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Vol. 289, Issue 2, 752-761, May 1999
Sanofi Recherche, 195 Toulouse Cedex, France
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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SR146131 is a potent and selective agonist at cholecystokinin subtype 1 (CCK1) receptors in vitro. The present study evaluates the activity of the compound in vivo. SR146131 completely inhibited gastric and gallbladder emptying in mice (ED50 of 66 and 2.7 µg/kg p.o., respectively). SR146131 dose dependently reduced food intake in fasted rats (from 0.1 mg/kg p.o.), in nonfasted rats in which food intake had been highly stimulated by the administration of neuropeptide Y (1-36) (from 0.3 mg/kg p.o.), in fasted gerbils (from 0.1 mg/kg p.o.), and in marmosets maintained on a restricted diet (from 3 mg/kg p.o.). SR146131 (10 mg/kg p.o.) also increased the number of Fos-positive cells in the hypothalamic paraventricular nucleus of rats. Locomotor activity of mice was reduced by orally administered SR146131 (from 0.3 mg/kg p.o.). When administered intrastriatally, SR146131 elicited contralateral turning behavior in mice. Furthermore, orally administered SR146131 (0.3-10 mg/kg), also reduced the levels of cerebellar cyclic GMP. Finally, SR146131 (0.1 µg/kg to 1 mg/kg, p.o.) significantly and dose dependently antagonized fluphenazine-induced mouth movements in rats. The CCK1 antagonist SR27897B prevented all the effects of SR146131. Conversely, SR146131 was unable to elicit any agonist or antagonist effects in a model of CCK2 receptor stimulation in vivo. SR146131 is a very potent and selective nonpeptide CCK1 agonist in vivo. SR146131 is more potent than any other CCK1 agonists reported to date. Because pharmacodynamic studies suggest that SR146131 should have a high absolute bioavailability, it may be a promising drug for the treatment of eating and motor disorders in humans.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cholecystokinin
(CCK) is a peptide that is secreted in response to food ingestion by
endocrine cells within the small intestine and is also abundant in the
brain (Crawley and Corwin, 1994
). CCK exerts its activity via two G
protein-coupled receptor subtypes, the CCK1 (or
CCKA) and the CCK2 (or
CCKB) receptors (Wank, 1998), which have been cloned
from several species. In rodents, the CCK1 receptor is located mainly in the periphery, particularly in pancreatic acinar cells, gallbladder, and ileal muscle (Crawley and Corwin, 1994),
but is also present in discrete regions of the central nervous system
(Crawley and Corwin, 1994).
In the periphery, via the CCK1 receptor, CCK
delays gastric emptying, modulates intestinal motility, stimulates
gallbladder contraction, increases bile secretion, and controls
pancreatic secretion (Crawley and Corwin, 1994). CCK also plays a key
role in satiety: CCK plasma concentrations, which depend on the
composition of meals, are increased after meals. CCK decreases food
intake in several species including humans (Gibbs et al., 1973;
Kissileff et al., 1981; Lieverse et al., 1994), and has been proposed
to act as a satiety signal (Gibbs et al., 1973) via
CCK1 receptor activation (Dourish et al., 1989).
The Otsuka Long Evans Tokushima Fatty (OLEFT) rat, which lacks
the CCK1 receptor, is insensitive to the
anorexigenic action of CCK (Moran et al., 1998).
CCK1 receptor antagonists increase food intake in
several species (Hewson et al., 1988; Moran et al., 1992), whereas
CCK1 agonists have the opposite effect (Asin et
al., 1992a,b; Elliott et al., 1994). These and other results suggest
that the CCK1 receptor regulates food intake by
increasing satiety signals (Jerzy et al., 1990; Reidelberger,
1992), and that CCK1 agonists can be used to
reduce food intake.
Experimental evidence also suggests the involvement of CCK in a number
of processes in the central nervous system (Crawley and Corwin, 1994).
In particular, the CCK1 receptor has been
implicated in the control of motor function. CCK1
agonists induce turning behavior when injected into the striatum (Worms
et al., 1986; Poncelet et al., 1993) and induce hypomotility after
systemic administration in rodents (Soar et al., 1989).
CCK1 receptors have also been implicated in the
control of cerebellar cyclic GMP (cGMP) levels (Poncelet et al., 1993),
a neurochemical parameter shown to vary with locomotor activity (Triner
et al., 1981).
CCK also inhibits neuroleptic-induced tardive dyskinesia in animals
(Stoessl et al., 1989), an effect reversed by
CCK1 antagonists (Van Kampen et al., 1996), and
the CCK analog ceruletide improves some cases of tardive dyskinesia and
chorea in humans (Hashimoto and Yanagisawa, 1990). This suggests that
CCK1 receptor stimulation may be beneficial in
some neurological disorders.
These observations on the effects of CCK have encouraged the search for
CCK1 agonists that could be of potential
therapeutic use, in particular for the treatment of eating disorders
and obesity. Peptide analogs of CCK were first synthesized and led to
the preparation of compounds with selective CCK1
agonist activity capable of reducing food intake in a range of species
(Asin et al., 1992 a,b; Holladay and Lin, 1992; Simmons et al.,
1998). However, these peptide derivatives had very limited activity
after oral administration. Nonpeptide compounds that might possess a
higher degree of oral bioavailability have also been described. Some
1,5 benzodiazepine derivatives have been found to be equipotent to CCK
as anorectic agents following oral administration (Aquino et al., 1996;
Hirst et al., 1996; Henke et al., 1997). However, in contrast to the
peptide CCK1 agonists, these compounds exhibited
a decreased affinity for the human CCK1 receptor.
SR146131 is a new, potent, and selective nonpeptide agonist of both human and rodent CCK1 receptors (Bignon et al., companion paper). In the present study we characterized the activity of SR146131 in a variety of experimental models involving CCK1 receptor stimulation in vivo. We focused on the effects of the compound on three main areas: the gastrointestinal system, the regulation of food intake in a number of different species, and the modulation of motor function, in particular of dyskinetic behavior. We also evaluated the effects of SR146131 on tests involving the activation of the CCK2 receptor to verify its selectivity in vivo. The results confirm that the compound exhibits a high potency and selectivity for the CCK1 receptor in vivo. Furthermore, SR146131 is very active after oral administration, and is therefore, in contrast to previously described CCK1 agonists, a better candidate for clinical development.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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All experimental procedures were approved by the "Comité d'Expérimentation Animale" (Animal Care and Use Committee) of Sanofi Recherche and were carried out in accordance with French legislation.
Drugs. SR146131 (2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid) and SR27897B (1-[2-[2-[4-(2(chloro-phenyl)-2 thiazolyl]-aminocarbonyl]-indoly]-acetic acid; Lintitript) were synthesized by Sanofi Recherche. Neuropeptide Y (NPY)(1-36) was obtained from Neosystem (Strasbourg, France). Pentagastrin was from Sigma-Aldrich (Milan, Italy). SR146131 was suspended in Tween 80 and water for oral administration and in DMSO plus water for i.v. injection, except where otherwise stated. SR27897B was suspended in Tween 80 and water for i.p. administration.
Gastric and Gallbladder Emptying in Mice. Female OF1 mice (Iffa Credo, France, 7-10 in each group) weighing 20 to 26 g were fasted for 18 h before the experiments.
For dose-response studies on gastric emptying, SR146131 was given 30 min (i.v.) or 1 h (p.o.) before a charcoal meal (0.3 ml/mouse of 10% charcoal powder, 5% gum arabic, and 1% carboxymethylcellulose suspension in distilled water) was administered. The mice were sacrificed by cervical dislocation 5 min later. The cardia and the pylorus were ligatured, the stomach removed, and the volume of the gastric content was estimated by the weight of the stomach (Gully et al., 1993). SR27897B (3-1000 µg/kg) or its vehicle were administered
i.p. 15 min before SR146131.
For time-course studies, SR146131 was given from 15 min to 7 h by
i.v. and oral routes before the charcoal meal was administered. Results
were expressed as the mean (±S.E.M.) of the weight of the stomach
(grams per kilogram of mouse body weight) for each dose group.
Statistical differences between drug- and vehicle-treated groups were
performed by Student's t test (when two groups were compared) or by ANOVA followed by Dunnett's test (for multiple comparisons).
For gallbladder emptying, the mice were sacrificed by cervical
dislocation 4 h after SR146131 administration, and the gallbladder was ligatured, removed, and weighed (Gully et al., 1993). SR27897B (1-300 µg/kg) or its vehicle was administered i.p. 15 min before SR146131. Results were expressed as the mean (±S.E.M.) of the weight
of the gallbladder (gram per kilogram of mouse body weight) for each
dose group. Statistical differences between groups were analyzed using
the Kruskall-Wallis test (heterogeneous variance) followed by the
Mann-Whitney test with the Holm correction (multiple comparisons).
ED50 and ID50 values were
estimated with their 95% CL, using the nonlinear logistic model
adjusted with the Levenberg-Marquardt algorithm.
Food Intake in Fasted Rats.
Over a period of 10 days before
the experiment, male Sprague-Dawley rats (200-230 g, Charles River
France, 9-14 per group) were fasted for 18 h, and allowed access
to food for only 6 h between 10 AM and 4 PM each day. Water was
available ad libitum. At the end of this adaptation phase, rats were
administered SR146131 (0.03-3 mg/kg p.o.). One hour after SR146131
administration, a weighed amount of food (A04 standard rat laboratory
diet; UAR, France) was introduced into the cage, and food intake was
measured (taking into account spillage) 1, 3, 6, and 23 h after
SR146131 administration. SR27897B (0.1 mg/kg) or its vehicle were
administered i.p. 15 min before SR146131. Results are expressed as the
mean ± S.E.M. for each treatment group. A dose × time ANOVA
(with repeated measures on the time factor) was conducted on cumulative
intakes across time following SR146131 administration. At each time
point, post hoc comparisons versus the control group were performed
using Dunnett's test. The determination of the
ED50 was made using the four-parameter logistic
model according to Ratkovsky and Reedy (1986). The adjustment was
obtained by nonlinear regression using the Lenvenberg-Marquardt
algorithm in RS/1 software (BBN Software Product Corporation,
Cambridge, MA).
Effect on NPY-Induced Feeding in Nonfasted Rats. Rats were treated with different doses of SR146131 (0.3, 1, and 3 mg/kg p.o.) or vehicle 2 h before an i.c.v. administration of NPY(1-36) or its vehicle. The peptide was solubilized in 0.9% NaCl with 1% BSA. The injection of NPY(1-36) (2.5 µg/5 µl) or vehicle was made free-hand into the lateral ventricle (i.c.v.) of nonrestrained rats by means of a 50-µl microsyringe with a 10-mm calibrated needle (final length below the skin: 3.5 mm). A known amount of food (A04 standard rat laboratory diet; UAR) was then immediately introduced into the cage, and food intake was measured (taking into account spillage) over the following 1, 2, 3, and 4 h. Statistical comparisons were performed using a dose × time ANOVA, followed by Dunnett's test, as described above.
Food Intake in Fasted Gerbils. Gerbils (60-80 g, CERJ, France) were fasted for 24 h before the test. On the day of test, groups of 10 gerbils were given SR146131 (0.1 or 1 mg/kg p.o.) or its vehicle and immediately placed individually in translucent boxes (8 × 11 × 24 cm) with a weighed amount of their regular food (A04 standard laboratory diet; UAR). The amount eaten by each gerbil was measured by weighing food (including spillage) before and then 1 h and 2 h after treatment. Statistical analysis was performed using ANOVA followed by Dunnett's test.
Food Intake in Marmosets. Marmosets (Callithrix Jacchus) were obtained from Sanofi Winthrop Ltd., Alnwick (UK). All the animals were domestically bred.
The marmosets were housed in a room maintained at a temperature of 24 ± 1°C and air conditioned to prevent overheating. The relative humidity was maintained at 50 to 60% by steam injection, and forced ventilation produced 12 air changes per hour, the incoming air being filtered through 5-µm filters. Timed switching provided fluorescent lighting for 12 h per day from 7 AM to 7 PM. This is preceded and followed by a short period of twilight, but a dim lighting was maintained throughout the night. Twelve male and 11 female adults, 350 to 500 g (3-5 years old), were housed individually or in single sex pairs, in a total of 17 cages. The cages (North Kent Plastic Cages Ltd., Dartford, Kent, UK) measured 76 cm high, 57 cm wide, and 61 cm deep, with a wire mesh front and floor, three wooden perches and an open-ended nesting shelf. Softwood shavings were provided as absorbent bedding in all cages. The animals were fed with a standard primate pellet diet (Mazzuri Primate; SDS, Vigny, France), and supplements of fresh fruits, boiled eggs, and vitamins disposed on a plate. During the week before the first treatment, and during the experimental session, food was available to the animals for 3 h only in the afternoon (water was available ad libitum). SR146131 was administered p.o. by gavage at the doses of 0.3, 1, 3, and 10 mg/kg in a volume of 1 ml/kg. Animals were treated acutely, twice a week (Tuesday and Thursday) with either SR146131 or its vehicle, according to a crossover design so that the effects of all treatments were evaluated on each animal. The animals were fasted overnight (from 20 h) before each experimental day. One hour after treatment, a preweighed amount of food was introduced into the cage for the following 3 h. After this time, residues were removed and spillage collected, and the animals' food intake was measured. For the cages containing two animals, the mean food intake was used. Differences between food intake after vehicle or each dose of SR146131 were assessed by Dunnett's test after ANOVA with repeated measures using RS/1 software (BBN Software Product Corporation).Expression of Fos in Paraventricular Nucleus of Rats. Male Sprague-Dawley rats (250-300 g, Charles River, France) received oral administrations of SR146131 (3.0 or 10 mg/kg) or the vehicle, and were sacrificed 2.5 h later as described below for the immunohistochemical detection of Fos. To examine the effects of the CCK1 receptor blockade on SR146131-induced Fos-like immunoreactivity, SR27897B (0.3 mg/kg) was injected i.p., 30 min before the oral administration of SR146131 (10 mg/kg) and rats were sacrificed 2.5 h after SR146131 treatment.
Rats were anesthetized with sodium pentobarbital (80 mg/kg i.p.) and perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were removed and allowed to postfix overnight in 4% paraformaldehyde. Fifty-micrometer coronal sections were cut from each brain using a vibratome. Immunohistochemistry was performed on free-floating tissue sections according to a standard avidin-biotin-peroxidase procedure using an anti-Fos sheep polyclonal antibody (Genosys Biotechnologies Inc., Cambridge, UK, CRB OA-11-824) directed against residues 2 to 16 of the N-terminal region of the Fos protein. Sections were rinsed with 0.02 M PBS and then pretreated for 10 min at room temperature with the same buffer containing 0.3% hydrogen peroxide. Next, they were rinsed in PBS three times, incubated with PBS containing 5% normal rabbit serum (Vector Laboratories, Burlingame, CA) for 1 h at room temperature, and placed for 72 h at 4°C in the Fos primary antiserum (diluted 1:8000) solution in PBS containing 1% normal rabbit serum and 0.3% Triton X-100. Thereafter, the sections were incubated successively with a biotinylated rabbit anti-sheep secondary antibody (Vector Laboratories; diluted 1:300) for 2 h at 37°C and with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories; diluted 1: 200) for 3 h at room temperature in PBS containing 0.1% Triton X-100. The reaction product was visualized with diaminobenzidine in the presence of nickel. Sections were mounted, air dried, dehydrated, and covered with acrytol. Omission of the primary antibody from the immunohistochemical procedure or preadsorption of this antibody with a synthetic peptide corresponding to the N-terminal antigenic sequence of the Fos protein (Genosys, OP-11-3210) eliminated Fos-like immunoreactivity. Sections processed for chromogenic detection of Fos-like immunoreactivity were imaged through a Leica DMIRBE microscope and the Fos-like immunoreactive signal was quantified with an image analysis system (Samba, Unilog; Grenoble, France) by counting the number of Fos-positive cells within a 1 mm2 area located at the level of the hypothalamic paraventricular nucleus (panel 25 of the atlas of Paxinos and Watson, 1986). Only visual objects that met the
appropriate size and optical density criteria (cells showing gray
levels between 0 and 160/170, total range 0-255), were automatically
counted as Fos-immunoreactive cells. For each animal, three serial
sections were counted bilaterally and the data averaged. Data from
treatment groups, presented as the mean ± S.E.M. number of
Fos-immunoreactive cells, were submitted to one-way ANOVA followed by
Dunnett's or Newman-Keuls' tests, as appropriate.
Locomotor Activity in Mice. Male Swiss CD1 mice (Charles River, France) weighing 19 to 29 g were used. Animals had free access to food (A04 standard laboratory diet; UAR) and water. Testing of locomotor activity was carried out in individual Perspex boxes (24 × 13 × 12 cm). The boxes were fitted with four pairs of infrared photocells, two pairs positioned in the front, and two in the back of the box. Activity was measured as the number of photocell interruptions. The locomotor cages were connected via a laboratory interface (Imetronic, Pessac, France) to a microcomputer system that recorded photocells count. SR146131 was administered p.o. at doses of 0.3 to 10 mg/kg. SR27897B was administered i.p. at the dose of 0.1 mg/kg. The compounds were prepared extemporaneously and administered in a volume of 10 ml/kg to groups of 10 mice. In each independent experiment, control mice received the corresponding vehicle.
Mice were placed singly in an activity cage 60 min after treatment with SR146131. SR27897B was administered 30 min before SR146131. Motility was assessed over a period of 30 min for SR146131. Results were expressed as the mean (±S.E.M.) of the locomotor scores for each dose group. Differences between control and treated groups were assessed by ANOVA followed by Dunnett's test. The ED50 was determined using the four-parameter logistic model of Ratkovsky and Reedy (1986). The adjustment was obtained by nonlinear regression using
the Levenberg-Marquardt algorithm in RS/1 software (BBN Software
Product Corporation).
Turning Behavior in Mice. SR146131 (0.01-1 pg) was solubilized in DMSO (1 mg/ml), diluted to the required concentrations with water, and injected (in 1 µl) into one striatum in awake, hand-restrained female CD1 mice (25-30 g; Charles River, France). After injection, the animals were placed individually in Plexiglas cages (10 × 10 × 15 cm). The number of complete contralateral rotations (away from the injection site) and ipsilateral rotations (toward the injection site) were visually recorded and cumulated over three 2-min periods (2-4, 5-7, and 8-10 min post injection), and the mean (±S.E.M.) number of turns in 6 min was calculated.
SR27897B was administered i.p. 30 min before intrastriatal injection of SR146131. Differences between control and treated groups were assessed by ANOVA, followed by Dunnett's test.Tardive Dyskinesia in Rats.
Two-month-old male Sprague-
Dawley rats (Charles River, France) were treated with fluphenazine
decanoate (25 mg/kg; Sanofi Winthrop) administered i.m. into the
hindlimb once every 3 to 4 weeks until the test sessions. In test
sessions, animals were allowed to habituate for at least 60 min in
Perspex boxes (23 × 13 × 13 cm). The duration of mouth
movements including vacuous nondirected chewing, tremor of the jaws,
grinding of the teeth, and tongue protrusions were continuously
recorded for 15 min (Stoessl et al., 1989). Values are the mean
(±S.E.M.) duration of abnormal movements during the 15-min observation
period. In the dose-response study, SR146131 was administered p.o. 60 min before the test period, 47 weeks following the initial injection of
fluphenazine to the rats. In the antagonism study, SR146131 was
administered p.o. and SR27897B i.p., 60 and 45 min, respectively,
before the test period, 67 weeks after the initial injection of
fluphenazine. Statistical analysis was performed using ANOVA, followed
by Dunnett's or Newman-Keuls' tests, as appropriate.
Determination of Cerebellar cGMP in Mice.
Experiments were
performed in the morning, and special care was taken to avoid stressful
manipulation. Male Swiss mice (18-20 g) were pretreated with SR146131
(0.1-10 mg/kg, p.o.) or its vehicle 60 min before sacrifice by focused
microwave irradiation (3.4 KW per cm 2/1.6 s; Sacron 8000, Sairem,
Villeurbanne, France). The cerebellar cortex was dissected and
homogenized with a polytron in 1.75 ml of ethanol. The homogenates were
centrifuged at 1500g at 4°C, and the supernatants
evaporated and stored at 
4°C until radioimmunoassay using kits with
succinyl-tyrosine [125I]methyl ester derivative
of cGMP radiolabeled antigen (NEN, Boston, MA). Protein contents were
measured by the method of Bradford (1976). SR27897B was injected i.p.
at various doses 30 min after SR146131 administration. The results are
expressed as a percentage of the control response. The effect of
SR146131 at various doses versus the control group, and the effect of
SR146131 + SR27897B versus SR146131 alone were tested using ANOVA
followed by Dunnett's or Newman-Keuls' tests, as appropriate.
Gastric Acid Secretion in Anesthetized Rats.
Gastric
secretion in anesthetized rats was measured by the stomach-lumen
perfusion technique of Ghosh and Schild (1958) with minor
modifications. Female Crl:CDBR rats (Charles River, Italy), weighing
220 ± 20 g were used. Rats were fasted for 24 h, then anesthetized with ethyl-urethane (1.2 g/kg, i.p.) and, after
laparatomy, the stomach was cannulated at the cardiac and pyloric ends
and perfused with warm saline (NaCl 154 mM, 37°C, pH 5.5) at a rate of 1 ml/min, using a peristaltic pump. Fractions of 5 ml were automatically collected and the pH of each fraction was determined (Amel 338 pH meter) and recorded by an automatic apparatus on which
samples from eight rats could be measured simultaneously. The pH values
were transformed into microequivalents of H+
secreted in the 5-min fraction.
treated µEq
H+/basal µEq H+)
The effect of SR146131 on a submaximal dose of pentagastrin (4 µg/kg, i.v.) was calculated as percentage of inhibition of pentagastrin effect. The results are expressed as mean ± S.E.M. Statistical analysis of the data was performed by paired Student's t test (when two groups were compared) or by paired one-way
ANOVA followed by Dunnett's test (for multiple comparisons).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Gastric and Gallbladder Emptying in Mice.
SR146131 completely
inhibited gastric emptying in mice at a very low dose after oral
administration (Fig. 1), with an
ED50 of 66 µg/kg (48-87, 95% CL). The
efficiency of SR146131 by this route was comparable with that obtained
after i.v. administration [ED50 of 38 µg/kg
(29-72, 95% CL)].
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AUC: AUCSR146131 AUCvehicle) was almost identical for
the two routes of administration. The ratio of the
AUC between the i.v. and oral routes was
estimated to be around 70%, suggesting that SR146131 should have a
high absolute bioavailability.
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Food Intake in Rats.
SR146131 induced a dose-related decrease
in food intake in fasted rats (Fig. 5).
This effect was highly significant from the dose 0.1 mg/kg p.o. on the
cumulative 3-h food intake, with an ED50 of 0.43 mg/kg (0.29-0.67, 95% CL). Food intake over the 23-h period was
significantly decreased by 42% and 63% at 1 and 3 mg/kg SR146131,
respectively. SR27897B (0.1 mg/kg i.p.) totally prevented the
hypophagic activity induced by 1 mg/kg of SR146131 (results not shown).
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Food Intake in Gerbils. Oral administration of SR146131 to gerbils reduced the food intake of these animals. At 1 h after treatment, there was a significant reduction (33%), of food intake at the dose of 1 mg/kg SR146131, but the effect of the dose of 0.1 mg/kg did not attain statistical significance at this time point. At 2 h after treatment, however, the reduction of food intake produced by both doses of SR146131 was statistically significant (33% and 45%, respectively).
Food Intake in Marmosets. Oral administration of SR146131 to marmosets significantly and dose dependently reduced the food intake of these animals (Table 1). The first active dose was 3 mg/kg p.o., which reduced the 3-h food intake by 23% (t = 4.28, P < .05), and this effect was somewhat greater at the dose of 10 mg/kg p.o. (28% reduction, t = 5.26, P < .05).
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Expression of Fos in Paraventricular Nucleus of Rats.
Oral
administration of SR146131 at the doses of 3 and 10 mg/kg (Fig.
7) increased the number of Fos-positive
cells in the hypothalamic paraventricular nucleus as compared with
vehicle-treated rats [number ± S.E.M.: vehicle: 48 ± 7 (n = 5); SR146131 (3 mg/kg): 75 ± 13 (n = 5); SR146131 (10 mg/kg): 181 ± 18 (n = 7)], while rats receiving SR27897B (0.3 mg/kg
i.p.) before oral administration of SR146131 (10 mg/kg, Fig. 7) had Fos
counts comparable with those of vehicle-treated rats [number ± SEM: SR27897B + SR146131: 51 ± 12 (n = 4)].
SR27897B (0.3 mg/kg) administered alone had no effect on Fos expression
(not shown). ANOVA yielded a significant main effect for drug
treatment [F(3,20) = 20.27, P < .01] and Dunnett's test showed that only animals treated with 10 mg/kg of
SR146131 differed significantly (p < .01) from
vehicle-treated rats with respect to the number of Fos-immunoreactive
cells. Post hoc Newman-Keuls test indicated that the pretreatment with
SR27897B significantly prevented (p < .01) the
Fos-inducing effect of SR146131 (10 mg/kg).
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Locomotor Activity in Mice. SR146131 induced a dose-related decrease in locomotor activity (Table 2). This effect was highly significant at the doses of 0.3 to 10 mg/kg p.o., with an ED50 of 0.7 mg/kg (0.2-1.4, 95% CL). SR27897B at 0.1 mg/kg i.p. totally antagonized the suppression of locomotor activity induced by 3 mg/kg p.o. of SR146131 (data not shown).
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Turning in Mice.
The unilateral striatal application of
SR146131 at 0.01, 0.03, 0.1, and 1 pg produced contralateral circling
behavior with a significant and dose-dependent effect (Fig.
8A). This was associated with a slight
but significant reduction of ipsilateral turns. In light of these
results, the antagonistic activity of SR27897B was studied on the
turning produced by 0.1 pg of SR146131. Figure 8B shows that SR27897B
dose dependently and significantly counteracted the turning induced by
SR146131, with an ID50 of 0.1 mg/kg i.p. (0.038-6.6, 95% CL); ipsilateral turns were restored only at the highest dose of SR27897B.
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Tardive Dyskinesia in Rats.
Figure
9A shows that SR146131 (0.1 µg/kg to 1 mg/kg, p.o.) significantly and dose dependently antagonized
fluphenazine-induced mouth movements with an ID50
of 12 µg/kg (0.6-386.6, 95% CL). Figure 9B shows that SR146131 (1 mg/kg p.o.) significantly antagonized (58%) the tardive dyskinesia.
SR27897B (0.3 mg/kg i.p.) did not significantly modify (26%) the
movements induced by fluphenazine, but significantly counteracted the
antagonism induced by SR146131.
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cGMP Levels in Cerebellar Cortex of Mice.
Oral administration
of SR146131 produced a dose-dependent decrease of cGMP levels in the
cerebellum in mice, from the dose of 1 mg/kg p.o. (Fig.
10), with an apparent maximum effect of
about 40% reduction. The effect of 10 mg/kg p.o. SR146131 on cGMP
levels was abolished by the administration of 3 µg/kg i.p. SR27897B
(Fig. 10).
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Gastric Acid Secretion in Anesthetized Rats. Pentagastrin (4 µg/kg i.v.) increased gastric acid secretion [t(3) = 8.02, P < .01] from control values of 1.3 ± 0.09 µEq H+ to 4.06 ± 0.36 µEq. In contrast, SR146131 (0.3 or 1 mg/kg i.v.) did not modify the secretion of gastric acid [t(4) = 0.74, P > .05 and t(2) = 1.59, P > .05, respectively]. At these same doses, SR146131 did not inhibit the increase [F(2,6) = 1.43, P > .05] of gastric acid secretion induced by pentagastrin (results not shown). Thus, SR146131 neither stimulated nor inhibited the CCK2 receptor in vivo.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The accompanying paper (Bignon et al., 1999) on SR146131 describes
the compound's effects as a potent and selective agonist at both human
and rodent CCK1 receptors in vitro. The present paper evaluated the drug's effects in vivo, and shows clearly that
SR146131 can mimic, in a variety of test systems and in several species, a wide range of the effects of sulfated cholecystokinin octapeptide (CCK8S), which have previously been attributed to the
stimulation of CCK1 receptors but not those
related to the stimulation of CCK2 receptors.
SR146131 inhibited gastric emptying in mice, and also decreased
gallbladder volume in this species after administration of low oral
doses. SR146131 also reduced food intake in two rodent and one primate
species. The compound reduced food intake in fasted rats, and in
nonfasted rats in which food intake had been highly stimulated by the
administration of NPY(1-36). Food intake was also reduced by oral
administration of SR146131 in fasted gerbils, and in marmosets
maintained on a restricted diet. Furthermore, SR146131 was able to
increase the number of Fos-positive cells in the hypothalamic
paraventricular nucleus of rats, indicating an activation of the
neurons in this nucleus, which is known to be part of the anatomical
pathway implicated in the regulation of food intake by CCK via
CCK1 receptors (McCann and Rogers, 1991; Mönnikes et al., 1997). In all the experimental models in which the effect of SR27897B was evaluated, the CCK1
receptor antagonist was able to antagonize the effects of SR146131.
SR146131 was also shown to act on a variety of parameters related to
locomotor control. When administered intrastriatally, SR146131, like
CCK-8S (Worms et al., 1986), elicited turning behavior in mice.
Locomotor activity of mice was reduced by orally administered SR146131.
Furthermore, orally administered SR146131 reduced the levels of
cerebellar cGMP, which have previously been shown to vary with
locomotor activity (Triner et al., 1981). However, the large
dose-dependent changes in locomotor activity were accompanied by modest
variations in cGMP levels. This indicates that the relationship between
these two phenomena is incomplete as already suggested by differences
in the ability of devazepide versus SR27897B to inhibit CCK-induced
hypolocomotion versus CCK-induced reduction in cerebellar cGMP levels.
(Poncelet et al., 1993). Finally, SR146131 (0.1 µg/kg to 1 mg/kg,
p.o.) significantly and dose dependently antagonized mouth movements
induced by long-term fluphenazine treatment, considered to be an animal
model of neuroleptic-induced tardive dyskinesia (Stoessl et al., 1989).
The CCK1 antagonist SR27897B prevented the
effects of SR146131, confirming that these effects of the compound,
like those of CCK8S in the same model (Van Kampen et al., 1996), were
effectively attributable to the stimulation of
CCK1 receptors.
In contrast to its efficacy in experimental models implicating the
CCK1 receptor, SR146131 was unable to elicit any
CCK-4-like effects in a model of CCK2 receptor
stimulation in vivo (the enhancement of gastric acid secretion in
rats). Furthermore, CCK2 agonists have been found
to induce signs of panic in humans and anxiety in rodents (van Megen et
al., 1996), but we were unable to find (our unpublished data)
any anxiogenic effects of SR146131 in the rat or in the marmoset, a
species in which the behavioral signs associated with anxiety are well
documented (Carey et al., 1992). Finally, SR146131 did not inhibit
CCK2 receptor-mediated events in vivo, because it
did not inhibit the effect of pentagastrin on gastric secretion.
Therefore, these data confirm that SR146131 retains its profile as a
potent and selective CCK1 receptor agonist in
vivo in a number of animal species.
Very few orally active nonpeptide CCK1 agonists
have been described: the 1.5 benzodiazepine derivatives GW7854 (Hirst
et al., 1996; Aquino et al., 1996) and the most recently described
GW5823 (Henke et al., 1997) bind to human CCK1
receptors with moderate affinity and selectivity over the
CCK2 receptor. In mice, by the oral route these
compounds stimulate gallbladder emptying with ED50s of 10 to 55 nmol/kg (approximately 5-25
µg/kg), which is comparable with the activity of SR146131 (which has
an ED50 of 2.7 µg/kg). GW5823 (Henke et al.,
1997) and GW7854 were effective in reducing food intake to 40% and
58%, respectively, of vehicle controls when given orally at a dose
of 10 µmol/kg (approximately 5 mg/kg). Although methodological
differences preclude a direct comparison, SR146131 would seem to be
more potent than these molecules for inhibiting food intake in rats,
because it significantly inhibited food intake from the dose of 100 µg/kg. These results also suggest that although the ability to
stimulate gallbladder emptying is a very sensitive and selective test
for the detection of CCK1 agonists, it cannot be
used to predict the potency of the compounds to inhibit feeding,
possibly due to the existence of a large quantity of spare receptors on
this tissue (Simmons et al., 1998). The potency of SR146131 by the i.v.
and oral routes is similar, and the duration of the inhibition of
gastric emptying in mice is also comparable by these routes, suggesting
that SR146131 should have a high absolute bioavailability. This does
not seem to be the case for GW5823 and several close analogs, which
suffer from poor oral bioavailability and a relatively high clearance
in the rat (Henke et al., 1997).
In view of the potency, selectivity, and oral bioavailability of
SR146131, this compound would appear to be one of the first nonpeptide
CCK1 receptor agonists worthy of consideration
for clinical development. The therapeutic field with which
CCK1 receptor stimulation has the most frequently
been associated has been that of obesity. For many years, evidence has
been accumulating on the physiological role of CCK as an important
satiety signal (Gibbs et al., 1973; Lee et al., 1994), a concept
supported by the observation that CCK has been found to efficiently
suppress food intake in humans (Kissileff et al., 1981; Lieverse et
al., 1994). Conversely, many studies indicate a role for NPY as a
physiological appetite stimulant (Stanley and Leibowitz, 1985;
Lee et al., 1994). The ability of SR146131 to reduce food intake in a
number of species at low oral doses and to powerfully block the
appetite-stimulant effects of NPY suggests that its anorexigenic
potential should be evaluated in humans.
Another possible field in which the effects of this compound might be
beneficial is the alleviation of the tardive dyskinesia that occurs as
a side effect of chronic neuroleptic administration. Although the new
generation of atypical antipsychotic agents may help to alleviate
concerns about tardive dyskinesia, this disorder remains a significant
clinical problem for both patients and physicians, because the older
antipsychotic drugs are still very frequently prescribed. Many cases of
tardive dyskinesia are mild, but patients with moderate to severe forms
of tardive dyskinesia present a great challenge and frequently require
medication to suppress their dyskinesias. A variety of suppressive
agents have been tried with limited success and no fully satisfactory
treatment strategy has as yet emerged (Egan et al., 1997). The present
results with SR146131 suggest that the selective stimulation of
CCK1 receptors may provide an effective means of
attenuating neuroleptic-induced tardive dyskinesias.
In conclusion, SR146131 is active at low oral doses in a wide variety of experimental models of CCK1 receptor activation. This pharmacological profile, together with the compound's high potency and selectivity for the human CCK1 receptor (Bignon et al., companion paper), suggests that SR146131 may be an excellent candidate for the treatment of clinical conditions such as obesity and tardive dyskinesia.
| |
Acknowledgments |
|---|
We thank Dr Danielle Gully for critical comments on the manuscript, Josephine Beuste, Roselyne Calassi, MariaGrazia Dozio, Claude Dufour, Michèle Fournier, MariaGiovanna Marongiu, Nadine Gonalons, Josette Guitard, Martine Keane, Massimo Meleri, Christine Menet, Yves Rouzier, and Jean-Jacques Storme for excellent technical support, and Marilyne Laborde and Danielle Lasserre for expert secretarial assistance. Christophe Agut and Noëlle Boussac are gratefully acknowledged for assistance with the statistical analysis of the data.
| |
Footnotes |
|---|
Accepted for publication December 23, 1998.
Received for publication October 14, 1998.
1 Current address: Sanofi Recherche, 371 rue du Professeur Joseph Blayac, 34184 Montpellier Cedex, France.
2 Current address: Sanofi Midy Research Center, via G.B. Piranesi, 38, 20137, Milan, Italy.
Send reprint requests to: Dr. Eric Bignon, Sanofi Recherche, Exploratory Research Department, 195 route d'Espagne, 31036 Toulouse Cedex, France. E-mail: eric.bignon{at}sanofi.com
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Abbreviations |
|---|
CCK, cholecystokinin; CCK8S, sulfated cholecystokinin octapeptide; NPY, neuropeptide Y.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) or its vehicle
(
) on gastric emptying after oral (A) and i.v. (B) administration as
a function of time. Charcoal suspension was given to mice at different
times after administration of SR146131. Weight of the stomach was
measured 5 min after charcoal administration. Results are expressed as
mean ± S.E.M. for each treatment group. See Materials and
Methods for statistical analysis. Student's t
test: *P < .05; **P < .01;
***P < .001 versus vehicle.
