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Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus
European Journal of Pharmacology 604 (2009) 132–137
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus Graeme L. Fraser d, Kalina Venkova b,c, Hamid R. Hoveyda d, Helmut Thomas d, Beverley Greenwood-Van Meerveld a,b,c,⁎ a
VA Medical Center, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA Department of Physiology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA d Tranzyme Pharma, Sherbrooke, QC, Canada, J1H 5N4 b c
a r t i c l e
i n f o
Article history: Received 10 June 2008 Received in revised form 21 November 2008 Accepted 9 December 2008 Available online 14 December 2008 Keywords: Ghrelin receptor TZP-101 Postoperative ileus Colonic transit Fecal pellet output (Rat)
a b s t r a c t Ghrelin, the natural ligand of the growth hormone secretagogue receptor (ghrelin receptor), is an orexigenic gut hormone with prokinetic action in the upper gastrointestinal tract. Previously we have shown in a rodent model of postoperative ileus that the synthetic ghrelin receptor agonist TZP-101 prevents the delay in gastric emptying and improves small intestinal transit. The goal of the present study was to investigate whether TZP-101 affects colonic transit and food intake in rats with postoperative ileus. Fasted rats were treated with morphine and subjected to laparotomy under isoflurane anesthesia. Following surgery the animals were placed in clean home cages and fecal pellet output and food intake were monitored for 48 h. TZP-101 or vehicle were administered as 3 i.v. bolus infusions at 0 h, 2 h and 4 h post-surgery. TZP-101 (0.03–1 mg/kg) dose-dependently decreased the time to first bowel movement and increased fecal pellet output measured at 12 h and 24 h post-surgery compared to the vehicle. The administration of TZP-101 was not associated with a significant alteration in food intake. In conclusion, this study provides the first experimental evidence that a novel ghrelin receptor agonist improves large bowel function in rats with postoperative ileus, suggesting that TZP-101 may be useful in the clinic to accelerate upper gastrointestinal transit and to shorten the time to the first bowel movement following surgery. Published by Elsevier B.V.
1. Introduction Postoperative ileus is the temporary impairment of gastrointestinal motility following abdominal surgery. The pathogenesis of prolonged ileus is positively correlated with the degree of surgical trauma and the use of opioids for pain management (Fotiadis et al., 2004; Artinyan et al., 2005). Surgical injury triggers local immune and nerve mediated mechanisms that inhibit coordinated gastrointestinal motility (Kalff et al., 1998; Boeckxstaens et al., 1999; Boeckxstaens, 2002; Bauer et al., 2002; de Jonge et al., 2003). In addition, exogenous opioids, administered to treat post-surgical pain, prolong the duration of ileus via the activation of peripheral opioid receptors inhibiting neurally mediated contractions as well as through direct effects on central opioid receptor signaling (Gmerek et al., 1986; GreenwoodVan Meerveld, 2007). Regardless of the abdominal site of surgical intervention, the spontaneous resolution of postoperative ileus ⁎ Corresponding author. VA Medical Center, Research Administration Rm. 151, 921 NE 13th street, Oklahoma City, OK 73104, USA. Tel.: +1 405 270 0501x3547; fax: +1 405 290 1719. E-mail address: [email protected] (B. Greenwood-Van Meerveld). 0014-2999/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.ejphar.2008.12.011
appears to be region-specific and is typically characterized by the initial recovery of the small intestine, followed by the stomach, and then the recovery of the colon (Graber et al., 1982; Waldhausen et al., 1990; Schippers et al., 1991). Since the first bowel movement is considered a clinical end point for the recovery of gastrointestinal function, assessment of the efficacy of new prokinetic drugs to reduce the time to first bowel movement following surgery in animal models is essential for the progress in postoperative therapy in humans. Ghrelin is an acylated peptide hormone secreted predominantly from endocrine cells in the gastric oxyntic mucosa (Kojima et al., 1999; Date et al., 2000). Ghrelin receptors regulating gastrointestinal function are expressed on vagal afferents (Sakata et al., 2003; Burdyga et al., 2006) and in the enteric nervous system (Dass et al., 2003; Xu et al., 2005), where ghrelin receptor expression appears to decrease distally from high levels in the stomach to lower levels in the colon (Dass et al., 2003; Kojima and Kangawa, 2005). The prokinetic actions of ghrelin on the stomach and small intestine arise from increased signaling through vagal afferents (Fujino et al., 2003; Murray et al., 2006) and direct activation of the enteric nervous system. Furthermore, previous studies have demonstrated that the administration of the ghrelin peptide or synthetic ghrelin receptor agonists restored
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gastric motility and accelerated upper gastrointestinal transit in various rodent models of ileus (Trudel et al., 2002; De Winter et al., 2004; Poitras et al., 2005; Sallam et al., 2007). A novel macrocyclic small molecule TZP-101 with peptidomimetic effects similar to the effects of ghrelin has been developed by Tranzyme Pharma for the treatment of gastrointestinal and metabolic disorders. The structure of TZP-101 features an 18-membered macrocycle containing 3 amide bonds and a secondary amine, as well as 4 stereogenic centers (Hoveyda et al., 2006; Sandham, 2008). Pharmacodynamic analyses suggested TZP-101 expresses activity at the ghrelin receptor (Lasseter et al., 2008). Using a rat model of postoperative ileus induced by abdominal surgery and morphine treatment we found that the synthetic ghrelin receptor agonist TZP-101 normalized gastric emptying and improved small intestinal transit (Venkova et al., 2007). Since patient recovery from postoperative ileus is actually assessed by the first bowel movement following surgery, an important study is to assess whether TZP-101 reduces the time to the first bowel movement in rats with postoperative ileus. Thus the current study describes a series of experiments to investigate whether systemic TZP-101 has an effect on fecal pellet output in rats with postoperative ileus. Although, there is no evidence to support a direct effect of ghrelin in the large intestine (Trudel et al., 2002; Edholm et al., 2004; Kitazawa et al., 2005; Bassil et al., 2005), we hypothesized that a ghrelin receptor agonist may shorten the overall transit time by stimulating gastric emptying and small intestinal transit. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (250–350 g) were purchased from Charles River Laboratories (Wilmington, MA) equipped with a
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polyurethane catheter implanted into the right jugular vein. The exterior end of the catheter was secured to the dorsal neck area and used for i.v. dosing of TZP-101 or vehicle. The catheters were flushed with heparinized saline once every 3–4 days but not on the day of the experiment. Rats that were not subjected to surgery and drug or vehicle treatment were equipped with catheters implanted into the proximal colon and used to infuse a dye marker for the measurement of colonic transit time. A total of 46 rats were used in the study. All animals were housed one per cage at standard conditions (21–22 °C, 12 h light/dark cycle, controlled humidity) with water and rat chow available ad libitum. At least one-week acclimation to the animal facility was allowed prior to the experiments. The experimental protocol and number of animals were approved by the Animal Care and Use Committees at the V.A. Medical Center and the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. 2.2. Abdominal surgery Prior to the experiments, rats were fasted for 20–22 h with free access to water. Postoperative ileus was induced by a surgical procedure described as “running of the bowel” (Kalff et al., 1998). Specifically, the rats were anesthetized with isoflurane (2–3% inhalation), the abdomen was shaved, disinfected and a midline incision was made to expose the abdominal viscera. The small intestine, the cecum and colon were exteriorized and inspected by gently pressing between two cotton applicators soaked in sterile saline. After completing the inspection, the intestines were covered with gauze soaked in saline and the abdomen was left open for 10 min. At the end of surgery 0.2 ml of dye (trypan-blue in saline) was carefully injected into the proximal colon (1 cm distal to the cecum) using a hypodermic needle. The viscera were then placed back into the
Fig. 1. Time-course of the changes in fecal pellet output, food intake and body weight in rats with postoperative ileus induced by morphine treatment followed by abdominal surgery compared to naïve rats. On the day of the experiment, all rats were fasted overnight and the naïve rats were placed in the observation cage supplied with food and water without been subjected to morphine treatment or surgery. Cumulative fecal output, expressed as the number of fecal pellets (A) or the stool weight (B), and food intake (C) were measured at 12 h. 24 and 48 h following the end of surgery. Body weight (D) was measured 24 h prior to fasting and at 24 h and 48 h of observation. Filled symbols represent individual experiments in rats with postoperative ileus while open symbols represent individual experiments in naïve rats. Mean values are from 6 naive rats and 8 rats with postoperative ileus. Statistical significance of differences was assessed by two-way ANOVA followed by Bonferonni's post test: ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001.
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2.4. Evaluation of food intake and body weight Food intake was also recorded at 3-h intervals during the first 12 h, and then every 12 h until the end of the 48-h observation period. Body weight was measured in fed rats 1 day prior to the experiment, in fasted rats before the surgery and at 24 h and 48 h post-surgery. 2.5. Experimental protocol
Fig. 2. Effect of TZP-101 on colonic transit time in rats with postoperative ileus. Results are presented as mean ± S.E.M. from 6–8 rats per group. The effects of increasing doses of 0.03–1 mg/kg i.v. TZP-101 were assessed for statistical significance by one-way ANOVA followed by Dunnett's post test for comparison to vehicle: ⁎⁎P b 0.01.
abdomen and the incision was closed with silk sutures. The whole procedure lasted 25–30 min and an additional period of 15 min was allowed for the animals to recover completely from the anesthesia before being treated with the drug or the vehicle. 2.3. Evaluation of colonic transit time and fecal pellet output Trypan blue, which is innocuous and non-absorbable by the gastrointestinal tract, was used to mark the fecal material and allow the measurement of colonic transit time (the time between the end of surgery and the appearance of the first dyed fecal pellet) in rats with postoperative ileus. A subgroup of rats not subjected to surgery, drug or vehicle treatment was fasted overnight and studied simultaneously with postoperative ileus rats as a reference to healthy controls. These animals had catheters implanted into the proximal colon to provide the infusion of the trypan blue marker. All rats were placed in clean cages for observation and fecal pellet output (pellet number and weight) was measured at 3-h intervals during the first 12 h, and then every 12 h until the end of the 48-h observation period.
To mimic the clinical situation in patients with abdominal surgery receiving opioid treatment for pain management, the rats received a subcutaneous injection of morphine (3 mg/kg s.c.) and were then subjected to “running of the bowel” surgery to induce ileus. Previously, we used this rat model to characterize the effect of TZP101 on the delay in upper gastrointestinal transit (Venkova et al., 2007). In the present experiments we evaluated the effects of TZP-101 on the post-surgical colonic transit time, fecal pellet output and food intake. TZP-101 or vehicle were dosed i.v. at 15 min, 2 h and 4 h postsurgery. The dose-response effect of increasing doses of TZP-101 (0.03–1 mg/kg) was evaluated in comparison to the effect of the vehicle. TZP-101 ((4R,7S,10R,13R)-7-Cyclopropyl-13-(4-fluorobenzyl)3-oxa-6,9,12,15-tetraaza-4,9,10-trimethyl-4,5,6,7,10,12,13,15,16,17,18undecahydro-1,2-benzocyclooctadecene-8,11,14-trione) was provided by Tranzyme Pharma (Sherbrooke, QC, Canada). TZP-101 is a smallmolecule agonist with potent binding affinity (Ki = 16 nM) and full agonist activity (EC50 = 29 nM, Emax = 111%) at the human recombinant ghrelin receptor (Fraser et al., 2008). A stock solution of 0.4 mg/ml TZP-101 in 9% (w/v) 2-hydroxypropyl-β-cyclodextrin was kept at 4 °C and used to prepare fresh dilutions for each experiment for 2–3 consecutive days. 2-Hydroxypropyl- β -cyclodextrin (Sigma Chemical Co, St. Louis, MO) dissolved in sterile saline was used in vehicle control experiments. Morphine sulfate was obtained from Baxter Healthcare Corp. (Dearfield, IL) and dissolved in sterile saline. 2.6. Statistical analysis The data are presented as mean± standard error of the mean (S.E.M.) for 6–8 rats per group. The difference between colonic transit time in
Fig. 3. Effect of TZP-101 on fecal output in rats with postoperative ileus. Cumulative fecal pellet output was assessed by the number of fecal pellets or stool weight measured at 12 h post-surgery (A) and 24 h post-surgery (B). Results are presented as mean ± S.E.M. from 6–8 rats per group. The effects of increasing doses of 0.03–1 mg/kg i.v. TZP-101 were evaluated for statistical significance by one-way ANOVA followed by Dunnett's post test for comparison to vehicle: ⁎P b 0.05, ⁎⁎P b 0.01.
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naïve rats and rats subjected to morphine treatment and surgery was evaluated by the nonparametric Mann-Whitney test. Differences in fecal pellet output, food intake and body weight were assessed for statistical significance at 12 h, 24 h and 48 h post-surgery using two-way ANOVA followed by Bonferroni's test for multiple comparisons. The effects of TZP-101 treatment at each post-surgical time period were determined by one-way ANOVA followed by Dunnett's test for comparison to the vehicle control. A probability level of P b 0.05 was considered significant. 3. Results 3.1. Effect of morphine and surgery on colonic transit and fecal pellet output Colonic transit time was significantly increased in rats with postoperative ileus induced by morphine treatment followed by “running of the bowel” in comparison to naive rats. The time to the appearance of the first fecal pellet was 13.2 ± 0.4 h in rats with postoperative ileus treated with vehicle and 5.5 ± 1.6 h in naïve rats (P b 0.01, Man-Whitney test). Cumulative fecal pellet output, measured by the number of fecal pellets (Fig. 1A) and the stool weight (Fig. 1B) was significantly decreased during the first 12 h and 24 h post-surgery in rats with postoperative ileus. However, the rats with ileus were able to compensate the deficit and at 48 h post-surgery there was no difference in the cumulative fecal pellet output between the naive and subjected to surgery groups. 3.2. Effect of morphine and surgery on food intake and body weight Both naïve rats and rats subjected to morphine treatment and surgery ingested approximately the same amount of food during the first 12 h, whereas the cumulative food intake was decreased at 24 h and 48 h post-surgery (Fig. 1C). The body weight was significantly reduced at 24 h and 48 h post-surgery in comparison to naïve rats (Fig. 1D). 3.3. Effect of TZP-101 on colonic transit and fecal pellet output in rats with postoperative ileus Treatment with TZP-101 (0.3–1 mg/kg) induced a dose-dependent decrease in colonic transit time, which was also the time to the first bowel movement following surgery. Maximal efficacy was achieved at doses of 0.3 and 1 mg/kg TZP-101 (Fig. 2). The same doses of 0.3 or 1 mg/kg TZP-101 induced a significant increase in stool output measured by the number of fecal pellets or the stool weight during the first 12 h and 24 h post-surgery (Fig. 3). Moreover, in the group of rats treated with 1 mg/kg TZP-101, the fecal pellet output at 24 h postsurgery was not significantly different compared to naïve rats (number of fecal pellets: 39 ± 5.5 vs. 54 ± 3.7, P N 0.05; stool weight: 9.25 ± 0.86 g vs. 9.63 ± 0.81 g, P N 0.05). 3.4. Effect of TZP-101 on food intake and body weight in rats with postoperative ileus TZP-101 had no significant effect on food intake measured cumulatively at 12 h, 24 h and 48 h post-surgery in rats with postoperative ileus (Fig. 4). The body weight measured at 24 h and 48 h post-surgery was similar in rats treated with vehicle or TZP-101 (Fig. 5). 4. Discussion Ghrelin and synthetic ghrelin receptor agonists, including TZP-101, have previously been shown to accelerate gastric emptying and small intestinal transit in animal models of ileus (Trudel et al., 2002; Poitras et al., 2005; Venkova et al., 2007). However, in patients a complete recovery from postoperative ileus depends on the functional coordi-
Fig. 4. Food intake in rats with postoperative ileus treated with increasing intravenous doses of 0.03–1 mg/kg TZP-101 or vehicle. Cumulative food intake was measured at 12 h post-surgery (A), 24 h post-surgery (B) and 48 h post-surgery (C). Results are mean ± S.E.M. from 6–8 rats per group. The effects of TZP-101 were not statistically significant when compared to the vehicle (one-way ANOVA, P N 0.05 for A, B and C).
nation of motility in the entire gastrointestinal tract, and the clinical resolution of postoperative ileus is typically confirmed by the first bowel movement. In the current study, we extended the evaluation of TZP-101 effects in rats with postoperative ileus, to include the recovery of colonic function following a standard surgical procedure known as “running of the bowel” (Kalff et al., 1998) and a single subcutaneous dose of morphine. This study provides the first evidence that a novel synthetic ghrelin receptor agonist significantly shortens colonic transit time and increases fecal pellet output in a model of postoperative ileus. Prior studies in animal models have emphasized the post-surgical delay in gastric emptying and small intestinal transit. Here, we show that postoperative ileus in rats is also characterized by a prolonged colonic transit time and decreased fecal pellet output over the initial 12–24 h period following surgery. In addition, rats with postoperative ileus show a decrease in food intake measured cumulatively during the first 48 h after surgery, and a decrease in body weight compared to naïve animals, not subjected to morphine treatment or surgery. Overall, these characteristics of the model correspond to the
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Fig. 5. Body weight in rats with postoperative ileus treated with increasing intravenous doses of 0.03–1 mg/kg TZP-101 or vehicle. Cumulative food intake was measured at 12 h post-surgery (A), 24 h post-surgery (B). Results are mean± S.E.M. from 6–8 rats per group. The effects of TZP-101 were not statistically significant when compared to the vehicle (oneway ANOVA, P N 0.05 for A and B).
symptoms in patients suffering from postoperative ileus (Kehlet and Holte, 2001) suggesting that the beneficial effects of TZP-101 in rats could be relevant to the clinical situation in patients following surgery. In our study, TZP-101 shortened colonic transit time and increased fecal pellet output over the same concentration range (0.3–1 mg/kg) previously demonstrated to accelerate gastric emptying in rats with postoperative ileus (Venkova et al., 2007). TZP-101 caused a parallel increase in the cumulative number and weight of fecal pellets produced during the first 12 h and 24 h post-surgery, indicating that the bowel response to TZP-101 should involve changes in motility rather that changes in colonic absorption or secretion. At a dose of 1 mg/kg, TZP-101 significantly reduced the time to the first bowel movement compared to vehicle-treated rats. Previous studies investigating the prokinetic effects of ghrelin in the rat, mouse and human failed to reveal a direct contractile effect on colonic motility. Studies in isolated colonic preparations have shown that ghrelin has no direct effects on contractility when administered in vitro (Edholm et al., 2004; Kitazawa et al., 2005; Bassil et al., 2005) and does not elicit changes in colonic motility in vivo following peripheral administration (Trudel et al., 2002). In the stomach, ghrelin was found to facilitate neurally mediated contractions activated by electrical field stimulation but such an effect was absent in the colon (Dass et al., 2003; Bassil et al., 2005). Regardless of the presence of ghrelin receptor immunoreactivity on colonic enteric neurons (Dass et al., 2003; Xu et al., 2005), in vivo experiments have indicated that peripheral ghrelin has no effect on colonic motility (Trudel et al., 2002; Ohno et al., 2006). Evidence for central, but not peripheral sites for the ghrelin receptors stimulating colonic motility were presented in a recent study (Shimizu et al., 2006) demonstrating that activation of ghrelin receptors in the lumbosacral spinal cord triggers coordinated propulsive contractions that empty the colorectum. However, since TZP-101 has extremely low blood-brain permeability (Fraser et al., 2008), it is highly unlikely that the improvement of colonic transit measured in the current study is due to stimulation of this central
neuronal pathway. Thus, the enhanced fecal pellet output and the shortening of the time to the first bowel movement most probably reflect the ability of TZP-101 to induce coordinated propulsive motility in the stomach and small intestine, promoting an indirect propulsive effect in the large bowel. Such a mechanism of TZP-101 action is in agreement with previous research indicating a prokinetic effect of ghrelin and synthetic ghrelin receptor agonists in the upper gastrointestinal tract in rat models of postoperative ileus (Trudel et al., 2002; Poitras et al., 2005; Sallam et al., 2007; Venkova et al., 2007), in mice with septic ileus (De Winter et al., 2004) and in patients with diabetic gastroparesis (Murray et al., 2005). Another important aspect of the physiological effects of ghrelin, is that increase in the plasma concentration of ghrelin elicits spontaneous food intake in rats and humans (Wren et al., 2001a,b). Thus it has been proposed that elevated levels of ghrelin stimulate the feeding center in the brain directly, by crossing the blood-brain barrier (Banks et al., 2002; Pan et al., 2006), and/or indirectly via peripheral activation of vagal afferents and the subsequent transmission of orexigenic signals to the brain (Date et al., 2002). We have shown that TZP-101 and ghrelin provoke comparable increases in spontaneous food intake in rats when administered directly into the brain (Fraser et al., 2005). Thus, it is noteworthy that TZP-101 had no effect on food intake in the current study, which is consistent with a recent report using ghrelin (Arnold et al., 2006) that challenges the importance of ghrelin receptor signaling through gut vagal afferents to transmit orexigenic signals to the brain (Date et al., 2002). In line with this evidence, it is not expected that TZP-101 would directly stimulate feeding centers in the brain due to its low blood-brain permeability, at least in the rodent model (Fraser et al., 2005; Fraser et al., 2008). However, in humans the action of TZP101 may include an increase in growth hormone release, since a recent pharmacokinetic/pharmacodynamic investigation in healthy volunteers showed that TZP-101 treatment increased blood growth hormone levels despite the minimal penetration of TZP-101 through the blood-brain barrier (Lasseter et al., 2008). This effect may be due to a TZP-101 action on the pituitary gland in humans, which remains outside the blood-brain barrier and most likely allows TZP-101 delivery to the anterior pituitary gland. In summary, the present results demonstrate that a novel synthetic, peripherally acting ghrelin receptor agonist TZP-101, which is currently in clinical development as a first-in-class treatment for postoperative ileus, improves colonic transit and shortens the time to the first bowel movement in rats with postoperative ileus. This observation extends our previous findings that TZP-101 restores gastric emptying and small intestinal transit in rats with postoperative ileus (Venkova et al., 2007) without modulating alternate aspects of ghrelin physiology such as growth hormone release and food intake (Fraser et al., 2008). Acknowledgements This study was funded by Tranzyme Pharma, Inc. Preliminary results were presented at the Digestive Disease Week, May 19–24, 2007, Washington, DC. References Arnold, M., Mura, A., Langhans, W., Geary, N., 2006. Gut vagal afferents are not necessary for the eating-stimulatory effect of intraperitoneally injected ghrelin in the rat. J. Neurosci. 26, 11052–11060. Artinyan, A., Nunoo-Mensah, J.W., Essani, R., Balasubramaniam, S., Gauderman, J., Gonzalez-Ruiz, C., Vukasin, P., Kaiser, A.M., Beart, R.W., 2005. Prolonged postoperative ileus: definition, risk factors and predictors in patients undergoing open colorectal surgery. Gastroenterology 128, A–979. Banks, W.A., Tschöp, M., Robinson, S.M., Heiman, M.L., 2002. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J. Pharmacol. Exp. Ther. 302, 822–827. Bassil, A.K., Dass, N.B., Murray, C.D., Muir, A., Sanger, G.J., 2005. Prokineticin-2, motilin, ghrelin and metoclopramide: prokinetic utility in mouse stomach and colon. Eur. J. Pharmacol. 524, 138–144.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus Graeme L. Fraser d, Kalina Venkova b,c, Hamid R. Hoveyda d, Helmut Thomas d, Beverley Greenwood-Van Meerveld a,b,c,⁎ a
VA Medical Center, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA Department of Physiology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA d Tranzyme Pharma, Sherbrooke, QC, Canada, J1H 5N4 b c
a r t i c l e
i n f o
Article history: Received 10 June 2008 Received in revised form 21 November 2008 Accepted 9 December 2008 Available online 14 December 2008 Keywords: Ghrelin receptor TZP-101 Postoperative ileus Colonic transit Fecal pellet output (Rat)
a b s t r a c t Ghrelin, the natural ligand of the growth hormone secretagogue receptor (ghrelin receptor), is an orexigenic gut hormone with prokinetic action in the upper gastrointestinal tract. Previously we have shown in a rodent model of postoperative ileus that the synthetic ghrelin receptor agonist TZP-101 prevents the delay in gastric emptying and improves small intestinal transit. The goal of the present study was to investigate whether TZP-101 affects colonic transit and food intake in rats with postoperative ileus. Fasted rats were treated with morphine and subjected to laparotomy under isoflurane anesthesia. Following surgery the animals were placed in clean home cages and fecal pellet output and food intake were monitored for 48 h. TZP-101 or vehicle were administered as 3 i.v. bolus infusions at 0 h, 2 h and 4 h post-surgery. TZP-101 (0.03–1 mg/kg) dose-dependently decreased the time to first bowel movement and increased fecal pellet output measured at 12 h and 24 h post-surgery compared to the vehicle. The administration of TZP-101 was not associated with a significant alteration in food intake. In conclusion, this study provides the first experimental evidence that a novel ghrelin receptor agonist improves large bowel function in rats with postoperative ileus, suggesting that TZP-101 may be useful in the clinic to accelerate upper gastrointestinal transit and to shorten the time to the first bowel movement following surgery. Published by Elsevier B.V.
1. Introduction Postoperative ileus is the temporary impairment of gastrointestinal motility following abdominal surgery. The pathogenesis of prolonged ileus is positively correlated with the degree of surgical trauma and the use of opioids for pain management (Fotiadis et al., 2004; Artinyan et al., 2005). Surgical injury triggers local immune and nerve mediated mechanisms that inhibit coordinated gastrointestinal motility (Kalff et al., 1998; Boeckxstaens et al., 1999; Boeckxstaens, 2002; Bauer et al., 2002; de Jonge et al., 2003). In addition, exogenous opioids, administered to treat post-surgical pain, prolong the duration of ileus via the activation of peripheral opioid receptors inhibiting neurally mediated contractions as well as through direct effects on central opioid receptor signaling (Gmerek et al., 1986; GreenwoodVan Meerveld, 2007). Regardless of the abdominal site of surgical intervention, the spontaneous resolution of postoperative ileus ⁎ Corresponding author. VA Medical Center, Research Administration Rm. 151, 921 NE 13th street, Oklahoma City, OK 73104, USA. Tel.: +1 405 270 0501x3547; fax: +1 405 290 1719. E-mail address: [email protected] (B. Greenwood-Van Meerveld). 0014-2999/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.ejphar.2008.12.011
appears to be region-specific and is typically characterized by the initial recovery of the small intestine, followed by the stomach, and then the recovery of the colon (Graber et al., 1982; Waldhausen et al., 1990; Schippers et al., 1991). Since the first bowel movement is considered a clinical end point for the recovery of gastrointestinal function, assessment of the efficacy of new prokinetic drugs to reduce the time to first bowel movement following surgery in animal models is essential for the progress in postoperative therapy in humans. Ghrelin is an acylated peptide hormone secreted predominantly from endocrine cells in the gastric oxyntic mucosa (Kojima et al., 1999; Date et al., 2000). Ghrelin receptors regulating gastrointestinal function are expressed on vagal afferents (Sakata et al., 2003; Burdyga et al., 2006) and in the enteric nervous system (Dass et al., 2003; Xu et al., 2005), where ghrelin receptor expression appears to decrease distally from high levels in the stomach to lower levels in the colon (Dass et al., 2003; Kojima and Kangawa, 2005). The prokinetic actions of ghrelin on the stomach and small intestine arise from increased signaling through vagal afferents (Fujino et al., 2003; Murray et al., 2006) and direct activation of the enteric nervous system. Furthermore, previous studies have demonstrated that the administration of the ghrelin peptide or synthetic ghrelin receptor agonists restored
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gastric motility and accelerated upper gastrointestinal transit in various rodent models of ileus (Trudel et al., 2002; De Winter et al., 2004; Poitras et al., 2005; Sallam et al., 2007). A novel macrocyclic small molecule TZP-101 with peptidomimetic effects similar to the effects of ghrelin has been developed by Tranzyme Pharma for the treatment of gastrointestinal and metabolic disorders. The structure of TZP-101 features an 18-membered macrocycle containing 3 amide bonds and a secondary amine, as well as 4 stereogenic centers (Hoveyda et al., 2006; Sandham, 2008). Pharmacodynamic analyses suggested TZP-101 expresses activity at the ghrelin receptor (Lasseter et al., 2008). Using a rat model of postoperative ileus induced by abdominal surgery and morphine treatment we found that the synthetic ghrelin receptor agonist TZP-101 normalized gastric emptying and improved small intestinal transit (Venkova et al., 2007). Since patient recovery from postoperative ileus is actually assessed by the first bowel movement following surgery, an important study is to assess whether TZP-101 reduces the time to the first bowel movement in rats with postoperative ileus. Thus the current study describes a series of experiments to investigate whether systemic TZP-101 has an effect on fecal pellet output in rats with postoperative ileus. Although, there is no evidence to support a direct effect of ghrelin in the large intestine (Trudel et al., 2002; Edholm et al., 2004; Kitazawa et al., 2005; Bassil et al., 2005), we hypothesized that a ghrelin receptor agonist may shorten the overall transit time by stimulating gastric emptying and small intestinal transit. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (250–350 g) were purchased from Charles River Laboratories (Wilmington, MA) equipped with a
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polyurethane catheter implanted into the right jugular vein. The exterior end of the catheter was secured to the dorsal neck area and used for i.v. dosing of TZP-101 or vehicle. The catheters were flushed with heparinized saline once every 3–4 days but not on the day of the experiment. Rats that were not subjected to surgery and drug or vehicle treatment were equipped with catheters implanted into the proximal colon and used to infuse a dye marker for the measurement of colonic transit time. A total of 46 rats were used in the study. All animals were housed one per cage at standard conditions (21–22 °C, 12 h light/dark cycle, controlled humidity) with water and rat chow available ad libitum. At least one-week acclimation to the animal facility was allowed prior to the experiments. The experimental protocol and number of animals were approved by the Animal Care and Use Committees at the V.A. Medical Center and the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. 2.2. Abdominal surgery Prior to the experiments, rats were fasted for 20–22 h with free access to water. Postoperative ileus was induced by a surgical procedure described as “running of the bowel” (Kalff et al., 1998). Specifically, the rats were anesthetized with isoflurane (2–3% inhalation), the abdomen was shaved, disinfected and a midline incision was made to expose the abdominal viscera. The small intestine, the cecum and colon were exteriorized and inspected by gently pressing between two cotton applicators soaked in sterile saline. After completing the inspection, the intestines were covered with gauze soaked in saline and the abdomen was left open for 10 min. At the end of surgery 0.2 ml of dye (trypan-blue in saline) was carefully injected into the proximal colon (1 cm distal to the cecum) using a hypodermic needle. The viscera were then placed back into the
Fig. 1. Time-course of the changes in fecal pellet output, food intake and body weight in rats with postoperative ileus induced by morphine treatment followed by abdominal surgery compared to naïve rats. On the day of the experiment, all rats were fasted overnight and the naïve rats were placed in the observation cage supplied with food and water without been subjected to morphine treatment or surgery. Cumulative fecal output, expressed as the number of fecal pellets (A) or the stool weight (B), and food intake (C) were measured at 12 h. 24 and 48 h following the end of surgery. Body weight (D) was measured 24 h prior to fasting and at 24 h and 48 h of observation. Filled symbols represent individual experiments in rats with postoperative ileus while open symbols represent individual experiments in naïve rats. Mean values are from 6 naive rats and 8 rats with postoperative ileus. Statistical significance of differences was assessed by two-way ANOVA followed by Bonferonni's post test: ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001.
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2.4. Evaluation of food intake and body weight Food intake was also recorded at 3-h intervals during the first 12 h, and then every 12 h until the end of the 48-h observation period. Body weight was measured in fed rats 1 day prior to the experiment, in fasted rats before the surgery and at 24 h and 48 h post-surgery. 2.5. Experimental protocol
Fig. 2. Effect of TZP-101 on colonic transit time in rats with postoperative ileus. Results are presented as mean ± S.E.M. from 6–8 rats per group. The effects of increasing doses of 0.03–1 mg/kg i.v. TZP-101 were assessed for statistical significance by one-way ANOVA followed by Dunnett's post test for comparison to vehicle: ⁎⁎P b 0.01.
abdomen and the incision was closed with silk sutures. The whole procedure lasted 25–30 min and an additional period of 15 min was allowed for the animals to recover completely from the anesthesia before being treated with the drug or the vehicle. 2.3. Evaluation of colonic transit time and fecal pellet output Trypan blue, which is innocuous and non-absorbable by the gastrointestinal tract, was used to mark the fecal material and allow the measurement of colonic transit time (the time between the end of surgery and the appearance of the first dyed fecal pellet) in rats with postoperative ileus. A subgroup of rats not subjected to surgery, drug or vehicle treatment was fasted overnight and studied simultaneously with postoperative ileus rats as a reference to healthy controls. These animals had catheters implanted into the proximal colon to provide the infusion of the trypan blue marker. All rats were placed in clean cages for observation and fecal pellet output (pellet number and weight) was measured at 3-h intervals during the first 12 h, and then every 12 h until the end of the 48-h observation period.
To mimic the clinical situation in patients with abdominal surgery receiving opioid treatment for pain management, the rats received a subcutaneous injection of morphine (3 mg/kg s.c.) and were then subjected to “running of the bowel” surgery to induce ileus. Previously, we used this rat model to characterize the effect of TZP101 on the delay in upper gastrointestinal transit (Venkova et al., 2007). In the present experiments we evaluated the effects of TZP-101 on the post-surgical colonic transit time, fecal pellet output and food intake. TZP-101 or vehicle were dosed i.v. at 15 min, 2 h and 4 h postsurgery. The dose-response effect of increasing doses of TZP-101 (0.03–1 mg/kg) was evaluated in comparison to the effect of the vehicle. TZP-101 ((4R,7S,10R,13R)-7-Cyclopropyl-13-(4-fluorobenzyl)3-oxa-6,9,12,15-tetraaza-4,9,10-trimethyl-4,5,6,7,10,12,13,15,16,17,18undecahydro-1,2-benzocyclooctadecene-8,11,14-trione) was provided by Tranzyme Pharma (Sherbrooke, QC, Canada). TZP-101 is a smallmolecule agonist with potent binding affinity (Ki = 16 nM) and full agonist activity (EC50 = 29 nM, Emax = 111%) at the human recombinant ghrelin receptor (Fraser et al., 2008). A stock solution of 0.4 mg/ml TZP-101 in 9% (w/v) 2-hydroxypropyl-β-cyclodextrin was kept at 4 °C and used to prepare fresh dilutions for each experiment for 2–3 consecutive days. 2-Hydroxypropyl- β -cyclodextrin (Sigma Chemical Co, St. Louis, MO) dissolved in sterile saline was used in vehicle control experiments. Morphine sulfate was obtained from Baxter Healthcare Corp. (Dearfield, IL) and dissolved in sterile saline. 2.6. Statistical analysis The data are presented as mean± standard error of the mean (S.E.M.) for 6–8 rats per group. The difference between colonic transit time in
Fig. 3. Effect of TZP-101 on fecal output in rats with postoperative ileus. Cumulative fecal pellet output was assessed by the number of fecal pellets or stool weight measured at 12 h post-surgery (A) and 24 h post-surgery (B). Results are presented as mean ± S.E.M. from 6–8 rats per group. The effects of increasing doses of 0.03–1 mg/kg i.v. TZP-101 were evaluated for statistical significance by one-way ANOVA followed by Dunnett's post test for comparison to vehicle: ⁎P b 0.05, ⁎⁎P b 0.01.
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naïve rats and rats subjected to morphine treatment and surgery was evaluated by the nonparametric Mann-Whitney test. Differences in fecal pellet output, food intake and body weight were assessed for statistical significance at 12 h, 24 h and 48 h post-surgery using two-way ANOVA followed by Bonferroni's test for multiple comparisons. The effects of TZP-101 treatment at each post-surgical time period were determined by one-way ANOVA followed by Dunnett's test for comparison to the vehicle control. A probability level of P b 0.05 was considered significant. 3. Results 3.1. Effect of morphine and surgery on colonic transit and fecal pellet output Colonic transit time was significantly increased in rats with postoperative ileus induced by morphine treatment followed by “running of the bowel” in comparison to naive rats. The time to the appearance of the first fecal pellet was 13.2 ± 0.4 h in rats with postoperative ileus treated with vehicle and 5.5 ± 1.6 h in naïve rats (P b 0.01, Man-Whitney test). Cumulative fecal pellet output, measured by the number of fecal pellets (Fig. 1A) and the stool weight (Fig. 1B) was significantly decreased during the first 12 h and 24 h post-surgery in rats with postoperative ileus. However, the rats with ileus were able to compensate the deficit and at 48 h post-surgery there was no difference in the cumulative fecal pellet output between the naive and subjected to surgery groups. 3.2. Effect of morphine and surgery on food intake and body weight Both naïve rats and rats subjected to morphine treatment and surgery ingested approximately the same amount of food during the first 12 h, whereas the cumulative food intake was decreased at 24 h and 48 h post-surgery (Fig. 1C). The body weight was significantly reduced at 24 h and 48 h post-surgery in comparison to naïve rats (Fig. 1D). 3.3. Effect of TZP-101 on colonic transit and fecal pellet output in rats with postoperative ileus Treatment with TZP-101 (0.3–1 mg/kg) induced a dose-dependent decrease in colonic transit time, which was also the time to the first bowel movement following surgery. Maximal efficacy was achieved at doses of 0.3 and 1 mg/kg TZP-101 (Fig. 2). The same doses of 0.3 or 1 mg/kg TZP-101 induced a significant increase in stool output measured by the number of fecal pellets or the stool weight during the first 12 h and 24 h post-surgery (Fig. 3). Moreover, in the group of rats treated with 1 mg/kg TZP-101, the fecal pellet output at 24 h postsurgery was not significantly different compared to naïve rats (number of fecal pellets: 39 ± 5.5 vs. 54 ± 3.7, P N 0.05; stool weight: 9.25 ± 0.86 g vs. 9.63 ± 0.81 g, P N 0.05). 3.4. Effect of TZP-101 on food intake and body weight in rats with postoperative ileus TZP-101 had no significant effect on food intake measured cumulatively at 12 h, 24 h and 48 h post-surgery in rats with postoperative ileus (Fig. 4). The body weight measured at 24 h and 48 h post-surgery was similar in rats treated with vehicle or TZP-101 (Fig. 5). 4. Discussion Ghrelin and synthetic ghrelin receptor agonists, including TZP-101, have previously been shown to accelerate gastric emptying and small intestinal transit in animal models of ileus (Trudel et al., 2002; Poitras et al., 2005; Venkova et al., 2007). However, in patients a complete recovery from postoperative ileus depends on the functional coordi-
Fig. 4. Food intake in rats with postoperative ileus treated with increasing intravenous doses of 0.03–1 mg/kg TZP-101 or vehicle. Cumulative food intake was measured at 12 h post-surgery (A), 24 h post-surgery (B) and 48 h post-surgery (C). Results are mean ± S.E.M. from 6–8 rats per group. The effects of TZP-101 were not statistically significant when compared to the vehicle (one-way ANOVA, P N 0.05 for A, B and C).
nation of motility in the entire gastrointestinal tract, and the clinical resolution of postoperative ileus is typically confirmed by the first bowel movement. In the current study, we extended the evaluation of TZP-101 effects in rats with postoperative ileus, to include the recovery of colonic function following a standard surgical procedure known as “running of the bowel” (Kalff et al., 1998) and a single subcutaneous dose of morphine. This study provides the first evidence that a novel synthetic ghrelin receptor agonist significantly shortens colonic transit time and increases fecal pellet output in a model of postoperative ileus. Prior studies in animal models have emphasized the post-surgical delay in gastric emptying and small intestinal transit. Here, we show that postoperative ileus in rats is also characterized by a prolonged colonic transit time and decreased fecal pellet output over the initial 12–24 h period following surgery. In addition, rats with postoperative ileus show a decrease in food intake measured cumulatively during the first 48 h after surgery, and a decrease in body weight compared to naïve animals, not subjected to morphine treatment or surgery. Overall, these characteristics of the model correspond to the
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Fig. 5. Body weight in rats with postoperative ileus treated with increasing intravenous doses of 0.03–1 mg/kg TZP-101 or vehicle. Cumulative food intake was measured at 12 h post-surgery (A), 24 h post-surgery (B). Results are mean± S.E.M. from 6–8 rats per group. The effects of TZP-101 were not statistically significant when compared to the vehicle (oneway ANOVA, P N 0.05 for A and B).
symptoms in patients suffering from postoperative ileus (Kehlet and Holte, 2001) suggesting that the beneficial effects of TZP-101 in rats could be relevant to the clinical situation in patients following surgery. In our study, TZP-101 shortened colonic transit time and increased fecal pellet output over the same concentration range (0.3–1 mg/kg) previously demonstrated to accelerate gastric emptying in rats with postoperative ileus (Venkova et al., 2007). TZP-101 caused a parallel increase in the cumulative number and weight of fecal pellets produced during the first 12 h and 24 h post-surgery, indicating that the bowel response to TZP-101 should involve changes in motility rather that changes in colonic absorption or secretion. At a dose of 1 mg/kg, TZP-101 significantly reduced the time to the first bowel movement compared to vehicle-treated rats. Previous studies investigating the prokinetic effects of ghrelin in the rat, mouse and human failed to reveal a direct contractile effect on colonic motility. Studies in isolated colonic preparations have shown that ghrelin has no direct effects on contractility when administered in vitro (Edholm et al., 2004; Kitazawa et al., 2005; Bassil et al., 2005) and does not elicit changes in colonic motility in vivo following peripheral administration (Trudel et al., 2002). In the stomach, ghrelin was found to facilitate neurally mediated contractions activated by electrical field stimulation but such an effect was absent in the colon (Dass et al., 2003; Bassil et al., 2005). Regardless of the presence of ghrelin receptor immunoreactivity on colonic enteric neurons (Dass et al., 2003; Xu et al., 2005), in vivo experiments have indicated that peripheral ghrelin has no effect on colonic motility (Trudel et al., 2002; Ohno et al., 2006). Evidence for central, but not peripheral sites for the ghrelin receptors stimulating colonic motility were presented in a recent study (Shimizu et al., 2006) demonstrating that activation of ghrelin receptors in the lumbosacral spinal cord triggers coordinated propulsive contractions that empty the colorectum. However, since TZP-101 has extremely low blood-brain permeability (Fraser et al., 2008), it is highly unlikely that the improvement of colonic transit measured in the current study is due to stimulation of this central
neuronal pathway. Thus, the enhanced fecal pellet output and the shortening of the time to the first bowel movement most probably reflect the ability of TZP-101 to induce coordinated propulsive motility in the stomach and small intestine, promoting an indirect propulsive effect in the large bowel. Such a mechanism of TZP-101 action is in agreement with previous research indicating a prokinetic effect of ghrelin and synthetic ghrelin receptor agonists in the upper gastrointestinal tract in rat models of postoperative ileus (Trudel et al., 2002; Poitras et al., 2005; Sallam et al., 2007; Venkova et al., 2007), in mice with septic ileus (De Winter et al., 2004) and in patients with diabetic gastroparesis (Murray et al., 2005). Another important aspect of the physiological effects of ghrelin, is that increase in the plasma concentration of ghrelin elicits spontaneous food intake in rats and humans (Wren et al., 2001a,b). Thus it has been proposed that elevated levels of ghrelin stimulate the feeding center in the brain directly, by crossing the blood-brain barrier (Banks et al., 2002; Pan et al., 2006), and/or indirectly via peripheral activation of vagal afferents and the subsequent transmission of orexigenic signals to the brain (Date et al., 2002). We have shown that TZP-101 and ghrelin provoke comparable increases in spontaneous food intake in rats when administered directly into the brain (Fraser et al., 2005). Thus, it is noteworthy that TZP-101 had no effect on food intake in the current study, which is consistent with a recent report using ghrelin (Arnold et al., 2006) that challenges the importance of ghrelin receptor signaling through gut vagal afferents to transmit orexigenic signals to the brain (Date et al., 2002). In line with this evidence, it is not expected that TZP-101 would directly stimulate feeding centers in the brain due to its low blood-brain permeability, at least in the rodent model (Fraser et al., 2005; Fraser et al., 2008). However, in humans the action of TZP101 may include an increase in growth hormone release, since a recent pharmacokinetic/pharmacodynamic investigation in healthy volunteers showed that TZP-101 treatment increased blood growth hormone levels despite the minimal penetration of TZP-101 through the blood-brain barrier (Lasseter et al., 2008). This effect may be due to a TZP-101 action on the pituitary gland in humans, which remains outside the blood-brain barrier and most likely allows TZP-101 delivery to the anterior pituitary gland. In summary, the present results demonstrate that a novel synthetic, peripherally acting ghrelin receptor agonist TZP-101, which is currently in clinical development as a first-in-class treatment for postoperative ileus, improves colonic transit and shortens the time to the first bowel movement in rats with postoperative ileus. This observation extends our previous findings that TZP-101 restores gastric emptying and small intestinal transit in rats with postoperative ileus (Venkova et al., 2007) without modulating alternate aspects of ghrelin physiology such as growth hormone release and food intake (Fraser et al., 2008). Acknowledgements This study was funded by Tranzyme Pharma, Inc. Preliminary results were presented at the Digestive Disease Week, May 19–24, 2007, Washington, DC. References Arnold, M., Mura, A., Langhans, W., Geary, N., 2006. Gut vagal afferents are not necessary for the eating-stimulatory effect of intraperitoneally injected ghrelin in the rat. J. Neurosci. 26, 11052–11060. Artinyan, A., Nunoo-Mensah, J.W., Essani, R., Balasubramaniam, S., Gauderman, J., Gonzalez-Ruiz, C., Vukasin, P., Kaiser, A.M., Beart, R.W., 2005. Prolonged postoperative ileus: definition, risk factors and predictors in patients undergoing open colorectal surgery. Gastroenterology 128, A–979. Banks, W.A., Tschöp, M., Robinson, S.M., Heiman, M.L., 2002. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J. Pharmacol. Exp. Ther. 302, 822–827. Bassil, A.K., Dass, N.B., Murray, C.D., Muir, A., Sanger, G.J., 2005. Prokineticin-2, motilin, ghrelin and metoclopramide: prokinetic utility in mouse stomach and colon. Eur. J. Pharmacol. 524, 138–144.
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