JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 JPET FastThis article Forward. has not beenPublished copyedited on and Marchformatted. 26, The 2009 final version as DOI:10.1124/jpet.109.152330 may differ from this version.
JPET #152330 - 1
The selective 5-HT1A antagonist, AZD7371 (robalzotan tartrate
monohydrate), inhibits visceral pain-related visceromotor, but not
autonomic cardiovascular, responses to colorectal distension in rats
E. Lindström, A. Ravnefjord, M. Brusberg, S. Hjorth, H. Larsson, V. Martinez
Biosciences, AstraZeneca R&D Mölndal, Mölndal, Sweden (EL, AR, MB, SH, Downloaded from HL, VM). Department of Cell Biology, Physiology and Immunology, Veterinary
School & School of Biosciences, Universidad Autónoma de Barcelona,
Barcelona, Spain (VM). jpet.aspetjournals.org at ASPET Journals on September 27, 2021
Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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Running title: AZD7371 and visceral pain in rats: Clinical significance
Text pages: 33
Figures: 6
Tables: 0
Words in abstract: 250
Words in introduction: 440
Words in discussion: 1485
Number of references: 40 Downloaded from
Address for correspondence Vicente Martinez
Unit of Physiology – Veterinary School jpet.aspetjournals.org Dept. Cell Biology, Physiology & Immunology
Universidad Autónoma de Barcelona
08193 – Bellaterra, Barcelona at ASPET Journals on September 27, 2021 Spain
Phone: +34 93 581 3834
FAX: +34 93 581 2006
e-mail: [email protected]
Abbreviations:
5-HT1A: 5-hydroxytryptamine 1A bpm: Beats per minute
CRD: Colorectal distension
ICV: Intracerebroventricular
VMR: Visceromotor response
Section of assignment: Gastrointestinal, Hepatic, Pulmonary, and Renal JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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Abstract
5-hydroxytryptamine 1A (5-HT1A) receptors have been suggested as a target for the treatment of irritable bowel syndrome (IBS). A recent clinical trial investigating the efficacy of the selective 5-HT1A antagonist AZD7371 showed no symptomatic improvement in IBS patients. We characterized the mechanisms mediating potential analgesic effects of AZD7371 [3(R)-(N,N- Downloaded from Dicyclobutylamino)-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide
(R,R)-tartrate monohydrate] in a model of colorectal distension (CRD)-induced
visceral pain in rats to understand its mechanism of action and the lack of jpet.aspetjournals.org clinical efficacy. Visceromotor and cardiovascular responses (telemetry) were assessed in conscious rats during noxious CRD (80 mmHg). Effects of
AZD7371 (3-300 nmol/kg, iv; 1-30 µmol/kg, po) and a reference 5-HT1A at ASPET Journals on September 27, 2021 antagonist, WAY-100635 (3-300 nmol/kg, iv), were assessed. Effects of intracerebroventricular (ICV) AZD7371 were also evaluated. Intravenous
AZD7371 or WAY-100635 and oral AZD7371 dose-dependently inhibited visceromotor responses to CRD (ED50s: 203 nmol/kg, 231 nmol/kg and 14
µmol/kg, respectively). In telemetrized rats, oral AZD7371 inhibited visceromotor responses to CRD without affecting the concomitant hypertensive and tachycardic responses. ICV AZD7371 did not affect visceromotor responses, while it inhibited micturition. None of the doses tested induced visible gross side-effects. AZD7371, likely acting at a spinal site, inhibited the visceromotor, but not the cardiovascular, responses to visceral pain in the CRD model in rats. While agents effective on multiple pain-related readouts in the
CRD model (e.g. pregabalin or clonidine) alleviate IBS symptoms, AZD7371, JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 4 effective on only one pain-related pseudo-affective readout, does not. Data from preclinical CRD models of visceral pain need to be interpreted cautiously as it relates to their clinical translational value. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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Introduction
Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by symptoms of abdominal pain or discomfort associated with altered bowel habits
(Azpiroz et al., 2007). Even if the etiology of IBS has been suggested to involve multiple factors, including, at least, abnormal central processing of gastrointestinal sensory signals, psychosocial disturbance, altered Downloaded from gastrointestinal motility, and visceral hypersensitivity (Drossman et al., 2002;
Azpiroz et al., 2007) the pathophysiology of the disease remains largely
unknown. The neurotransmitter and gut mucosal signaling molecule 5- jpet.aspetjournals.org hydroxytrypamine (5-HT, serotonin) plays a central role in normal gastrointestinal tract function through its modulatory effects on gut motility,
intestinal secretion, and visceral sensitivity (Mawe et al., 2006, Gershon and at ASPET Journals on September 27, 2021
Tack, 2007). Correspondingly, there is evidence that serotonin-mediated signaling is altered in individuals with IBS (Mawe et al., 2006). Indeed most of the treatments for IBS to date have been designed to modulate serotonin signaling, such are the 5-HT3 antagonist alosetron and the 5-HT4 partial agonist
(and 5-HT1B1 and 5-HT2B receptor antagonist) tegaserod (Spiller, 2008). Other serotonin receptors have been proposed as potential targets for the treatment of
IBS, including 5-HT1A, 5-HT2 and 5-HT7 (Danzebrink and Gebhart, 1991; Coelho et al., 1998, 2001; Sivarao et al., 2004; Tonini et al., 2005). For instance, it has been shown that the 5-HT1A antagonist, WAY-100635, inhibits pain-related responses to colorectal distension (CRD) in rats (Coelho et al., 1998) and 5-HT7 might affect sensitivity through the modulation of colonic compliance (Tonini et al., 2005). JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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The aims of the present study were to gain insight into the effects and mechanism of action of the selective, competitive, 5-HT1A receptor antagonist
AZD7371 tartrate monohydrate (robalzotan tartrate monohydrate, NAD299, here termed AZD7371) (Johansson et al., 1997) on visceral pain-related visceromotor and autonomic cardiovascular responses in a model of colorectal distension-induced visceral pain in conscious rats. AZD7371 was initially developed as a potential treatment for depression and anxiety disorders Downloaded from
(Mucke, 2000). However, based on previous observations with the 5-HT1A antagonist WAY-100635 (Coelho et al., 1998) and also in part of the preclinical
observations presented in the present report, it was suggested that AZD7371 jpet.aspetjournals.org would be effective in reducing abdominal pain and discomfort in patients with
IBS. In addition, because mental stress, anxiety, and depression have been
implicated in the pathophysiology of IBS (Mayer et al., 2001), it was speculated at ASPET Journals on September 27, 2021 that the potential anxiolytic properties of AZD7371 might provide further benefits in IBS patients. Nevertheless, the results obtained in a clinical study in IBS patients showed no symptomatic improvement over placebo, leading to discontinuation of development (Drossman et al., 2008). Therefore, we also aimed to understand the discrepancy between the positive preclinical findings and the negative clinical observations with AZD7371. JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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Methods
Animals
Adult female Sprague–Dawley rats (Harlan, The Netherlands; 250–300 g) were used. The rats were allowed to acclimatize to the animal facility for at least 1 week after arrival. Rats were housed in groups of five, unless otherwise stated, in an enriched environment with free access to food (Standard pellets, Downloaded from R3, Lactamin, Sweden) and water under controlled conditions of temperature
(21 °C) and humidity (50%) on a 12:12 h light–dark cycle. The phase of the
estrous cycle was not taken into consideration in the current study. All jpet.aspetjournals.org experiments were approved by the local animal ethics review committee in
Göteborg, Sweden. All procedures were in accordance with current European
laws concerning animal experimentation. at ASPET Journals on September 27, 2021
Surgical preparation
Implantation of radio transmitters When assessing cardiovascular responses, a telemetric system was used. Rats were anesthetized with a mixture (2 ml kg/kg, i.p.) of ketamin (88 mg/kg; Ketalar® Vet; Pfizer AB, Täby,
Sweden) and xylazin (5 mg/kg; Rompun® Vet; Bayer AG, Leverkusen,
Germany) and were surgically equipped with intraperitoneal radio transmitters
(PhysioTel® C50-PXT, DSI, St. Paul, MN, USA). The catheter of the transmitter was inserted into the abdominal aorta and fixed with tissue adhesive
(Vetbond®, 3M, St. Paul, MN, USA) for blood pressure measurements. The animals recovered from surgery in a quiet and dim post-op room for 24 h and received also antibiotic (Bactrim®, Roche, Basel Switzerland) and analgesic JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 8
(Finadyne®, Shering-Plough, NJ, USA) treatment. Thereafter, a 7- to 10-day recovery period was allowed before starting any experimental procedures.
Implantation of intracerebroventricular (ICV) canulae For ICV administrations rats with chronically implanted ICV canulae were used. Rats were anesthetized with a mixture (2 ml kg/kg, i.p.) of ketamin (88 mg/kg;
Ketalar® Vet; Pfizer AB, Täby, Sweden) and xylazin (5 mg/kg; Rompun® Vet; Downloaded from Bayer AG, Leverkusen, Germany) and the ICV guide canulae were stereotaxically implanted into the right lateral brain ventricle according to
coordinates in the Paxinos and Watson’s atlas (Paxinos and Watson, 1998), i.e. jpet.aspetjournals.org from the bregma posterior -0.8 mm; lateral -1.5 mm and dorsoventral -3.5 mm, as previously described (Martinez and Taché, 2001). After surgery animals
were housed in individual cages with direct bedding and allowed a 7 day at ASPET Journals on September 27, 2021 recovery period before starting any experiment.
Colorectal distension (CRD)
Rats were habituated to Bollmann cages (Plexi-glass tubes, length 18 cm, diameter 6 cm, AstraZeneca, Mölndal, Sweden) 30 min per day for three consecutive days prior to experiments, to reduce motion artifacts and confounding effects due to stress-related responses.
A 3 cm polyethylene balloon (made in-house) with connecting catheter
(PE-50) was inserted in the distal colon, 2 cm from the base of the balloon to the anus, during light isoflurane anesthesia (Forene®, Abbott Scandinavia AB,
Solna, Sweden). The catheter was fixed to the tail with tape. At the same time, if needed, an intravenous catheter (Neoflon®, Becton Dickinson AB, Helsingborg, JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 9
Sweden) was inserted in the tail vein for vehicle or compound administration.
The intravenous catheter was flushed with 0.2 ml of heparin (50 IE/KY/ml, Leo
Pharma, Ballerup, Denmark). The balloons were connected to pressure transducers (P-602, CFM-k33, 100 mmHg, Bronkhorst HI-TEC, Veenendal, The
Netherlands). Rats were allowed to recover from sedation in the Bollmann cages for at least 15 min before the start of experiments.
A customized barostat (AstraZeneca, Mölndal, Sweden) was used to Downloaded from manage air inflation and balloon pressure control. A customized computer software (PharmLab on-line 5.0) running on a standard computer was used to
control the barostat and to perform data collection. The distension paradigms jpet.aspetjournals.org generated by the barostat were achieved by generating pulse patterns on an analog output channel. For the assessment of visceral pain responses, the CRD
paradigm consisted of repeated phasic distensions, 12 times at 80 mmHg, with at ASPET Journals on September 27, 2021 a pulse duration of 30 s at 5 min intervals. This protocol has been used before to assess noxious responses to colorectal distension in rats (Käll et al., 2007;
Martinez et al., 2007; Brusberg et al., 2008, 2009a; Lindström et al., 2008;
Ravnefjord et al., 2008).
Data collection and analysis
The analog input channels were sampled with individual sampling rates, and digital filtering was performed on the signals. The balloon pressure signals were sampled at 50 samples/s. A highpass filter at 1 Hz was used to separate the contraction-induced pressure changes from the slow varying pressure generated by the barostat. A resistance in the airflow between the pressure generator and the pressure transducer further enhanced the pressure variations JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 10 induced by abdominal contractions of the animal. Data analysis was performed using pre-designed automatic analysis paradigms. Hence, manual analysis and potential bias by the investigator was avoided. Data represent the average rectified value of the highpass-filtered balloon pressure signals, calculated for
30 s before the pulse (i.e. baseline response) and for the duration of the pulse, and is given in arbitrary units. When calculating the magnitude of the highpass- filtered balloon pressure signals, the first and last seconds of each pulse were Downloaded from excluded since these reflect artifact signals produced by the barostat during inflation and deflation and do not originate from the animal.
jpet.aspetjournals.org
Drugs
AZD7371 [3(R)-(N,N-Dicyclobutylamino)-8-fluoro-3,4-dihydro-2H-1-
benzopyran-5-carboxamide (R,R)-tartrate monohydrate, robalzotan tartrate at ASPET Journals on September 27, 2021 monohydrate, NAD299; AstraZeneca R&D] (Johansson et al., 1997) and WAY-
100635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-(2- pyridyl)cyclohexanecarboxamide maleate salt; Sigma-Aldrich] (Fletcher et al.,
1993) were dissolved in 0.9% saline solution at the appropriate concentration.
Saline solution was used as vehicle control.
Experimental protocols
Dose-related effects of intravenous AZD7371 and WAY-100635 and oral
AZD7371 on visceromotor responses to CRD AZD7371 (3, 10,
30, 100 or 300 nmol/kg; equivalent approximately to 1.5, 5, 15, 50 and 150
µg/kg, respectively), WAY-100635 (3, 10, 30, 100 or 300 nmol/kg; equivalent to approximately to 1.3, 4.2, 13, 42 and 130 µg/kg, respectively) or vehicle (0.9% JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 11 saline solution, 1 ml/kg) was administered i.v. between distensions 3 and 4 of the CRD protocol.
In a separate experiment, the dose-related effect of oral AZD7371 was also characterized. In this case, AZD7371 (1, 3, 10 or 30 µmol/kg; equivalent approximately to 0.5, 1.5, 5 and 15 mg/kg) or vehicle (0.9% saline solution, 2 ml/kg) was administered orally (p.o.) 30 min before starting the CRD procedure.
In all experiments, each rat received both vehicle and a dose of Downloaded from compound on different occasions, with at least 4 days between experiments.
Hence, each rat served as its own vehicle control. Experiments were performed
in a counterbalanced cross-over fashion in which vehicle and different doses of jpet.aspetjournals.org compounds were tested during the same experiment, and repeated in several occasions.
at ASPET Journals on September 27, 2021
Effects of repetitive oral dosing with AZD7371 on visceromotor responses to
CRD Separate groups of rats received a daily oral dose of
AZD7371 (30 µmol/kg) or vehicle (2 ml/kg) for 9 consecutive days.
Visceromotor responses (VMRs) to CRD were assessed before starting any treatment and on days 1, 5 and 9 of treatment (30 min after the administration of the corresponding treatment).
Effects of AZD7371 on autonomic cardiovascular responses to CRD
Telemetrized animals were used for this experiment. Animals were dosed i.v. with either AZD7371 (100 or 300 nmol/kg, n=4) or vehicle (1 ml/kg, n=4) between distensions 3 and 4 of the CRD procedure. In this case, cardiovascular parameters (blood pressure and heart rate) and VMRs to CRD were assessed JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 12 simultaneously. The same group of animals was used in these studies, with a 4 day interval between the two treatments.
Effects of ICV AZD7371 on visceromotor responses to CRD and micturition
Rats with chronically implanted ICV canulae were used for these experiments. The ICV injection was performed in lightly restrained animals using a 28 ga injection canula, 1 mm longer than the guide canula, connected to Downloaded from a 50 µl Hamilton syringe by a PE-50 catheter filled with distilled water. A small air bubble (1 μl) was drawn at the distal end of the PE-50 catheter to separate
μ
the injecting solution from the water and for visual inspection of the 10 l jpet.aspetjournals.org injection (Martinez and Taché, 2001). Either AZD7371 (1 µg/rat) or vehicle
(sterile saline, 10 µl/rat) were injected slowly, over a 30 s period, between
distensions 3 and 4 of the CRD paradigm. At the same time, a piece of pre- at ASPET Journals on September 27, 2021 weighed absorbent paper was placed under each Bollmann cage to collect any urine excreted during the remaining experimental time. Differences in weight of the absorbent paper before and after the procedure were taken as a measure of micturition. All animals received two ICV injections, vehicle and treatment, with an interval of 4-5 days. At the end of the experiments, at the time of euthanasia, the correct location of the canula into the lateral ventricle was verified by injecting 10 μl of dye (0.1% toluidine blue). Visualization of dye on the wall of the lateral ventricle indicates correctness of the ICV injections.
Plasma levels of AZD7371
Separate groups of animals were used for the determination of plasma levels of AZD7371. Animals were dosed orally with AZD7371 (30 µmol/kg, 2 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 13 ml/kg, n=3) and blood samples were obtained 15, 30, 60, 75, 90, 120, 180, 240 and 350 min post-dosing in order to determine the complete pharmacokinetic profile of AZD7371 following oral administration. Total plasma levels of
AZD7371 were determined using standard HPLC combined with mass spectroscopy procedures (limit of quantification: 1 nmol/l).
Statistical analysis Downloaded from Data are expressed as mean±S.E.M. Differences between two groups were assessed by paired or unpaired Student’s t-test, as appropriate.
Differences between multiple groups were determined by a repeated or non- jpet.aspetjournals.org repeated measures one-way analysis of variance (ANOVA), as appropriate, followed, when necessary, by a Student-Newman-Keuls multiple comparisons
test. ED50 values were determined by nonlinear regression to a sigmoidal at ASPET Journals on September 27, 2021 equation with variable slope (Prism, version 4.01; GraphPad Software Inc., San
Diego, CA). Data were considered statistically significant when P was <0.05. JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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Results
Dose-related effects of AZD7371 and WAY-100635 on visceromotor responses to CRD
CRD at 80 mmHg induced a VMR manifested as a 4-fold increase in the activity of the abdominal musculature, compared to basal activity (basal:
0.05±0.003; first distension at 80 mmHg: 0.20±0.02; P<0.05; pooled data from Downloaded from 36 animals). In animals treated with vehicle the response to CRD increased by
164±20% from the first to the last distension indicating the development of
acute mechanical hyperalgesia (Fig. 1). AZD7371 (3, 10, 30, 100 or 300 jpet.aspetjournals.org nmol/kg, n=8 for each), administered iv between distensions 3 and 4, inhibited in a dose-dependent manner the response to CRD with an ED50 of 203 nmol/kg
2
(95% confidence interval: 109 - 377 nmol/kg, r =0.3281; Fig. 1A). The overall at ASPET Journals on September 27, 2021 response to CRD during distensions 4th to 12th was inhibited by 9±6 %, 4±10 %,
30±14 % (P<0.05), 41±10 % (P<0.05) and 48±10 % (P<0.05) at 3, 10, 30, 100 or 300 nmol/kg, respectively [F(5,70)=3.752; P=0.0046]. Similar effects were observed with WAY-100635 [3, 10, 30, 100 or 300 nmol/kg, n=4-8 for each;
F(5,66)=3.395; P=0.0086], which inhibited the response to CRD with an ED50 of
231 nmol/kg (95% confidence interval: 113 - 473 nmol/kg, r2=0.2722; Fig. 1B).
Similarly, oral AZD7371 (1, 3, 10 or 30 µmol/kg) inhibited the response to
CRD with an ED50 of 14 µmol/kg (95% confidence interval: 9 - 23 nmol/kg, r2=0.4543). Analgesic effects of AZD7371 were visible from the first distension.
In vehicle-treated animals, the activity of the abdominal musculature increased
5-fold during the first CRD (basal: 0.03±0.002; 1st CRD: 0.17±0.02; n=30;
P<0.05) while after AZD7371 the response was reduced to 0.13±0.02, JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
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0.11±0.02 (P=0.099), 0.12±0.04 (P=0.077) and 0.07±0.006 (P<0.05) at 1, 3, 10 and 30 µmol/kg, respectively (n=7-8 for each dose). After oral AZD7371, the overall response to CRD (distensions 1 to 12) was inhibited by 25±8 %, 22±12
%, 41±7% (P<0.05) and 55±8% (P<0.05) at 1, 3, 10 and 30 µmol/kg, respectively (Fig. 2). At 30 µmol/kg, total plasma levels of AZD7371 at the time interval corresponding to the CRD procedure ranged between 2.21±0.35 µmol/l
(30 min post-dosing) and 0.75±0.06 µmol/l (90 min post-dosing) as determined Downloaded from in a satellite group of animals (Fig. 3). The calculated Tmax after oral dosing
(0.33±0.08 h) corresponded to the start of the CRD procedure.
Although not systematically assessed, no gross side effects were jpet.aspetjournals.org observed at the doses tested after either intravenous or oral administration.
Based on these observations, the doses of 300 nmol/kg iv and 30
µmol/kg po were selected for further studies. at ASPET Journals on September 27, 2021
Effects of repetitive oral dosing with AZD7371 on visceromotor responses to CRD
Visceromotor responses to CRD were of similar magnitude in all animals before starting any treatment. Daily treatment with vehicle for 9 consecutive days (2 ml/kg/day, n=8) did not affect the response to CRD assessed on days 1, 5 and
9 [F(3,7)=0.07723, P=9716; Fig. 4]. In AZD7371-treated animals, after a single oral dose (30 µmol/kg, n=8) the response to CRD was reduced by 43.4±9 % compared with the response observed before starting the treatment procedure
(P<0.05; Fig. 4). Similar degree of inhibition was observed after 5 or 9 days of repeated daily treatment [42.6±11.8 % and 44.7±10.0 %, respectively; both
P<0.05 vs the VMR before treatment; F(3,7)=8.75, P=0.0033; Fig. 4]. JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 16
Effects of ICV AZD7371 on visceromotor responses to CRD and micturition
Visceromotor responses to CRD were of similar magnitude after the ICV administration of vehicle or AZD7371 (AUC for distensions 4-12; vehicle:
2.42±0.39; AZD7371: 2.58±0.54; n=7 for each, Fig. 5A).
Urine production during the 45 min post-treatment, corresponding o the Downloaded from time-interval of the CRD procedure, was reduced by 32 % after ICV AZD7371, when compared with the responses observed after vehicle administration ( Fig.
5B). The inhibitory effects of AZD7371 on micturition were clear in 6 out of the 7 jpet.aspetjournals.org animals tested. Nevertheless, due to the variability imposed by one of the animals, statistical significance was not reached.
at ASPET Journals on September 27, 2021
Effects of AZD7371 on visceromotor and autonomic cardiovascular responses to CRD in telemetrized rats
In telemetrized rats, CRD induced a VMR, similar in magnitude to that described above, and a simultaneous increase in blood pressure (mean increase: 16±6 mmHg) and heart rate (mean increase: 23±7 beats per minute, bpm). While, as described above, the VMR to CRD increased over time with consecutive distensions (106±21 % increase from the first to the last distension;
P<0.05), cardiovascular responses remained relatively stable along the CRD protocol (Fig. 6). AZD7371 (100 or 300 nmol/kg), administered iv between distensions 3 and 4, inhibited the VMR to CRD by 27 % and 30 %, respectively
(AUC for distensions 4-12; vehicle: 4.69±0.77; 100 nmol/kg: 3.46±0.50; 300 nmol/kg: 3.32±0.35; n=4 for each; P<0.05; Fig. 6A). In the same animals, JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 17
AZD7371 did not affect neither the hypertensive (mean increase in blood pressure; vehicle: 18±5 mmHg; 100 nmol/kg; 18±6 mmHg; 300 nmol/kg: 19±4 mmHg; P>0.05; Fig. 6B) nor the tachycardic responses elicited by CRD (mean increase in heart rate; vehicle: 37±8 bpm; 100 nom/kg: 40±10 bpm; 300 nmol/kg: 34±6 bpm; P>0.05; Fig. 6C). AZD7371, per se, did not affect neither resting (between distensions) blood pressure (vehicle: 126±2 mmHg; 100 nom/kg: 129±2 mmHg; 300 nmol/kg: 129±1 mmHg) nor resting (between Downloaded from distensions) heart rate (vehicle: 397±3 bpm; 100 nmol/kg: 410±7 bpm; 300 nmol/kg: 402±5 bpm) jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 18
Discussion
This study shows that the selective 5-HT1A antagonist, AZD7371, inhibited pain- related VMRs in a model of mechanically (CRD)-induced visceral pain in rats, likely through a peripheral or spinal mechanism. Interestingly, autonomic cardiovascular responses, generated concomitantly with the VMRs as part of the response to pain, were not affected by AZD7371. Downloaded from Pseudo-affective VMRs to CRD are considered a valid surrogate maker of visceral pain in rodents and are widely used in pharmacological studies
assessing the potential analgesic effects of compounds on visceral pain (Ness jpet.aspetjournals.org and Gebhart, 1988; Käll et al., 2007; Brusberg et al., 2008, 2009a; Lindström et al., 2008; Ravnefjord et al., 2008). Results obtained here show that AZD7371
dose-dependently inhibited the VMRs to CRD after systemic administration. at ASPET Journals on September 27, 2021
After iv administration, AZD7371 and the 5-HT1A antagonist WAY-100635 displayed similar potency and efficacy. This, together with the previously reported analgesic effects of WAY-100635 in several pain models, including
CRD-evoked visceral pain (Coelho et al., 2001; Wei and Pertovaara, 2006), and the high selectivity of AZD7371 for 5-HT1A receptors (Johansson et al., 1997) indicate that the effects observed are likely to correspond to a blockade of 5-
HT1A receptors.
On the other hand, AZD7371 failed to affect the pseudo-affective cardiovascular autonomic responses associated to pain, at doses significantly inhibiting concomitant VMRs. Pseudo-affective autonomic cardiovascular responses (changes in heart rate and blood pressure) have been characterized as a component of the pain response (Ness and Gebhart, 1988; Sivarao et al., JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 19
2007; Brusberg et al., 2008, 2009b; Lindström et al., 2008; Ravnefjord et al.,
2008), likely generated at higher CNS centers, primarily in the brainstem, where the primary integration of ascending pain-related signals is likely to occur and autonomic cardiovascular centers are located (Martinez et al., 2006; Loewy and
McKellar, 1980). Nevertheless, cardiovascular responses have been used to a lesser extent than the VMR as a surrogate marker for pain. The lack of effect of
AZD7371 on pain-related cardiovascular changes is difficult to interpret, Downloaded from particularly since VMRs were inhibited simultaneously. This finding was rather unexpected since, in the same experimental conditions we have consistently
observed that agents attenuating the VMR to CRD also inhibited autonomic jpet.aspetjournals.org cardiovascular responses to a similar degree (Brusberg et al., 2008, 2009b,
Lindström et al., 2008; Ravnefjord et al., 2008). In the case of AZD7371, this
might suggest that the effects on VMRs do not reveal a true analgesic effect of at ASPET Journals on September 27, 2021 the compound but a motor-related effect, leading to the inhibition of the motor reflex implicated in the generation of contractions of the skeletal musculature of the abdominal wall during CRD. Such a motor inhibitory effect could take place at peripheral sites, directly in the muscle, or at a spinal level, modulating the activity of the efferent motor pathways. Indeed, several reports implicate spinal
5-HT1A receptors in the modulation of spinal motor reflexes; resulting in both the inhibition of withdrawal monosynaptic spinal reflexes and the enhancement of motor activity (Clarke and Ward, 2000; Hedo et al., 2002; Zimmer et al., 2006;
Gajendiran, 2008). Therefore, it is possible that the inhibitory effects exerted by
AZD7371, and by WAY-100635, on the VMRs to CRD are due to the 5-TH1A- dependent modulation of motor reflexes at a spinal level, without interference with the afferent processing of pain signals. Nevertheless, no obvious motor- JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 20 related side-effects were noted in the animals at the doses tested. In addition, the fact that pain-related cardiovascular responses were not affected by
AZD7371 suggests that the compound neither blocks pain pathways at supraspinal sites nor at peripheral afferent sites. The consequence of this will be that pain-related afferent information is still integrated at central levels and, therefore, might lead to the modulation of, at least, some pain-related efferent activity. Further studies using, for example, emerging animal imaging Downloaded from technologies might help to understand the effects of AZD7371 on brain activation and how the compound might affect the central integration of sensory
signals elicited by visceral pain. jpet.aspetjournals.org
Alternatively, the lack of effects of AZD7371 on cardiovascular responses might be interpreted as an inadequate central exposure after peripheral
systemic administration. However, this is unlikely to happen since AZD7371 at ASPET Journals on September 27, 2021 freely enters the brain upon peripheral administration, as previously demonstrated (Larsson et al. 1998; Johansson et al., 1997; Farde et al., 2000;
Andrée et al., 2003; Hjorth, unpublished observations). In addition, in the present experiments, ICV AZD7371 was effective at inhibiting micturition, a well-characterized, 5-HT1A- and centrally-mediated effect of AZD7371 (Pehrson et al., 2002; Yoshiyama et al., 2003); thus indicating that the lack of effects of
ICV AZD7371 on pseudo-affective VMRs is likely not due to an ineffective blockade of central 5-HT1A receptors. Moreover, the lack of effects of AZD7371 on CRD responses after ICV administration, reported here, reinforces the view that inhibitory effects of the compound on VMRs are exerted, likely, at a spinal location and are independent of any effect at supraspinal sites. JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 21
Overall, the present observations might help to understand the negative results obtained recently in a clinical trial with AZD7371 assessing symptoms improvement in IBS patients (Drossman et al., 2008). In that study, AZD7371 failed to show any symptomatic improvement over a 12-week treatment period.
This, together with the undesirable profile observed (associated to the presence of CNS-related side-effects), lead to discontinuation of development (Drossman et al., 2008). It is worth pointing out that Drossman et al., (2008) did not assess Downloaded from the possible effects of AZD7371 on pain/discomfort thresholds during experimental CRD and assessed only symptomatic changes. Although the
relationship between visceral hypersensitivity and IBS symptoms is not jpet.aspetjournals.org completely clear, the lack of effects on pain/discomfort symptoms in IBS patients partially agrees with the preclinical observations presented here and
suggests that the analgesic-like effects observed when assessing pseudo- at ASPET Journals on September 27, 2021 affective VMRs during CRD in rats are probably not sufficient to confidently demonstrate true analgesic effects of the compound. Alternatively, the CNS profile of side-effects reported in humans (Drossman et al., 2008) might mask a positive analgesic effect, leading to a negative outcome as it relates to the reporting of symptoms. Finally, species-related differences in 5-HT1A receptor expression and distribution between rat and humans and their role in modulating visceral sensitivity may offer an additional explanation to these discrepant results.
The implications of the current observations might be of importance in the selection of compounds intended for clinical development for the treatment of IBS, at least for compounds targeting primarily visceral pain/discomfort. This is particularly important in light of the failure of AZD7371 to prove clinical JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 22 efficacy in the clinical trial in IBS patients mentioned above (Drossman et al.,
2008). For instance, other compounds, tested in the CRD model in the same experimental conditions described here, with proven clinical efficacy for the treatment of visceral pain, such as clonidine or pregabalin, not only reduced
VMRs to CRD but simultaneously reduced the concomitant hypertensive and tachycardic responses associated to visceral pain (Viramontes et al., 2001;
Houghton et al., 2007; Brusberg et al., 2008, 2009b; Ravnefjord et al., 2008). Downloaded from This suggests that animal data derived from the CRD model has to be interpreted carefully as it relates to its clinical translation to humans. This might
imply that multiple pain readouts should be tested at preclinical level as to jpet.aspetjournals.org support, with enough confidence, the progression into man of new chemical entities targeting visceral pain/discomfort. One could also consider that VMRs
mainly reflect the animal’s attempt to expel the balloon by increasing at ASPET Journals on September 27, 2021 intraabdominal pressure and are not valid as a marker of pain. Therefore, alternative surrogate markers, such as cardiovascular parameters, should be used to assess the efficacy of new drugs targeting visceral pain. Nevertheless, we can not exclude the possibility that AZD7371, because of its characteristic pharmacological profile, represents a unique case in its clinical translation to humans. AZD7371, as mentioned above, was developed as a potential treatment for depression and anxiety disorders (Mucke, 2000). According to the side-effect profile described in the clinical trial carried out with AZD7371 in IBS patients (Drossman et al., 2008), it can not be excluded that central effects of the compound not directly implicated in pain processing might interfere with any beneficial effects on pain perception leading to a negative outcome as it relates to pain-related symptomatology. Unfortunately no other studies, such as JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 23 experimental colorectal balloon distension for the assessment of visceral sensitivity, have been performed in humans as to further understand the mechanism of action of AZD7371 and the negative outcome as it relates to symptoms improvement in IBS patients.
In summary, the present results show that the selective 5-HT1A antagonist, AZD7371, inhibited pain-related visceromotor, but not cardiovascular autonomic, responses in a model CRD-induced visceral pain in Downloaded from rats. This suggests that AZD7371 might not show a complete inhibitory profile on pseudo-affective responses in rats, which might explain its failure to improve
pain/discomfort in IBS patients in a recent clinical trial. From these jpet.aspetjournals.org observations, we suggest that multiple pain-related read-outs should be monitored in preclinical models of visceral pain as to increase the clinical
translational value of preclinical data as it relates to the visceral analgesic at ASPET Journals on September 27, 2021 effects of compounds. JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 24
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Footnotes
This work was supported by AstraZeneca R&D Mólndal (Mólndal, Sweden). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 31
Legends for Figures
Fig. 1 Dose-related effects of AZD7371 and WAY-100635 on visceromotor responses to CRD in rats. Either AZD7371 (3 – 300 nmol/kg),
WAY-100635 (3 – 300 nmol/kg) or vehicle (1 ml/kg) was administered iv between distensions 3 and 4 of the CRD protocol (see methods for details).
Data correspond to the overall response to CRD for distensions 4 to 12 (AUC) Downloaded from (mean±SEM, n = 4 – 8 for each dose and compound). *: P<0.05 vs vehicle.
Fig. 2 Dose-related effects of oral AZD7371 on visceromotor responses jpet.aspetjournals.org to CRD in rats. Either AZD7371 (1 – 30 µmol/kg) or vehicle (2 ml/kg) were administered orally 30 min before starting the CRD protocol (see methods for
details). A: Response to repetitive noxious CRD (12 distensions at 80 mmHg) in at ASPET Journals on September 27, 2021 the different experimental groups. The broken line corresponds to the mean basal activity of the abdominal musculature between distensions. B: Overall response to CRD for distensions 1 to 12 (AUC) of the data included in panel A.
Data are mean±SEM of n = 7 – 8 for each dose and compound. *: P<0.05 vs vehicle.
Fig. 3 Plasma levels of AZD7371 after oral administration in rats. Data are mean±SEM (n=3) and represents total plasma levels of AZD7371 after a single oral dose (30 µmol/kg) at time 0. Pharmacokinetic parameters calculated for AZD7371 are included. The shadowed area indicates the time interval after oral dosing at which CRD experiments were performed.
JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 32
Fig. 4 Effects or repetitive oral dosing with AZD7371 on the visceromotor response to CRD in rats. Animals were treated orally with AZD7371 (30
µmol/kg/day) or vehicle (2 ml/kg/day) during 9 consecutive days. Responses to
CRD were assessed before treatment, after a single dose, after a 5-day treatment and at the end of the treatment period (9 days). Data are mean±SEM of 8 animals per group. *: P<0.05 vs response before treatment (day 0).
Downloaded from Fig. 5 Effects of intracerebroventricular (ICV) AZD7371 on visceromotor responses to CRD and urinary output in rats. A: Responses to CRD. AZD7371
(1 µg/rat) or vehicle (10 µl/rat) was administered ICV between distension 3 and jpet.aspetjournals.org
4 of the CRD protocol (arrow). Broken lines with open symbols represent the basal activity of the abdominal musculature. Data are mean mean±SEM of 7
animals per group, treated with vehicle and AZD7371 in separate experiments. at ASPET Journals on September 27, 2021
B: Urinary output in the same animals included in A during the time corresponding to distensions 4 to 12 of the CRD protocol. Each point represents an individual animal (open symbols with broken lines). The black symbols with errors represent the mean±SEM of each group. Notice how, compared with vehicle treatment, AZD7371 inhibited micturition in all animals except one.
Fig. 6 Effects of intravenous AZD7371 on visceromotor and autonomic cardiovascular responses to CRD in telemetrized rats. A: Visceromotor responses to CRD. The broken line corresponds to the mean basal activity of the abdominal musculature between distensions. B: Changes in mean arterial blood pressure (Δ mmHg) during CRD. C: Changes in heart rate (Δ beats per minute, bpm) during CRD. AZD7371 or vehicle was administered iv between JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #152330 - 33 distension 3 and 4 of the CRD protocol (arrow). Data are mean mean±SEM of 4 animals treated with vehicle and AZD7371 in separate experiments. *: P<0.05 vs corresponding response in the vehicle-treated group. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on March 26, 2009 as DOI: 10.1124/jpet.109.152330 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021