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Salutary Effects of Tachykinin Receptor Antagonists in a Rat Model of Postoperative Ileus1
Journal of Surgical Research 133, 197–202 (2006) doi:10.1016/j.jss.2006.01.006
Salutary Effects of Tachykinin Receptor Antagonists in a Rat Model of Postoperative Ileus 1 R. Ciechanownicz, M.D.,* J. Sein-Anand, M.D., Ph.D.,* Z. Chodorowski, M.D., Ph.D.,* M. Bitel, M.D.,† J. Petrusewicz, Ph.D.,† and R. P. Korolkiewicz, M.D., Ph.D.†,2 *Department of Internal Medicine and Toxicology, Medical University of Gdan´sk, De˛binki 7, 80-211 Gdan´sk, Poland; and †Department of Pharmacology, Medical University of Gdan´sk, De˛bowa 23, 80-204 Gdan´sk, Poland Submitted for publication September 20, 2005
Background. Postoperative ileus (PI) is a common surgical complication treated mainly with supportive measures. Tachykinins control gastrointestinal motility and modulate somatic and visceral pain sensation; therefore, the effect of tachykinin receptor antagonists in a rat model of PI using NK 1–3 antagonists, SR140333, SR48968, and SR142801, was investigated. Materials and methods. Intestinal transit was measured as Evans blue migration after varied nociceptive stimuli: skin incision (SI), laparotomy (LAP), or laparotomy plus gut manipulation (L ⴙ M) in anesthetized rats. Results. Diethyl ether anesthesia and SI did not influence the intestinal transit of the dye in comparison to untreated animals—UN: 61.17 ⴞ 5.47, 62.10 ⴞ 8.30, and 56.70 ⴞ 4.10 cm, respectively. In contrast LAP and L ⴙ M have significantly reduced intestinal motility to 26.40 ⴞ 2.07 and 9.70 ⴞ 1.15 cm, respectively. SR140333 (3–30 g/kg), SR48968 (1–30 g/kg), and SR142801 (3– 10 g/kg) reversed the additional inhibitory effects of gut manipulation subsequent to LAP dose-dependently, the dye transit returning with the use of the most effective antagonist doses up to 25.28 ⴞ 1.08, 21.70 ⴞ 0.19, and 25.0 ⴞ 1.34 cm. The combinations of submaximal doses of NK 1 and NK 3, NK 2 and NK 3 and NK 1, and NK 2 and NK 3 antagonists were not more effective than a single-agent regimen. On the other hand SR140333 and SR48968 (NK 1 ⴙ NK 2 antagonists) acted additively, the intestinal transit reaching 26.60 ⴞ 0.85
1 Results were presented in part at a Brain-Gut Axis in Gastrointestinal System. Basic and Clinical Aspects Symposium, Kraków, Poland, November 2003. 2 To whom correspondence and reprint requests should be addressed at Department of Pharmacology, Medical University of Gdan´sk, De˛bowa 23, 80-204 Gdan´sk, Poland. E-mail: rokor@amg. gda.pl.
cm. SR140333, SR48968, and SR142801 have not affected the intestinal passage in UN rats or those undergoing SI or LAP. Conclusions. SR140333, SR48968, and SR142801 exert a salutary action on suppressed gut motility following surgical manipulation of the gut, the combination of NK 1 and NK 2 antagonists being most beneficial. © 2006 Elsevier Inc. All rights reserved.
Key Words: postoperative ileus; rats; tachykinin receptor antagonists. INTRODUCTION
Postoperative ileus (PI) is a spontaneously reversible, transient inhibition of propulsive bowel motility occurring after major surgical interventions and especially those that involve an open technique abdominal operation leading to an increased morbidity and higher hospitalization costs [1]. Due to an unclear etiology, supportive measures remain the cornerstone of the current therapy despite achieving some progress with multimodal postsurgical rehabilitation programs and the introduction of peripherally acting opioid antagonists [1, 2]. It seems that the inhibitory neuronal arches, with afferent capsaicin-sensitive unmyelinated fibers and efferent adrenergic and nitrergic neurons, play a prominent role in the development of PI. In concert with this hypothesis, thoracic epidural anesthesia improved PI outcome in humans, whereas the inhibition of nitric oxide synthase (NOS) reversed the intestinal transit migration in rodents [3, 4]. The enteric nervous system produces a number of inhibitory (e.g., NO, VIP, CO) and stimulatory neurotransmitters (e.g., substance P) [5]. Tachykinins belong to the largest peptide family with three main subtypes of
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receptors present in rat intestinal smooth muscle cells: NK 1, NK 2, and NK 3 [6]. Major tachykinins such as substance P (SP) and neurokinins A and B (NKA and NKB) bind to all of these the receptors with varied degrees of affinity [7]. Tachykinins are most frequently an excitatory transmitters in the gut; however, their net effect depends on the animal species, gut segment, the profile of activated receptors, and other mediators acting at target sites. The main sources of neuronal tachykinins in the gut are the intrinsic enteric neurons of myenteric and the submucosal plexuses together with the extrinsic primary afferent fibers. Moreover many C-afferent fibers possess tachykinin receptors that can be sensitized by mechanical stimulation or an induction of an inflammatory process [8]. NK 1, NK 2, and NK 3 receptors are involved in the modulation of visceral hypersensitivity evoked by colorectal distension or inflammation [9, 10] Octreotide, VIP, SP, and CGRP antagonists ameliorate the postsurgical inhibition of gastric and small intestine motility in dogs and rats, implying that multiple tachykinin substances are involved in the pathogenesis of PI [11, 12]. The activity of tachykinins in the gut and the availability of potent synthetic tachykinin receptor antagonists, SR140333, SR48968, and SR142801, enabled a study of the effect of NK 1–3 receptor blockade in a rat model of PI. Due to conflicting reports in the literature, it was investigated whether in a combination of NK 1–3 receptor antagonists could potentially exert an additive salutary effect on the small bowel motility following a surgical insult. MATERIALS AND METHODS Surgical Protocol The Bioethics Committee of the Medical University of Gdan´sk approved the experimental procedures. Male albino Wistar rats (180 –250 g) were fasted for 48 h, maintaining free access to tap
water. Rats were randomly divided into four groups: one control group (untreated animals) and three groups exposed to surgical insult under diethyl ester anesthesia as previously described [3]. Rats from the second group underwent skin incision after having abdominal hair shaven and skin disinfected with 70% ethanol. In the third group, the animals underwent laparotomy. Animals from the fourth group were exposed to laparotomy and subsequent gut evisceration followed by mechanical stimulation of cecum and small intestine. Briefly, the small intestine and cecum were pulled out of the abdominal cavity and gently spread between two layers of damp, sterile gauze. The small intestine was carefully touched by two wet cotton bud applicators from cecum upward, until the duodenal end of the intestine was reached. This procedure was repeated six times within 10 min, and then the cecum and small intestine were returned to the abdominal cavity and the surgical wound was sutured. After the operation, the rats recovered for 1 h. Recovery time was based on the findings of the pilot experiments that diethyl ester anesthesia affects gastrointestinal (GI) motility for approximately 1 h. Subsequently, all animals received 0.15 ml Evans blue via an orogastric tube and 30 min later the animals were sacrificed by cardiotomy under deep ether anesthesia. The small intestines were excised and, to avoid tissue stretching, gently laid on corkboard for measurements, which consisted of establishing the most distal point of dye migration from the pylorus. A blinded observer, unaware of the treatment the animals were receiving, performed measurements. The intestinal transit was measured from the pylorus to the most distal point of dye migration. Experimental details are depicted in Fig. 1.
The Effects of Tachykinin Receptor Antagonists on the Intestinal Transit Initially the effects of intraperitoneally (i.p.) injected SR 140333 (3–100 g/kg), SR 48968 (1–30 g/kg), and SR142801 (3–30 g/kg) were investigated in untreated or anesthetized rats and animals exposed to skin incision, laparotomy, laparotomy, and gut manipulation. Subsequently, a combination of submaximal doses of NK 1 ⫹ NK 2 (SR140333 ⫹ SR48968; 10 ⫹ 3 g/kg), NK 1 ⫹ NK 3 (SR140333 ⫹ SR142801; 10 ⫹ 6 g/kg), NK 2 ⫹ NK 3 (SR48968 ⫹ SR142801; 3 ⫹ 6 g/kg), or NK 1 ⫹ NK 2 ⫹ NK 3 receptor antagonists (SR 140333 ⫹ SR48968 ⫹ SR142801; 10 ⫹ 3 ⫹ 6 g/kg) were investigated in rats undergoing laparotomy followed by gut manipulation. Respective controls in each experimental group received an equal volume of saline (0.9% NaCl) instead of test article. All tested agents or saline was administered 1.5 h before surgical procedures.
FIG. 1. The effects of NK 1–3 antagonists, SR140333 (3–100 g/kg), SR48968 (1–30 g/kg), SR142801(3–30 g/kg), administered separately or in combination were tested on the intestinal transit of Evans blue, after surgical intervention in rats subjected to different nociceptive stimuli: skin incision (SI), laparotomy (L), or laparotomy followed by gut handling (L ⫹ M). Respective controls in each experimental group received an equal volume of saline (0.9% NaCl) instead of test article. All tested agents or saline were administered 1.5 h before surgical procedures.
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Drugs SR 140333, SR 48968, and SR 142801 were a generous gift of Dr. X. Emonds-Alt, Sanofi-Synthelabo, France. SR 140333, SR 48968, and SR 142801 were dissolved in a small volume of 70% ethanol. All agents were adjusted to the desired concentration with saline. Evans blue was obtained from Sigma-Aldrich (Poznan´, Poland) and 50 mg of the dye was dissolved in 1 ml saline. Saline was purchased from Fresenius Kabi (Kutno, Poland) and diethyl ether was purchased from Polskie Odczynniki Chemiczne SA (Gliwice, Poland).
Statistical Analysis The length of small intestine between animals in all experimental groups was not statistically different in the course of this study (data not shown); hence the distance covered by Evans blue was expressed in centimeters and the results were presented in the text as a mean value ⫾ standard error of the mean (SEM) for the number of rats mentioned in each group. Results were compared using one-way analysis of variance (ANOVA) plus Bonferroni post-ANOVA test. Two-tailed P values of less than 0.05 were taken to indicate significant difference.
RESULTS The Effects of Ether Anesthesia and Surgical Intervention on the Intestinal Transit
Evans blue migrated over a distance of 62.10 ⫾ 8.33 cm out of a total length of 112 ⫾ 3.67 cm of the small intestine in the untreated conscious rats (n ⫽ 5). Ether anesthesia and skin incision have not influenced the intestinal transit of Evans blue reaching 61.17 ⫾ 5.47 of 108 ⫾ 1.05 cm and 56.70 ⫾ 4.10 of 102 ⫾ 5.66 cm, respectively (n ⫽ 6 and 5). In contrast, both laparotomy and laparotomy followed by gut manipulation significantly reduced intestinal motility (Fig. 2); the dye mi-
FIG. 2. Small intestinal Evans blue transit in rats: untreated (UN), undergoing ether anesthesia (ET), skin incision (SI), laparotomy (LAP), and laparotomy followed by gut manipulation (L ⫹ M). Results are shown as centimeter migration of the dye and are presented as a mean result ⫾ SEM of a number of experiments performed in different animals (n ⫽ 5– 6). **UN, **ET, or *SI versus LAP (P ⬍ 0.01 or P ⬍ 0.05, respectively); ###L ⫹ M versus UN, ET, SI (P ⬍ 0.001); !LAP versus L ⫹ M (P ⬍ 0.05).
FIG. 3. The effects of SR140333 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–7). *Control (untreated rats) versus animals pretreated with 6 g/kg of SR140333 (P ⬍ 0.05); ***controls versus animals pretreated with 10 or 30 g/kg of SR140333 (P ⬍ 0.001).
grated 26.40 ⫾ 0.93 of 108 ⫾ 4.43 cm in the former group (n ⫽ 5) and 9.70 ⫾ 2.56 of 104 ⫾ 3.72 cm in the latter group (n ⫽ 5). The Effects of SR 140333, SR 48968, and SR142801 on the Intestinal Transit
SR140333 (3–30 g/kg), SR48968 (1–30 g/kg), and SR142801 (3–10 g/kg) reversed the additional inhibitory effects of gut manipulation subsequent to LAP in a dose-dependent manner. The intestinal passage reached 25.28 ⫾ 1.08, 21.70 ⫾ 0.19, and 25.0 ⫾ 1.34 cm with the most effective doses of the antagonists (Figs. 3–5). On the other hand, SR 140333, SR 48968, and SR142801 used in the most effective doses have not
FIG. 4. The effects of SR48968 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–7). *Control (untreated rats) versus animals pretreated with 3 g/kg of SR48968 (P ⬍ 0.05); ***controls versus animals pretreated 10 or 30 g/kg of SR48968 (P ⬍ 0.001).
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FIG. 5. The effects of SR142801 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–7). ***Controls (untreated animals) versus animals pretreated with 10 g/kg of SR142801 (P ⬍ 0.001).
affected the intestinal propulsion in untreated rats or animals subjected to skin incision or laparotomy (Fig. 6). Submaximal doses of SR140333 (10 g/kg), SR48968 (3 g/kg), and SR142801 (6 g/kg) substantially reversed the additional inhibitory effects of gut manipulation subsequent to LAP, so that the intestinal dye migration amounted to 18.58 ⫾ 0.86, 17.14 ⫾ 0.50, and 16.80 ⫾ 0.73 cm in comparison to untreated animals, 11.30 ⫾ 0.54 cm. The combination of NK 1 and NK 2 antagonists, SR140333 ⫹ SR48968, was additive, the passage of Evans blue reaching 26.60 ⫾ 0.35 cm, a value significantly different from the single-agent regimens. In contrast, the combinations of NK 2 ⫹ NK 3, NK 1 ⫹ NK 3, or NK 1 ⫹ NK 2 ⫹ NK 3 blockers proved to be no more effective than each of the test drugs given alone (Fig. 7), the dye progressing over 17.92 ⫾ 0.58 cm
FIG. 6. The effects of SR140333 (30 g/kg), SR48968, and SR142801 (10 g/kg) on the small intestinal transit of Evans blue in untreated rats (UN) or animals subjected to skin incision (SI) or laparotomy (LAP). Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–15).
FIG. 7. The effects of the combinations of SR140333, SR48968, and SR142801 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–9). *Control (untreated rats) versus animals pretreated with SR140333, SR48968, SR142801, and SR140333 ⫹ SR48968 ⫹ SR142801 (P ⬍ 0.05); **control (untreated rats) versus animals pretreated with SR140333 ⫹ SR48968 (P ⬍ 0.01); #animals pretreated with either SR140333 or SR48968 versus rats given SR140333 ⫹ SR48968 (P ⬍ 0.05); !!control (untreated rats) versus animals pretreated with SR48968 ⫹ SR142801 and SR140333 ⫹ SR142801 (P ⬍ 0.01).
(SR48968 ⫹ SR142801), 19.10 ⫾ 1.24 cm (SR140333 ⫹ SR142801), and 20.92 ⫾ 1.81 cm (SR140333 ⫹ SR48968 ⫹ SR142801). DISCUSSION
To the best of our knowledge we are the first to demonstrate an additive salutary effect of NK 1 and NK 2 receptor antagonists in the PI model in rats, even though a combination of NK 1 and NK 2 antagonists had been more effective than each of the agents given alone in visceral hyperalgesia or urinary bladder hyperactivity [10, 13]. In the course of this study three different nociceptive stimuli were used: skin incision, laparotomy, and laparotomy followed by a subsequent gut manipulation. Laparotomy decreased the intestinal dye passage by 57% compared to untreated rats or animals exposed only to ether anesthesia. This effect has been further potentiated by intestinal manipulation, the inhibition reaching almost 84%. Thus it seems that inhibition of gut motility following gastrointestinal surgery results mainly from the activation of mechanisms evoked by laparotomy and gut manipulation, whereas skin incision or ether anesthesia has no marked effect on the intestinal transit [3]. SR140333, SR48968, and SR142801 did not exert a marked effect on the movement of Evans blue in untreated rats or animals subjected to skin incision. In contrast, they reversed the additional inhibition of in-
CIECHANOWNICZ ET AL.: TACHYKININS AND POSTOPERATIVE ILEUS
testinal transit evoked by evisceration and gut manipulation in a dose-dependent manner. These results are consistent with data showing that NK 1–3 antagonists do not affect peristalsis in guinea pig and dog under normal conditions [14, 15], whereas they exert a protective action against the inhibition of gastrointestinal motility invoked by colorectal distension or intestinal manipulation [9, 16, 17]. In contrast, Herrscher showed that the administered combination of NK 1 receptor and CGRP antagonists abolish the protective activity of each agent given separately to prevent the postoperative gastric motility inhibition [18]. The discrepancies could at least partially be accounted for by methodological differences. In light of the results of our experiments, it is not possible to pinpoint the exact locus of action or discuss the molecular mechanisms of the prevention of PI development by SR140333, SR48968, and SR142801. However one can speculate that the protective effect of tachykinin blockers may result from the alteration of transmission or processing of nociceptive stimuli at different levels, ranging from the primary afferents innervating the gut to the central nervous system [19 –22]. While interpreting the acquired data in clinical context, one should realize that despite having some advantages the current model possesses significant drawbacks. On the one hand, it seems that the inhibition of small intestinal propulsion corresponds well to the suppression of spike activity and the absence of migrating motor complex [23, 24], whereas on the other hand it provides a mixture of gastric emptying and small intestinal propulsion. The relationship of these two phenomena could not be defined in this experimental setting; furthermore, the effective duration of PI in humans depends largely on the return of colonic motility [1, 2]. However the methodological shortcomings might be offset to some degree by the important results of clinical experiments demonstrating a considerable benefit of SR142801 in patients with schizophrenia, panic, and schizoactive disorders [25, 26]. Similarly SR 48968 protected against a tachykinin-induced uterine contraction in near-term pregnant women or bronchoconstriction in mild asthmatics [27, 28]. Both agents were well tolerated. Therefore we conclude that the present data support the potential utility of SR140333, SR48968, and SR142801 in the prevention of PI.
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Zagorodnyuk V, Santicioli P, Maggi CA, et al. Evidence that tachykinin NK1 and NK2 receptors mediate NANC excitation and contraction in the circular muscle of guinea-pig duodenum. Br J Pharmacol 1995;115:237.
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Yamamoto T, Yaksh TL. Stereospecific effects of a nonpeptidic NK1 selective antagonist, CP-96,345: antinociception in the absence of motor dysfunction. Life Sci 1991;49:1955.
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ACKNOWLEDGMENTS
We thank the Medical University of Gdan´sk for financial contributions to this study. We are grateful to Dr. X. Emonds-Alt, Sanofi-Synthelabo, France for the generous donation of SR140333, SR48968, and SR142801.
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Gaudreau GA, Plourde V. Role of tachykinin NK1, NK2 and NK3 receptors in the modulation of visceral hypersensitivity in the rat. Neurosci Lett 2003;351:59.
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McLean PG, Picard C, Garcia-Villar R, et al. Effects of nematode infection on sensitivity to intestinal distension: role of tachykinin NK2 receptors. Eur J Pharmacol 1997;337:279.
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Ruwart MJ, Klepper MS, Rush BD. Adrenergic and cholinergic contributions to decreased gastric emptying, small intestinal transit, and colonic transit in the postoperative ileus rat. J Surg Res 1980;29:126.
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Kelley MC, Hocking MP, Marchand SD, et al. Ketorolac prevents postoperative small intestinal ileus in rats. Am J Surg 1993;165:107.
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Kronenberg G, Berger P, Tauber RF, et al. Randomized, doubleblind study of SR142801 (Osanetant). A novel neurokinin-3 (NK3) receptor antagonist in panic disorder with pre- and posttreatment cholecystokinin tetrapeptide (CCK-4) challenges. Pharmacopsychiatry 2005;38:24.
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Meltzer HY, Arvanitis L, Bauer D, et al. Meta-Trial Study Group. Placebo-controlled evaluation of four novel compounds for the treatment of schizophrenia and schizoaffective disorder. Am J Psychiatry 2004;161:975.
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Patak EN, Ziccone S, Story ME, et al. Activation of neurokinin NK2 receptors by tachykinin peptides causes contraction of uterus in pregnant women near term. Mol Hum Reprod 2000; 6:549.
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van Schoor J, Joos GF, Chasson BL, et al. The effect of the NK2 tachykinin receptor antagonist SR48968 (saredutant) on neurokinin-A-induced bronchoconstriction in asthmatics. Eur Respir J 1998;12:17.
Salutary Effects of Tachykinin Receptor Antagonists in a Rat Model of Postoperative Ileus 1 R. Ciechanownicz, M.D.,* J. Sein-Anand, M.D., Ph.D.,* Z. Chodorowski, M.D., Ph.D.,* M. Bitel, M.D.,† J. Petrusewicz, Ph.D.,† and R. P. Korolkiewicz, M.D., Ph.D.†,2 *Department of Internal Medicine and Toxicology, Medical University of Gdan´sk, De˛binki 7, 80-211 Gdan´sk, Poland; and †Department of Pharmacology, Medical University of Gdan´sk, De˛bowa 23, 80-204 Gdan´sk, Poland Submitted for publication September 20, 2005
Background. Postoperative ileus (PI) is a common surgical complication treated mainly with supportive measures. Tachykinins control gastrointestinal motility and modulate somatic and visceral pain sensation; therefore, the effect of tachykinin receptor antagonists in a rat model of PI using NK 1–3 antagonists, SR140333, SR48968, and SR142801, was investigated. Materials and methods. Intestinal transit was measured as Evans blue migration after varied nociceptive stimuli: skin incision (SI), laparotomy (LAP), or laparotomy plus gut manipulation (L ⴙ M) in anesthetized rats. Results. Diethyl ether anesthesia and SI did not influence the intestinal transit of the dye in comparison to untreated animals—UN: 61.17 ⴞ 5.47, 62.10 ⴞ 8.30, and 56.70 ⴞ 4.10 cm, respectively. In contrast LAP and L ⴙ M have significantly reduced intestinal motility to 26.40 ⴞ 2.07 and 9.70 ⴞ 1.15 cm, respectively. SR140333 (3–30 g/kg), SR48968 (1–30 g/kg), and SR142801 (3– 10 g/kg) reversed the additional inhibitory effects of gut manipulation subsequent to LAP dose-dependently, the dye transit returning with the use of the most effective antagonist doses up to 25.28 ⴞ 1.08, 21.70 ⴞ 0.19, and 25.0 ⴞ 1.34 cm. The combinations of submaximal doses of NK 1 and NK 3, NK 2 and NK 3 and NK 1, and NK 2 and NK 3 antagonists were not more effective than a single-agent regimen. On the other hand SR140333 and SR48968 (NK 1 ⴙ NK 2 antagonists) acted additively, the intestinal transit reaching 26.60 ⴞ 0.85
1 Results were presented in part at a Brain-Gut Axis in Gastrointestinal System. Basic and Clinical Aspects Symposium, Kraków, Poland, November 2003. 2 To whom correspondence and reprint requests should be addressed at Department of Pharmacology, Medical University of Gdan´sk, De˛bowa 23, 80-204 Gdan´sk, Poland. E-mail: rokor@amg. gda.pl.
cm. SR140333, SR48968, and SR142801 have not affected the intestinal passage in UN rats or those undergoing SI or LAP. Conclusions. SR140333, SR48968, and SR142801 exert a salutary action on suppressed gut motility following surgical manipulation of the gut, the combination of NK 1 and NK 2 antagonists being most beneficial. © 2006 Elsevier Inc. All rights reserved.
Key Words: postoperative ileus; rats; tachykinin receptor antagonists. INTRODUCTION
Postoperative ileus (PI) is a spontaneously reversible, transient inhibition of propulsive bowel motility occurring after major surgical interventions and especially those that involve an open technique abdominal operation leading to an increased morbidity and higher hospitalization costs [1]. Due to an unclear etiology, supportive measures remain the cornerstone of the current therapy despite achieving some progress with multimodal postsurgical rehabilitation programs and the introduction of peripherally acting opioid antagonists [1, 2]. It seems that the inhibitory neuronal arches, with afferent capsaicin-sensitive unmyelinated fibers and efferent adrenergic and nitrergic neurons, play a prominent role in the development of PI. In concert with this hypothesis, thoracic epidural anesthesia improved PI outcome in humans, whereas the inhibition of nitric oxide synthase (NOS) reversed the intestinal transit migration in rodents [3, 4]. The enteric nervous system produces a number of inhibitory (e.g., NO, VIP, CO) and stimulatory neurotransmitters (e.g., substance P) [5]. Tachykinins belong to the largest peptide family with three main subtypes of
197
0022-4804/06 $32.00 © 2006 Elsevier Inc. All rights reserved.
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receptors present in rat intestinal smooth muscle cells: NK 1, NK 2, and NK 3 [6]. Major tachykinins such as substance P (SP) and neurokinins A and B (NKA and NKB) bind to all of these the receptors with varied degrees of affinity [7]. Tachykinins are most frequently an excitatory transmitters in the gut; however, their net effect depends on the animal species, gut segment, the profile of activated receptors, and other mediators acting at target sites. The main sources of neuronal tachykinins in the gut are the intrinsic enteric neurons of myenteric and the submucosal plexuses together with the extrinsic primary afferent fibers. Moreover many C-afferent fibers possess tachykinin receptors that can be sensitized by mechanical stimulation or an induction of an inflammatory process [8]. NK 1, NK 2, and NK 3 receptors are involved in the modulation of visceral hypersensitivity evoked by colorectal distension or inflammation [9, 10] Octreotide, VIP, SP, and CGRP antagonists ameliorate the postsurgical inhibition of gastric and small intestine motility in dogs and rats, implying that multiple tachykinin substances are involved in the pathogenesis of PI [11, 12]. The activity of tachykinins in the gut and the availability of potent synthetic tachykinin receptor antagonists, SR140333, SR48968, and SR142801, enabled a study of the effect of NK 1–3 receptor blockade in a rat model of PI. Due to conflicting reports in the literature, it was investigated whether in a combination of NK 1–3 receptor antagonists could potentially exert an additive salutary effect on the small bowel motility following a surgical insult. MATERIALS AND METHODS Surgical Protocol The Bioethics Committee of the Medical University of Gdan´sk approved the experimental procedures. Male albino Wistar rats (180 –250 g) were fasted for 48 h, maintaining free access to tap
water. Rats were randomly divided into four groups: one control group (untreated animals) and three groups exposed to surgical insult under diethyl ester anesthesia as previously described [3]. Rats from the second group underwent skin incision after having abdominal hair shaven and skin disinfected with 70% ethanol. In the third group, the animals underwent laparotomy. Animals from the fourth group were exposed to laparotomy and subsequent gut evisceration followed by mechanical stimulation of cecum and small intestine. Briefly, the small intestine and cecum were pulled out of the abdominal cavity and gently spread between two layers of damp, sterile gauze. The small intestine was carefully touched by two wet cotton bud applicators from cecum upward, until the duodenal end of the intestine was reached. This procedure was repeated six times within 10 min, and then the cecum and small intestine were returned to the abdominal cavity and the surgical wound was sutured. After the operation, the rats recovered for 1 h. Recovery time was based on the findings of the pilot experiments that diethyl ester anesthesia affects gastrointestinal (GI) motility for approximately 1 h. Subsequently, all animals received 0.15 ml Evans blue via an orogastric tube and 30 min later the animals were sacrificed by cardiotomy under deep ether anesthesia. The small intestines were excised and, to avoid tissue stretching, gently laid on corkboard for measurements, which consisted of establishing the most distal point of dye migration from the pylorus. A blinded observer, unaware of the treatment the animals were receiving, performed measurements. The intestinal transit was measured from the pylorus to the most distal point of dye migration. Experimental details are depicted in Fig. 1.
The Effects of Tachykinin Receptor Antagonists on the Intestinal Transit Initially the effects of intraperitoneally (i.p.) injected SR 140333 (3–100 g/kg), SR 48968 (1–30 g/kg), and SR142801 (3–30 g/kg) were investigated in untreated or anesthetized rats and animals exposed to skin incision, laparotomy, laparotomy, and gut manipulation. Subsequently, a combination of submaximal doses of NK 1 ⫹ NK 2 (SR140333 ⫹ SR48968; 10 ⫹ 3 g/kg), NK 1 ⫹ NK 3 (SR140333 ⫹ SR142801; 10 ⫹ 6 g/kg), NK 2 ⫹ NK 3 (SR48968 ⫹ SR142801; 3 ⫹ 6 g/kg), or NK 1 ⫹ NK 2 ⫹ NK 3 receptor antagonists (SR 140333 ⫹ SR48968 ⫹ SR142801; 10 ⫹ 3 ⫹ 6 g/kg) were investigated in rats undergoing laparotomy followed by gut manipulation. Respective controls in each experimental group received an equal volume of saline (0.9% NaCl) instead of test article. All tested agents or saline was administered 1.5 h before surgical procedures.
FIG. 1. The effects of NK 1–3 antagonists, SR140333 (3–100 g/kg), SR48968 (1–30 g/kg), SR142801(3–30 g/kg), administered separately or in combination were tested on the intestinal transit of Evans blue, after surgical intervention in rats subjected to different nociceptive stimuli: skin incision (SI), laparotomy (L), or laparotomy followed by gut handling (L ⫹ M). Respective controls in each experimental group received an equal volume of saline (0.9% NaCl) instead of test article. All tested agents or saline were administered 1.5 h before surgical procedures.
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Drugs SR 140333, SR 48968, and SR 142801 were a generous gift of Dr. X. Emonds-Alt, Sanofi-Synthelabo, France. SR 140333, SR 48968, and SR 142801 were dissolved in a small volume of 70% ethanol. All agents were adjusted to the desired concentration with saline. Evans blue was obtained from Sigma-Aldrich (Poznan´, Poland) and 50 mg of the dye was dissolved in 1 ml saline. Saline was purchased from Fresenius Kabi (Kutno, Poland) and diethyl ether was purchased from Polskie Odczynniki Chemiczne SA (Gliwice, Poland).
Statistical Analysis The length of small intestine between animals in all experimental groups was not statistically different in the course of this study (data not shown); hence the distance covered by Evans blue was expressed in centimeters and the results were presented in the text as a mean value ⫾ standard error of the mean (SEM) for the number of rats mentioned in each group. Results were compared using one-way analysis of variance (ANOVA) plus Bonferroni post-ANOVA test. Two-tailed P values of less than 0.05 were taken to indicate significant difference.
RESULTS The Effects of Ether Anesthesia and Surgical Intervention on the Intestinal Transit
Evans blue migrated over a distance of 62.10 ⫾ 8.33 cm out of a total length of 112 ⫾ 3.67 cm of the small intestine in the untreated conscious rats (n ⫽ 5). Ether anesthesia and skin incision have not influenced the intestinal transit of Evans blue reaching 61.17 ⫾ 5.47 of 108 ⫾ 1.05 cm and 56.70 ⫾ 4.10 of 102 ⫾ 5.66 cm, respectively (n ⫽ 6 and 5). In contrast, both laparotomy and laparotomy followed by gut manipulation significantly reduced intestinal motility (Fig. 2); the dye mi-
FIG. 2. Small intestinal Evans blue transit in rats: untreated (UN), undergoing ether anesthesia (ET), skin incision (SI), laparotomy (LAP), and laparotomy followed by gut manipulation (L ⫹ M). Results are shown as centimeter migration of the dye and are presented as a mean result ⫾ SEM of a number of experiments performed in different animals (n ⫽ 5– 6). **UN, **ET, or *SI versus LAP (P ⬍ 0.01 or P ⬍ 0.05, respectively); ###L ⫹ M versus UN, ET, SI (P ⬍ 0.001); !LAP versus L ⫹ M (P ⬍ 0.05).
FIG. 3. The effects of SR140333 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–7). *Control (untreated rats) versus animals pretreated with 6 g/kg of SR140333 (P ⬍ 0.05); ***controls versus animals pretreated with 10 or 30 g/kg of SR140333 (P ⬍ 0.001).
grated 26.40 ⫾ 0.93 of 108 ⫾ 4.43 cm in the former group (n ⫽ 5) and 9.70 ⫾ 2.56 of 104 ⫾ 3.72 cm in the latter group (n ⫽ 5). The Effects of SR 140333, SR 48968, and SR142801 on the Intestinal Transit
SR140333 (3–30 g/kg), SR48968 (1–30 g/kg), and SR142801 (3–10 g/kg) reversed the additional inhibitory effects of gut manipulation subsequent to LAP in a dose-dependent manner. The intestinal passage reached 25.28 ⫾ 1.08, 21.70 ⫾ 0.19, and 25.0 ⫾ 1.34 cm with the most effective doses of the antagonists (Figs. 3–5). On the other hand, SR 140333, SR 48968, and SR142801 used in the most effective doses have not
FIG. 4. The effects of SR48968 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–7). *Control (untreated rats) versus animals pretreated with 3 g/kg of SR48968 (P ⬍ 0.05); ***controls versus animals pretreated 10 or 30 g/kg of SR48968 (P ⬍ 0.001).
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FIG. 5. The effects of SR142801 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–7). ***Controls (untreated animals) versus animals pretreated with 10 g/kg of SR142801 (P ⬍ 0.001).
affected the intestinal propulsion in untreated rats or animals subjected to skin incision or laparotomy (Fig. 6). Submaximal doses of SR140333 (10 g/kg), SR48968 (3 g/kg), and SR142801 (6 g/kg) substantially reversed the additional inhibitory effects of gut manipulation subsequent to LAP, so that the intestinal dye migration amounted to 18.58 ⫾ 0.86, 17.14 ⫾ 0.50, and 16.80 ⫾ 0.73 cm in comparison to untreated animals, 11.30 ⫾ 0.54 cm. The combination of NK 1 and NK 2 antagonists, SR140333 ⫹ SR48968, was additive, the passage of Evans blue reaching 26.60 ⫾ 0.35 cm, a value significantly different from the single-agent regimens. In contrast, the combinations of NK 2 ⫹ NK 3, NK 1 ⫹ NK 3, or NK 1 ⫹ NK 2 ⫹ NK 3 blockers proved to be no more effective than each of the test drugs given alone (Fig. 7), the dye progressing over 17.92 ⫾ 0.58 cm
FIG. 6. The effects of SR140333 (30 g/kg), SR48968, and SR142801 (10 g/kg) on the small intestinal transit of Evans blue in untreated rats (UN) or animals subjected to skin incision (SI) or laparotomy (LAP). Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–15).
FIG. 7. The effects of the combinations of SR140333, SR48968, and SR142801 on the small intestinal transit of Evans blue following laparotomy and gut manipulation. Results are shown as centimeter migration of Evans blue and are presented as mean results ⫾ SEM (n ⫽ 5–9). *Control (untreated rats) versus animals pretreated with SR140333, SR48968, SR142801, and SR140333 ⫹ SR48968 ⫹ SR142801 (P ⬍ 0.05); **control (untreated rats) versus animals pretreated with SR140333 ⫹ SR48968 (P ⬍ 0.01); #animals pretreated with either SR140333 or SR48968 versus rats given SR140333 ⫹ SR48968 (P ⬍ 0.05); !!control (untreated rats) versus animals pretreated with SR48968 ⫹ SR142801 and SR140333 ⫹ SR142801 (P ⬍ 0.01).
(SR48968 ⫹ SR142801), 19.10 ⫾ 1.24 cm (SR140333 ⫹ SR142801), and 20.92 ⫾ 1.81 cm (SR140333 ⫹ SR48968 ⫹ SR142801). DISCUSSION
To the best of our knowledge we are the first to demonstrate an additive salutary effect of NK 1 and NK 2 receptor antagonists in the PI model in rats, even though a combination of NK 1 and NK 2 antagonists had been more effective than each of the agents given alone in visceral hyperalgesia or urinary bladder hyperactivity [10, 13]. In the course of this study three different nociceptive stimuli were used: skin incision, laparotomy, and laparotomy followed by a subsequent gut manipulation. Laparotomy decreased the intestinal dye passage by 57% compared to untreated rats or animals exposed only to ether anesthesia. This effect has been further potentiated by intestinal manipulation, the inhibition reaching almost 84%. Thus it seems that inhibition of gut motility following gastrointestinal surgery results mainly from the activation of mechanisms evoked by laparotomy and gut manipulation, whereas skin incision or ether anesthesia has no marked effect on the intestinal transit [3]. SR140333, SR48968, and SR142801 did not exert a marked effect on the movement of Evans blue in untreated rats or animals subjected to skin incision. In contrast, they reversed the additional inhibition of in-
CIECHANOWNICZ ET AL.: TACHYKININS AND POSTOPERATIVE ILEUS
testinal transit evoked by evisceration and gut manipulation in a dose-dependent manner. These results are consistent with data showing that NK 1–3 antagonists do not affect peristalsis in guinea pig and dog under normal conditions [14, 15], whereas they exert a protective action against the inhibition of gastrointestinal motility invoked by colorectal distension or intestinal manipulation [9, 16, 17]. In contrast, Herrscher showed that the administered combination of NK 1 receptor and CGRP antagonists abolish the protective activity of each agent given separately to prevent the postoperative gastric motility inhibition [18]. The discrepancies could at least partially be accounted for by methodological differences. In light of the results of our experiments, it is not possible to pinpoint the exact locus of action or discuss the molecular mechanisms of the prevention of PI development by SR140333, SR48968, and SR142801. However one can speculate that the protective effect of tachykinin blockers may result from the alteration of transmission or processing of nociceptive stimuli at different levels, ranging from the primary afferents innervating the gut to the central nervous system [19 –22]. While interpreting the acquired data in clinical context, one should realize that despite having some advantages the current model possesses significant drawbacks. On the one hand, it seems that the inhibition of small intestinal propulsion corresponds well to the suppression of spike activity and the absence of migrating motor complex [23, 24], whereas on the other hand it provides a mixture of gastric emptying and small intestinal propulsion. The relationship of these two phenomena could not be defined in this experimental setting; furthermore, the effective duration of PI in humans depends largely on the return of colonic motility [1, 2]. However the methodological shortcomings might be offset to some degree by the important results of clinical experiments demonstrating a considerable benefit of SR142801 in patients with schizophrenia, panic, and schizoactive disorders [25, 26]. Similarly SR 48968 protected against a tachykinin-induced uterine contraction in near-term pregnant women or bronchoconstriction in mild asthmatics [27, 28]. Both agents were well tolerated. Therefore we conclude that the present data support the potential utility of SR140333, SR48968, and SR142801 in the prevention of PI.
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ACKNOWLEDGMENTS
We thank the Medical University of Gdan´sk for financial contributions to this study. We are grateful to Dr. X. Emonds-Alt, Sanofi-Synthelabo, France for the generous donation of SR140333, SR48968, and SR142801.
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