in EC cells of the gastro- defecation. Population-based studies have shown that the pre- intestinal tract, and stimulation of TRPA1 activates gastrointestinal valence of constipation is up to 30% of the population in developed motility (Doihara et al., 2009b; Kojima et al., 2009; Nozawa et al., countries (Drossman et al., 1993; Everhart et al., 1989; Longstreth 2009). The above present TRPA1 as a potential novel therapeutic et al., 2006; Pare et al., 2001). Current drug treatments for target for constipation. constipation are based on traditional drugs, such as osmotic or Here, we investigated the therapeutic potential of (E)-[7-fluoro- secretory laxatives and bulking agents, and fail to offer sufficient 1-(2-methylpropyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]acetic improvement in condition due to poor efficacy, late onset, and acid (ASP7663; Fig.1), a newly synthesized TRPA1 agonist, as abdominal side effects such as pain, cramps and bloating (Johnson, a potential anti-constipation agent. 2006; Jones et al., 2002). As such, an anti-constipation drug with good efficacy and no abdominal side effects is awaited. TRPA1 is a member of the TRP channel family and is activated 2. Materials and methods by various physiochemical stimuli, including cold temperatures and pungent natural chemicals such as aryl isothiocyanate (AITC) 2.1. In vitro studies and cinnamaldehyde (Bandell et al., 2004; Bautista et al., 2005; 2þ fl Jordt et al., 2004). Functional studies of TRPA1 have mainly 2.1.1. Ca in ux assay Following a previously described method (Nozawa et al., 2009), we examined the TRPA1 agonist potential of ASP7663 for human,

n rat and mouse TRPA1. Brieﬂy, HEK293 cells expressing human, Correspondence to: Drug Discovery Research, Astellas Pharma Inc., 21Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Tel.: þ81 29 863 7063; fax: þ81 29 852 2955. rat, or mouse TRPA1 were seeded into black-walled, clear-base E-mail address: [email protected] (R. Kojima). 96-well poly-D-lysine-coated plates in 100 μL of DMEM media

anus using a silicon-tipped rod. After recovery from the anesthesia, the time required for bead evacuation was measured. Loperamide or clonidine and ASP776 were administered subcutaneously 30 min before and orally 3 min before bead insertion, respectively. In the intravenous administration study, ASP7663 was administered into the tail vein 3 min before bead insertion. In the antagonist study, HC- 030010andASP7663wereorallyadministrated 45 and 30 min before bead insertion, respectively. Further characterization of the prokinetic effects of ASP7663 was conducted in vagotomized mice. Vagotomy was performed following the method of Ghia et al. with minor modiﬁcations (Ghia et al., 2006). Mice were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg), and ventral and dorsal truncal branches of the subdiaphragmatic vagi were cut apart. After a one-week recovery period, the mice were used for bead transit experiments.

Fig. 1. Chemical structure of ASP7663.

2.2.2. Abdominal pain with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin To assess the influence of ASP7663 on abdominal pain, we at densities of 16,000, 20,000, and 20,000 cells per well, respec- evaluated the effect on colorectal distension (CRD)-induced pain tively. The cells were cultured overnight at 37 1C in an atmosphere behavior by the method of Fioramonti et al. with small modifications of 5% CO2. On the day of measurement, cells were incubated for 2 h (Fioramonti et al., 2003). Briefly, male Wistar rats (280–350 g; Japan at room temperature in Hank's Balanced Salt Solution containing SLC, Inc.) were lightly anesthetized with 5% isoflurane, and a 5-cm 20 mmol/L HEPES pH 7.4 and 0.02% CHAPS (HBSS–HEPES–CHAPS) long latex balloon was carefully inserted into the colorectal area via with the addition of the calcium indicator Fluo-4 AM at 2 μmol/L. the anus and positioned such that the end of the balloon was 1 cm The cells were washed twice with 80 μL of HBSS–HEPES–CHAPS, from the anus. The balloon catheter was then fixed in place by and 80 μL of the buffer was added prior to the assay. taping to the base of the tail, and the rats were placed in individual Plates were placed into the fluorometric imaging plate reader cages. After recovery from anesthesia, CRD was loaded using a (FLIPR; Molecular Devices Corporation Japan, Tokyo, Japan) to computerized barostat (DISTENDER SERIES IIRs; G&J Electronics measure cell fluorescence. After addition of ASP7663, the change Inc.,Ontario,Canada).Theballoonwasinflated progressively in 15- in fluorescence was monitored for 30 min, and the differences mmHg steps from 15 to 60 mmHg, with each step of inflation lasting between maximum and minimum values in the fluorescent signal 5 min with 5 min intervals. The number of abdominal contractions were noted. during inflation was visually counted for each step. ASP7663 or vehicle was orally or intravenously administered 5 min before the fl 2.1.2. 5-HT release assay start of the in ation. QGP-1 cells obtained from HSRRB (HSRRB JCRB0183) were seeded at 3 105 cells/mL/well in 24-well plates and cultured for 3 days. The cells were rinsed 3 times with HBSS containing 2.3. Drugs 0.5% FBS and 2 μmol/L fluoxetine (Tocris, Ellisville, MO, USA). The HBSS–FBS–fluoxetine was then removed and replaced with ASP7663 and HC-030010 were synthesized at Astellas Pharma 200 μL HBSS–FBS–fluoxetine containing ASP7663 and incubated Inc. (Tokyo, Japan). Loperamide hydrochloride (loperamide) and for 60 min at 37 1C. The assay buffer was collected and centrifuged clonidine hydrochloride (clonidine) were purchased from Sigma- for 5 min to remove any detached cells. The supernatants were Aldrich (St. Louis, MO, USA). ASP7663 was dissolved in dimethyl- – – collected and stored at 80 1C until 5-HT measurement. The 5-HT sulfoxide (DMSO) and diluted with HBSS HEPES CHAPS in the 2 þ – –fl concentration was measured using an enzyme immunoassay (EIA) Ca assay or with HBSS FBS uoxetine in the 5-HT release assay. kit (Immunotech, Marseille, France) as per the manufacturer's In the animal studies, ASP7663 was dissolved in 10% dimethylfor- instructions. Absorbance at 405 nm in each well was measured mamide and 10% Tween 80/distilled water or 0.5% methylcellulose using a spectrophotometer (SpectraMaxs Molecular Devices Cor- (MC) in the oral administration, and 5% dimethylformamide poration Japan, Tokyo, Japan). and 5% cremophore/saline in the intravenous administration. HC-030010 was suspended in 0.5% MC. Loperamide and clonidine were dissolved in 1% Tween 80/saline and saline, respectively. 2.2. In vivo studies

All animal experimental procedures were approved by the 2.4. Statistical analyses Committee for Animal Experiments of Astellas Pharma Inc. The – animals were housed under a 12-h light dark cycle in a controlled- The EC value of each experiment was calculated by Sigmoid- 7 1 50 temperature environment (23 2 C) with food and water available Emax non-linear regression analysis. The geometric mean and the ad libitum. 95% confidence intervals (95% CI) of 4 individual experiments performed in triplicate were calculated. In the animal studies, 2.2.1. Drug-induced constipation models all data are expressed as the mean7standard error of the mean Effects of ASP7663 in drug-induced constipation models were (S.E.M.). The significance of differences between two groups was evaluated by the bead transit method described previously (Kojima determined using the unpaired Student's t-test, whereas that et al., 2009). Briefly, male ddY mice (Japan SLC, Inc., Shizuoka, Japan; among more than two groups was determined using Dunnett's weighing 27–30 g) were fasted overnight with free access to water multiple comparison test. Statistical significance was defined as prior to the experiments. Under light ether anesthesia, a glass bead Po0.05. In the case of multiple-point comparisons, Bonferroni's (3 mm in diameter) was inserted into the distal colon 2 cm above the type adjustment was made. 290 R. Kojima et al. / European Journal of Pharmacology 723 (2014) 288–293

3. Results clonidine (0.01 mg/kg) or loperamide (0.3 mg/kg) significantly delayedbeadexpulsion(Fig. 4). Orally administered ASP7663 shor- 3.1. Ca2 þ influx assay tened the prolonged bead expulsion time caused by loperamide, and this effect was statistically significant at doses of 0.3 and 1 mg/kg ASP7663 concentration dependently increased intracellular (Fig. 4A). ASP7663 also improved the delayed bead transit induced Ca2 þ concentration in human, rat, and mouse TRPA1 expressed by clonidine, an effect that was statistically significant at doses of in HEK293 cells in a similar manner, with respective EC50 values 1and3mg/kg(Fig. 4B).ASP7663,however,didnotimprovethe (95% confidence interval [CI]) of 0.51 (0.40–0.66), 0.54 (0.41–0.72), loperamide-induced delay in bead transit by intravenous adminis- and 0.50 (0.41–0.63) μmol/L (Fig. 2). Of note, the increases in tration (Fig. 5A). The improvement effect of oral ASP7663 was intracellular Ca2 þ induced by 10 μmol/L ASP7663 were abolished abolished entirely in vagotmized mice (Fig. 5B) and inhibited by oral by pretreatment with 10 μmol/L HC-030031, a TRPA1 antagonist pretreatment with HC-030031 100 mg/kg (Fig. 5C). (Fig. 2). 3.4. Abdominal pain 3.2. 5-HT release assay We evaluated the effect of ASP7663 on abdominal pain using the ASP7663 concentration-dependently stimulated 5-HT release CRD model. The number of abdominal contractions was increased from QGP-1 cells, a lineage of TRPA1-expressing EC cells (Doihara with colorectal pressure level (15–60 mmHg). Compared with vehicle fi et al., 2009b), with an EC50 value of 72.5 (52.6–99.9) μmol/L treatment, ASP7663 at oral doses of 1 and 3 mg/kg signi cantly (Fig. 3). reduced the number of abdominal contractions evoked during CRD at pressures of 30, 45, and 60 mmHg (Fig. 6A). ASP7663 also reduced the 3.3. Colonic transit number of abdominal contractions by intravenous treatment (Fig. 6B). No behavior indicative of pungency was observed after administration We examined the anti-constipation efficacy of ASP7663 using two of ASP7663. different constipation models in mice. Subcutaneous treatment with 4. Discussion

In the present experiments, we showed that ASP7663 concentration-dependently increased intracellular Ca2 þ concentration in human, rat, and mouse TRPA1-expressing HEK293 cells at the same potency, and that the stimulating effects of ASP7663 were abolished by HC-030031. These results indicate that ASP7663 is a potent TRPA1 agonist with no species difference. We also confirmed that ASP7663 had no or only slight affinity to more than 60 receptors, including channels and enzymes like TRPV1 (see Supplemental table S1 and Supplemental figure S1). We previously reported that QGP-1, a human pancreatic endocrine cell line, highly expresses bothTRPA1andtryptophanhydro- xylase and releases 5-HT upon stimulation with TRPA1 agonists such as AITC and cinnamaldehyde (Doihara et al., 2009b). These features are in good accordance with those of EC cells (Nozawa et al., 2009), indicating that QGP-1 is a good model for investigating the functional Fig. 2. Activating effects of ASP7663 on human, rat, and mouse TRPA1. (A) ASP7663 significance of the 5-HT-releasing activity of TRPA1 agonists. The increases the intracellular Ca2 þ concentration in HEK293 cells expressing human, 5-HT-releasing activity of ASP7663 observed in the present study rat and mouse TRPA1. HC-030031 (10 mmol/L) effects on ASP7663 (10 mmol/L) induced an increase in levels of intracellular Ca2 þ . Data are expressed as mean indicated that ASP7663 has the potential to release 5-HT from EC values7S.E.M. (n¼4). cells in the gut wall. Because peripheral 5-HT is mainly synthesized in EC cells and plays an important role in regulating gastrointestinal motility (Gershon, 2003; Spiller, 2007), we investigated ASP7663 effects on gastrointestinal motility in vivo. Although irritable bowel syndrome with constipation (IBS-C) and chronic constipation (CC) are the most common subtypes of functional constipations (Cash et al., 2007), the two differ substantially in terms of gastrointestinal motility, with IBS-C being spastic whereas CC is atonic. We previously characterized loperamide- and clonidine- induced delay in mouse colonic transit models as spastic and atonic constipation models, respectively (Kojima et al., 2009). In the present study, ASP7663 was effective in both models, suggesting its efficacy against both IBS-C and CC. While improvement of constipation in mice was observed immediately (3 min) after oral administration of ASP7663, it is unlikely that the compound reached the colon within such a short span time. Further, intravenous administration of ASP7663 did not affect the delay in colonic transit in mice, suggesting ASP7663 effects on colonic propulsion were not evoked via systemic blood Fig. 3. Effects of ASP7663 on 5-HT released from human QGP-1 cells. ASP7663 fl releases 5-HT from QGP-1 cells in a concentration-dependent manner. Data are ow. These results suggest that orally administered ASP7663 acts expressed as mean values7S.E.M. (n¼4). on the luminal surface of the upper gastrointestine, where EC cells R. Kojima et al. / European Journal of Pharmacology 723 (2014) 288–293 291

Fig. 4. Effects of orally administered ASP7663 in loperamide- and clonidine-induced constipation models in mice. ASP7663 improves delays in colonic propulsion induced by loperamide (A) and by clonidine (B). All values are represented as mean7S.E.M (n¼10). #, Po0.05 compared with the control group (Student's t-test); n, Po0.05 compared with the vehicle group (Dunnett's test).

Fig. 5. Effects of ASP7663 in the loperamide-induced constipation model in mice. (A) Effects of intravenous administration of ASP7663. (B) Effects of oral administration of ASP7663 in vagotomized mice. (C) Effects of oral administration of ASP7663 in HC-030031-pretreated mice. All values are presented as the mean7S.E.M (n¼5 or 6). #, Po0.05 compared with the control group (Student's t-test); n, Po0.05 compared with the vehicle group (Dunnett's test); ♭, between loperamide and its vehicle treated groups without ASP7663 treatment (Student's t-test).; $, Po0.05 between ASP7663 and its vehicle treated groups with loperamide (Student's t-test).

Fig. 6. Inhibitory effects of ASP7663 on colorectal distension in rat. ASP7663 decreased the number of abdominal contractions induced by increasing the pressure of colorectal distension. All values are presented as the mean7S.E.M (n¼6 or 8). n, Po0.0178 compared with the vehicle group (Dunnett's test (A) or Student's t-test(B) with Bonferroni correction). 292 R. Kojima et al. / European Journal of Pharmacology 723 (2014) 288–293 are abundant (Gershon and Tack, 2007; Rindi et al., 2004), in the direct effect on the afferent nerve may be expected. TRPV1, constipation models. Further, the efficacy of ASP7663 in mice was another member of the TRP family, is well known to be expressed abolished by the transection of the vagus nerves, which are known in the peripheral afferent nerve, with desensitization of TRPV1 by to act as an afferent pathway for the gastrocolic reflex by activating agonists inducing analgesic effects (Bhave et al., 2002; Vyklicky 5-HT from EC cells (Gershon and Tack, 2007; Lang, 1999). We have et al., 2008). Like TRPV1, TRPA1 is expressed in afferent nerves previously reported that AITC delays gastric emptying in rats and and reported to contribute to mechano- and chemo-sensations in that the effect of AITC is abolished with treatment of a 5-HT3 the colon (Brierley et al., 2009; La et al., 2011). Therefore, the antagonist or tryptophan hydroxylase inhibitor (Doihara et al., analgesic effect of ASP7663 on CRD-induced abdominal pain may 2009a). These results indicate TRPA1 agonists release 5-HT in the be mediated by direct desensitization of TRPA1 located on afferent upper gastrointestinal tract. Taken together, these results argue nerve terminal rather than 5-HT-mediated mechanisms. The pre- that orally administered ASP7663 stimulates 5-HT release from EC cise mechanisms underlining the analgesic effect of TRPA1 agonist cells located at the luminal surface of the upper gastrointestinal on abdominal pain remain to be elucidated. tract, and that the released 5-HT activates the vagal afferent nerve In summary, we showed here that ASP7663 is a potent TRPA1 to trigger the gastrocolic reflex. agonist with 5-HT-releasing efficacy. The compound enhanced Abdominal pain or discomfort is a common side effect of colonic motility and exerted analgesic effects against abdominal currently used anti-constipation agents (Johnson, 2006; Tack pain in animal models. Our present results therefore indicate et al., 2011). Bulk laxatives increase the water content in stools, that ASP7663 can be a novel constipation-improving agent with whereas osmotic agents attract water from the gastrointestinal abdominal analgesic effects. wall (Stephen and Cummings, 1979). These properties are sup- posed to increase the volume of gastrointestinal contents and cause bloating and abdominal cramping. Stimulant laxatives Appendix A. Supplementary material directly stimulate the colonic luminal surface and cause abdominal discomfort and cramps (Bengtsson and Ohlsson, 2005), and IBS-C Supplementary data associated with this article can be found in patients frequently complain of abdominal pain (Saito et al., 2002). the online version at http://dx.doi.org/10.1016/j.ejphar.2013.11.020. Moreover, currently available drugs fail to mitigate abdominal pain in constipation patients (Whitehead et al., 2011). We therefore References considered effects on abdominal pain as a criterion when evaluat- ing novel anti-constipation agents. The CRD model in rat is Bandell, M., Story, G.M., Hwang, S.W., Viswanath, V., Eid, S.R., Petrus, M.J., commonly used to evaluate drug effects on abdominal pain and Earley, T.J., Patapoutian, A., 2004. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41, 849–857. discomfort (Kiso et al., 2001; Liang et al., 2005). In this study, Bautista, D.M., Movahed, P., Hinman, A., Axelsson, H.E., Sterner, O., Hogestatt, E.D., ASP7663 showed a potent analgesic effect in this model which, in Julius, D., Jordt, S.E., Zygmunt, P.M., 2005. Pungent products from garlic activate addition to its anti-constipation effect, makes ASP7663 an appeal- the sensory ion channel TRPA1. Proc. Natl. Acad. Sci. USA 102, 12248–12252. Bautista, D.M., Jordt, S.E., Nikai, T., Tsuruda, P.R., Read, A.J., Poblete, J., Yamoah, E.N., ing agent. 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