Download a distribution study with 14c-otilonium bromide in the rat: evidence

0090-9556/00/2806-0643–647$03.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
DMD 28:643–647, 2000 /1832/829067
Vol. 28, No. 6
Printed in U.S.A.
A DISTRIBUTION STUDY WITH 14C-OTILONIUM BROMIDE IN THE RAT: EVIDENCE FOR
SELECTIVE TROPISM FOR LARGE INTESTINE AFTER ORAL ADMINISTRATION
STEFANO EVANGELISTA, PASCAL COCHET, NORBERT BROMET, MARCO CRISCUOLI,
AND
CARLO ALBERTO MAGGI
Menarini Ricerche S.P.A., Firenze, Italy (S.E., M.C., C.A.M.) and Biotec Centre, Orleans, France (P.C., N.B.)
(Received for publication November 9, 1999; accepted March 9, 2000)
This paper is available online at http://www.dmd.org
ABSTRACT:
found in trace amounts in the liver. The presence of radioactivity in
the GI walls reflected the transit kinetics of drug-enriched contents. The radioactivity in large intestine walls was measurable at
otilonium bromide concentrations in the range of micromole equivalents/kg, from 4 to 8 h after drug administration. Total body
radioactivity recovery was 95, 101, 24, and 9% at 1.5, 4, 8, and 24 h,
respectively. In conclusion, orally administered 14C-otilonium bromide is poorly absorbed systemically, as indicated by the very low
plasma radioactivity levels, but it is able to effectively penetrate
into the large intestine walls, a recognized target for drugs oriented toward irritable bowel syndrome therapy.
Otilonium bromide (diethylmethyl{[(octyloxy-2 benzamido)-4
benzoyloxy]2ethyl}ammonium bromide) is a quaternary ammonium
salt possessing gastrointestinal (GI)1 spasmolytic properties, and used
world-wide for the treatment of irritable bowel syndrome (IBS). A
recent meta-analysis of double-blind randomized placebo-controlled
trials of various smooth muscle relaxants (Poynard et al., 1994) has
ranked otilonium bromide among the leading treatments in IBS when
global assessment, pain relief, and absence of side effects were taken
into account.
These clinical results reinforce the hypothesis of the marked selectivity of otilonium bromide toward the colon. Previous experimental
studies have shown that otilonium bromide spasmolytic action, regardless of the nature of the agonist, was preferentially exerted in
intestinal muscles, rather than in vascular or respiratory smooth muscle preparations (Manzini et al., 1984). In vitro studies have indicated
that the colon is more sensitive than other GI segments to the relaxing
action of otilonium bromide (Maggi and Meli, 1983; Maggi et al.,
1985). This drug possesses a potent antimuscarinic and calcium antagonistic effect (Maggi et al., 1983b; Gandia et al., 1996; Evangelista
et al., 1998), but is free of the typical systemic side effects of this drug
type (Scarpignato et al., 1980; Maggi et al., 1983a). Moreover, when
orally administered at doses that produce spasmolytic effects in hu-
mans, it was devoid of both central and peripheral atropine-like side
effects (Sutton et al., 1997). All these data suggest negligible systemic
absorption of otilonium bromide, whereas in vitro studies performed
using the 14C-labeled compound showed preferential binding to GI
tract tissues (Amenta et al., 1991). This study was carried out to
determine the quantitative and temporal tissue distribution of 14Cotilonium bromide in rats, after a single oral dose that is similar to the
therapeutic dose currently administered in IBS treatment (Battaglia et
al., 1998).
Materials and Methods
Animals and Treatments. Twenty male Sprague-Dawley rats (Janvier, Le
Genest St. Isle, France), 240 to 260 g, were used. They were housed in a
temperature- and humidity-controlled room and fasted 18 h before the experiment. Each animal received 2 mg/kg of a mixture of 14C-otilonium bromide
(specific activity 58 mCi/mmol; Amersham Pharmacia Biotech, Courtaboeuf,
France) and unlabeled compound (Spasmomen; Menarini Pharmaceuticals,
Firenze, Italy) dissolved in a sterile physiological vehicle and slowly delivered
to the stomach through a gastric cannula (5 ml/kg). The nominal radioactive
dose was 150 ␮Ci/kg.
Blood Sampling and Animal Euthanasia. Blood samples were drawn into
heparinized pipettes from the retro-orbital sinus at 1.5, 4, 8, and 24 h after
dosing. Plasma was separated by centrifugation (3000 rpm for 10 min) from an
aliquot of each blood sample. At each sampling time, after blood collection,
five animals were sacrificed by excess anesthesia with diethyl ether, and
1
Abbreviations used are: GI, gastrointestinal; IP, imaging plate; IBS, irritable
processed according to the different study protocols below.
bowel syndrome; LQL, low quantitation limit; LSC, liquid scintillation counting;
Quantitative Radioluminography (QRLG). After sacrifice, the 12 aniPSL, photostimulated luminescence; QRLG, quantitative radioluminography.
mals (three at a time) to be studied with the QRLG technique (Ullberg and
Larsson, 1981) were prepared for cryosectioning by immersion of the suitably
Send reprint requests to: Dr. Stefano Evangelista, Preclinical Development,
restrained body in a cooling medium at ⫺70°C (eutectic mixture of heptane
Menarini Ricerche spa, Via Sette Santi 1, 50131 Firenze, Italy. E-mail:
and solid carbon dioxide) for 45 min. Frozen rats were stored at ⫺20°C until
[email protected]
embedding into a carboxymethylcellulose matrix (2% in water) and cryosec643
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017
The aim of this study was to determine the plasma levels and the
tissue distribution of otilonium bromide, measured as total radioactivity, after oral administration of 2 mg/kg of 14C-labeled drug to
rats. Radioactivity levels were very low in the plasma (ranging from
2.7 ng Eq/ml at 1.5 h to 0.6 ng Eq/ml at 24 h) as compared with
those found in the gastrointestinal (GI) tract, indicating negligible
systemic otilonium bromide absorption. Results from both quantitative radioluminography of whole body tissue distribution and
radioassay of dissected parts of the GI tract carried out with liquid
scintillation counting clearly demonstrate the presence of radioactive compounds in the walls of the GI tract at all sacrifice times.
In the other tissues and organs examined, radioactivity was only
644
EVANGELISTA ET AL.
TABLE 1
Radioactivity concentrations [Mean and S.D. (n ⫽ 5), expressed as nanogram
equivalents otilonium bromide per milliliter in blood and plasma samples at
various times after 2 mg/kg p.o. of 14C-otilonium bromide
Blood
Plasma
Time
Mean
S.D.
Mean
S.D.
5.5
3.2
5.3
2.0
3.4
0.8
0.3
0.2
2.7
1.3
0.9
0.6
0.6
0.4
0.3
0.1
h
1.5
4
8
24
mg) was then incubated at 60°C for 48 h with Soluene350 (1 ml) in a
scintillation vial. Pico-Fluor (15 ml) was added 6 h before counting. Each rat
carcass was digested by incubating with 250 ml of 1 N NaOH at 60°C for 72 h;
then 1 ml of solution was mixed with 15 ml of Pico-Fluor 12 h before counting.
Radioactivity was counted in a liquid scintillation spectrometer (1900 CA;
Packard). Blank biological medium treated like other tissues and fluids gave no
significant radioactivity signal.
The low quantitation limit (LQL) was set at 25 dpm/100 mg blood (ca. 0.11
nCi/g, i.e., 1.4 ng Eq otilonium bromide/g) and 20 dpm/300 mg plasma (ca.
0.03 nCi/g, i.e., 0.4 ng Eq otilonium bromide/g). For samples from the GI tract,
the LQL was defined as 25 dpm/200 mg. Approximate radioactivity elimination half-lives were calculated by linear regression analysis of a semilogarithmic plot of concentration values (from the peak on) versus time.
Results
Blood and Plasma Levels. As shown in Table 1, very low levels of
radioactivity were reached in rat plasma and blood after oral administration of 14C-otilonium bromide. In whole blood, radioactivity
concentrations were comparable between 1.5 and 8 h, and declined at
24 h after administration. Plasma sample levels gradually declined
from 1.5 h after administration (2.7 ⫾ 0.6 ng Eq/ml) to values that
were close to the LQL at the last sampling time (0.6 ⫾ 0.1 ng Eq/ml).
Distribution of 14C-Otilonium Bromide in Whole Animal by
Radioluminography. The digitalized images obtained from QRLG
show the whole body location of 14C-otilonium bromide and any
labeled metabolites. The presence of radioactivity is indicated in these
radioluminograms by the blackened areas (the higher the radioactivity
concentration, the darker the area). Figure 1 shows examples obtained
at the four different sampling times.
At the first sacrifice time (1.5 h; Fig. 1A), radioactivity was mainly
detected in the small intestine contents, at a value above the upper
quantitation limit (⬎25 ␮g Eq/g). High radioactivity was also measured in the stomach and cecum contents (7.22 and 5.59 ␮g Eq/g,
respectively), the stomach mucosa (2.22 ␮g Eq/g), and the small
intestine wall (1.60 ␮g Eq/g). Lower levels were recorded in the
stomach and cecum walls (0.27 and 0.74 ␮g Eq/g, respectively). Only
trace amounts were observed in the liver (0.04 ␮g Eq/g) and in the
colon contents (0.02 ␮g Eq/g) whereas all other tissues, organs, or
biological fluids were free of any labeling (i.e., radioactivity level ⬍
LQL or 0.01 ␮g Eq/g).
Four hours after administration (Fig. 1B), radioactivity levels were
still high in the stomach (11.38 ␮g Eq/g), had increased in colon and
cecum contents (both ⬎25 ␮g Eq/g, respectively), and in cecum and
colon walls (0.75 and 3.18 ␮g Eq/g), but had declined in the small
intestine contents, in the stomach mucosa and wall, and in the small
intestine wall (5.50, 0.66, 0.17, and 0.48 ␮g Eq/g, respectively). The
liver showed a similar concentration to the previous sacrifice time
(0.03 ␮g Eq/g). Levels of activity in the other tissues examined were
always below the quantitation limit (⬍0.010 ␮g Eq/g).
At 8 h after drug administration (Fig. 1C), a general decrease in
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017
tion of blocks. Animals were sliced using a bright cryomicrotome (Instrument
Company Ltd., Huntingdon, England). Thirty sagittal sections of 30-␮m thickness were collected and fixed on an adhesive support. Slices were dried at
⫺20°C in a cryostat for 24 h. Twelve lyophilized sections were selected for
evaluating the concentrations of 14C-otilonium bromide-associated radioactivity in duplicate or triplicate in every tissue/organ.
The bioimaging analyzer system (BAS 1800; Fuji, Tokyo, Japan) was used
for the QRLG, using imaging plates (IPs), which accumulate and store radioactive energy (Mori and Hamaoka, 1994). To quantify radioluminograms, a
calibrating scale of 11 radioactivity levels (range 0.7–1849 nCi/g) was prepared by the addition of 14C-otilonium bromide to rat whole blood. After liquid
scintillation counting (LSC) of each standard, precooled blood samples containing 14C-otilonium bromide were poured into 0.7-mm-diameter pores
drilled in a block of carboxymethylcellulose matrix maintained at ⫺20°C.
Sections of 30-␮m thickness were obtained by cryomicrotomy and processed
in the same way as the previous ones. The tapes with lyophilized material were
attached to rigid supports and identified with radiolabeled ink (100 ␮Ci/ml).
The IPs were exposed to whole body freeze-dried sections of rats together with
radiolabeled blood standards. To improve resolution, close contact between
histological sections and IP was ensured using X-ray cassettes. At the end of
exposure (72 h), each IP was inserted into the image reading unit and scanned
with a laser beam (100 ␮m of pixel resolution). Radioactivity was detected and
quantified from 16-bit numbered images using TINA Fuji software, and results
were expressed as photostimulated luminescence (PSL) units corrected for
background per unit area (PSL/S, i.e., [PSL-background]/mm2). The PSL/S
data were directly proportional to the amount of radioactivity that had penetrated the sample. A calibration curve (linear regression analysis of logtransformed PSL values versus blood radioactivity concentrations) was plotted
for each IP, allowing transformation of PSL/S values into international radioactivity units.
The assay limits for QRLG were defined by the upper and lower PSL/S
values of the standard calibration curves and associated LSC counts. Each
standard curve was plotted between 1849 and 0.71849 nCi/g. Expressed as
equivalents of the test drug, these limits corresponded to 25.35 and 0.01 ␮g
Eq/g, respectively.
The following tissues or organs were quantitatively studied with QRLG:
adrenal gland, aorta wall, blood (heart cavity), bone marrow (femur and
vertebras), brown fat, cecum (contents and walls), cartilage, cerebellum, cerebrum, choroid plexus, colon (contents and walls), epididymis, epididymal
fat, eye, Harderian glands, hypophysis, kidney (cortex and medulla), liver,
lungs, muscle (skeletal), myocardium, pancreas, perirenal fat, salivary glands
(submaxillary), seminal vesicles (membrane and content), skin plus hair, small
intestine (contents and wall), spinal cord, spleen, stomach (contents, mucosa,
and wall), sublingual glands (mucous glands), testis, thymus, thyroid, tongue,
and uveal tract.
Radioassay Study in the GI Tract. For this study, eight animals were used
(two animals per sacrifice time). The abdomen was carefully opened with
scissors and the alimentary tract was removed from the abdominal cavity. Ten
different samples were obtained from the GI tract: stomach wall, stomach
contents, duodenum wall, jejunum wall, ileum wall, small intestine contents,
cecum wall, cecum contents, colon plus rectum walls, and contents of colon
plus rectum. The contents of stomach, small intestine, cecum, and colon plus
rectum were separately expelled by manual pressure, and mixed with a large
amount of distilled water in a 100-ml plastic container. All tissues were then
rinsed with a small amount of water, blotted dry, weighed, and scissor-minced
in a 20-ml polyethylene vial. The weighed carcasses (without the GI tract),
sampled tissues, and macerated contents were stored at ⫺20°C until used for
radioactivity analysis.
Sample Processing and Radioactivity Measurements. Blood (100 mg)
was digested in a scintillation vial with 1 ml of Soluene350/isopropanol
mixture (1:1, v/v) at 50°C for 30 min; then 30% H2O2 (0.5 ml) was added
before additional incubation at room temperature and at 50°C (30 ⫹ 30 min).
Pico-Fluor (15 ml; Packard) was added 2 h before counting. Plasma (200 mg)
was mixed with 6 ml of Pico-Fluor in a scintillation vial and counted. Weighed
tissue samples were digested with 1 N NaOH (10 ml) at 60°C for 48 h; an
aliquot of the solution (250 mg) was then mixed with 15 ml of Pico-Fluor in
a scintillation vial 6 h before counting. GI contents, diluted and weighed, were
homogenized with an Ultra-Turrax blender; an aliquot of the suspension (250
645
OTILONIUM BROMIDE TISSUE DISTRIBUTION
14
C-otilonium bromide at 1.5 (A), 4 (B), 8 (C), and 24 h (D) after the administration of 2 mg/kg p.o. of
radioactivity concentrations was observed, with the exception of colon
contents, where a higher value than the upper quantification limit was
still found. The second highest levels were associated with the cecum
contents (13.09 ␮g Eq/g). The small intestine contents, the cecum, and
colon walls had values of 1.47, 1.10, and 1.07 ␮g Eq/g, respectively.
The stomach mucosa (0.69 ␮g Eq/g) and the small intestine wall (0.20
␮g Eq/g) were somewhat less radioactive. Very low radioactivity
levels (⬍0.03 ␮g Eq/g) were found in the stomach wall and contents
and in the liver. As expected, no radioactivity was again found in
other organs.
At the last sacrifice time (24 h, Fig. 1D), radioactivity had significantly decreased in all sections of the GI tract. The colon and cecum
contents were again found to be the samples that were richest in
labeled compound(s) (5.56 and 2.07 ␮g Eq/g, respectively). However,
the colon wall (0.32 ␮g Eq/g), as well as the stomach mucosa (0.23 ␮g
Eq/g) and the small intestine contents (0.26 ␮g Eq/g), still showed
marked levels. Weaker activities were found in the cecum walls (0.06
␮g Eq/g) and in the small intestine wall (0.02 ␮g Eq/g). No radioactivity was detectable at this time in the liver and other tissues.
Distribution of 14C-Otilonium Bromide in GI Tract by Direct
Radioassay. The temporal distribution of radioactivity in GI contents
and walls, as assessed by LSC, is shown in Table 2 and Fig. 2, where
data are reported as a percentage of the administered dose or as
microgram equivalents of otilonium bromide per gram, respectively.
At the first sampling time (1.5 h), most of the administered radioactive
dose (82%) was recovered in the GI contents: 12% in the stomach,
65% in the small intestine, and 5% in the cecum. A considerable part
14
C-otilonium bromide.
TABLE 2
Radioassays of 14C-otilonium bromide (expressed as mean percentage of
administered dose) in the GI tract at various times after 2 mg/kg p.o. of 14Cotilonium bromide
Sacrifice Time
GI Tissue or Contents
Stomach wall
Stomach contents
Duodenum wall
Jejunum wall
Ileum wall
Small intestine contents
Cecum wall
Cecum contents
Colon and rectum walls
Colon and rectum contents
Carcass without gastrointestinal tract
Total recovery
1.5 h
4h
8h
24 h
1.02
12.30
0.11
5.56
3.64
65.14
0.02
4.76
0.01
0.11
2.58
95.24
0.07
2.73
0.04
0.86
0.21
10.91
0.40
36.25
1.12
48.58
0.51
101.68
0.01
0.05
0.01
0.12
0.03
0.74
0.54
12.06
0.41
9.44
0.24
23.65
0.04
0.03
0.01
0.10
0.03
0.53
0.09
4.52
0.08
3.60
0.13
9.16
(9%) was also associated with the GI membranes. Jejunum, ileum, and
stomach walls showed the highest concentrations of labeled compound with 5.7, 11.1, and 3.4 ␮g Eq/g, respectively (Fig. 2). Much
lower amounts were present in duodenum, colon-rectum, and cecum
walls. An additional 3% of the dose was measured in the carcass, and
the overall radioactivity recovery was close to 95% (Table 2).
Four hours after drug administration, about 99% of radioactivity
was associated with the GI contents (3% in the stomach, 11% in the
small intestine, 36% in the cecum, and 49% in the colon and rectum;
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017
FIG. 1. Tissue distribution of
646
EVANGELISTA ET AL.
14
C-otilonium bromide in the walls of stomach, duodenum, jejunum, ileum, cecum, and colon-rectum after the administration of 2
mg/kg p.o. of 14C-otilonium bromide.
Table 2). The remainder was mainly detected in the colon-rectum and
jejunum walls, where the radioactivity concentration reached 4.0 and
0.9 ␮g Eq/g, respectively (Fig. 2).
At 8 h after drug administration, only 24% of administered radioactivity was still present in the body (Table 2). It was mainly associated with the contents of the large intestine (22%). As shown in Fig.
2, measurable radioactivity levels were still found in the walls of the
cecum (2.0 ␮g Eq/g) and colon-rectum (1.2 ␮g Eq/g).
At 24 h after drug administration, 9% of the dose was recovered in
sampled tissues and contents (Table 2). The radioactivity was mainly
present in the large intestine contents, but the concentration in the
various intestinal wall samples was still quantifiable (0.33 and 0.30
␮g Eq/g in the cecum and colon-rectum walls, respectively). Approximate apparent half-lives for the elimination of radioactive compound(s) from the GI walls were 2.5 h for the stomach, 4.0 h for the
small intestine, 6.3 h for the cecum, and 6.5 h for the colon.
Discussion
The results presented above indicate that otilonium bromide has
poor systemic absorption, as witnessed by the very low values of total
radioactivity in blood and plasma after oral administration of the
14
C-labeled compound to rats. In fact, the plasma levels found after a
dose of the drug (2 mg/kg p.o.) that is close to that used in humans to
treat IBS (Battaglia et al., 1998) are always at least 1000 times lower
than those reached in the walls of the large intestine. Moreover, these
plasma values (Cmax ⫽ 2.7 ng Eq/␮l) are far below the otilonium
bromide concentration (0.56 ␮g/ml or 1 ␮M) needed to exert a
spasmolytic activity through its combined calcium channel blocking,
tachykinin NK2 receptor antagonist, and antimuscarinic activity
(Maggi et al., 1983b; Gandia et al., 1996; Evangelista et al., 1998;
Santicioli et al., 1999).
Two studies in humans have shown that otilonium bromide plasma
levels after oral administration were undetectable (Signorini et al.,
1984; Sutton et al., 1997). The plasma levels reached in this study
substantiate the concept that otilonium bromide spasmolytic activity
occurs through local and selective activity in the intestine. On the
other hand, preliminary studies carried out with a higher dose (50
mg/kg p.o.) that is able to block stimulated GI motility in dogs
(Giachetti, 1991), have shown that 14C-otilonium bromide was concentrated in the intestine wall and that the radioactivity persisted up to
24 h from administration of the drug (from 77 to 5 ␮g Eq/g).
In this study, the radioactivity was again found almost exclusively
in the GI tract. Other organs, with the exception of liver, were not
radioactive at all and no blood-brain barrier transfer was recorded.
The weak radioactivity measured in the liver is in agreement with the
major role of this organ in the excretion of circulating otilonium
bromide; about 85% in i.v. administered drug is excreted through the
bile in rats (I.D. Capel and J.W. Daniel, unpublished data, on file at
Menarini Ricerche). Overall, these data are in agreement with preclinical studies (Scarpignato et al., 1980; Maggi et al., 1983a) and
clinical studies (Poynard et al., 1994; Sutton et al., 1997) showing that
otilonium bromide lacks the typical side effects of other antimuscarinic and calcium-blocking drugs.
Regarding tissue distribution, in accordance with the movement of
the gut contents across the GI tract, otilonium bromide-related radioactivity was first observed in the stomach and the small intestine
walls, from which it faded away in a relatively short time. Peak levels
in the large intestine tissues were attained between 4 and 8 h from
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017
FIG. 2. Temporal distribution of
OTILONIUM BROMIDE TISSUE DISTRIBUTION
dosing, and declined slowly, still being high at 24 h after drug
administration.
It is noteworthy that the concentrations reached (and maintained for
a fairly long time) in these tissues are in the range of those known to
exert spasmolytic activity in in vitro studies, i.e., 1 to 5 ␮M (0.6 –3
␮g/ml) (Maggi et al., 1983b; Gandia et al., 1996; Evangelista et al.,
1998; Santicioli et al., 1999). Previous in vitro studies have shown that
otilonium bromide accumulates in the inner layer of the colonic
circular muscle and submucosa (Amenta et al., 1991). At this level,
otilonium bromide has been shown to inhibit the contractility induced
by three main receptors for excitatory transmitters, i.e., muscarinic
and tachykinin NK1 and NK2 receptors (Santicioli et al., 1999), and to
bind with competitive interaction calcium channels and muscarinic
receptors at micromolar concentrations (Evangelista et al., 1998). This
myorelaxant effect exerted by otilonium bromide at the concentrations
found in the GI tract is thought to influence pain sensation and
impaired visceral sensitivity, which are leading signs of IBS.
References
Amenta F, Baroldi P, Ferrante F, Napoleone P and Meli A (1991) Autoradiographic localisation
of otilonium bromide binding sites in the rat gastrointestinal tract. Arch Int Pharmacodyn Ther
311:5–19.
Battaglia G, Morselli-Labate AM, Camarri E, Francavilla A, De Marco F, Mastropaolo G and
Naccarato R (1998) Octylonium bromide in the irritable bowel syndrome: A double-blind,
placebo controlled, 15-week study in a large number of patients. Aliment Pharmacol Ther
12:1003–1010.
Evangelista S, Giachetti A, Chapelain B, Neliat G and Maggi CA (1998) Receptor binding profile
of otilonium bromide. Pharmacol Res 38:111–117.
Gandia L, Lopez MG, Villaroya M, Gilabert JA, Cardenas A, Garcia AG and Borges R (1996)
Blocking effects of otilonium on Ca2⫹ channels and secretion in rat chromaffin cells. Eur
J Pharmacol 298:199 –205.
Giachetti A (1991) Pharmacological studies on otilonium bromide. Ital J Gastroenterol 23
(Suppl 1):56 –59.
Maggi CA, Grimaldi G, Volterra G and Meli A (1983a) Octylonium bromide: A new spasmolytic
agent, devoid of atropine-like side-effects. Drugs Exp Clin Res 9: 235–242.
Maggi CA, Manzini S and Meli A (1983b) Octylonium bromide: A smooth muscle relaxant
which interferes with calcium ions mobilisation. Arch Int Pharmacodyn Ther 264:305–323.
Maggi CA, Manzini S and Meli A (1985) Regional selectivity of calcium blockers at intestinal
level. Arch Int Pharmacodyn Ther 276:202–221.
Maggi CA and Meli A (1983) Assessment of potential selectivity of antispasmodics for the
various sections of the gastrointestinal tract of the rat as a guideline for their clinical use. Arch
Int Pharmacodyn Ther 262:221–230.
Manzini S, Maggi CA and Meli A (1984) System and organ-selectivity of smooth muscle
relaxants on in vitro spontaneously contracting preparations. Arch Int Pharmacodyn Ther
270:50 – 60.
Mori K and Hamaoka T (1994) Imaging plate autoradiography system. Tanpakushitsu Kakusan
Koso 39:1877–1887.
Poynard T, Naveau S, Mory B and Chaput JC (1994) Meta-analysis of smooth muscle relaxants
in the treatment of irritable bowel syndrome. Aliment Pharmacol Ther 8: 499 –510.
Santicioli P, Zagorodnyuk V, Renzetti AR and Maggi CA (1999) Antimuscarinic, calcium
channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in
the circular muscle of guinea-pig colon. Naunyn Schmiedebergs Arch Pharmacol 359:
420 – 427.
Scarpignato C, Coruzzi G, Zappia L and Bertaccini G (1980) Azione spasmolitica dell’ottilonio
bromuro sul tratto gastrointestinale e in vivo. Il Farmaco 5:249 –257.
Signorini C, Tosoni S, Ballerini R, Chinol M and Mannucci C (1984) A study of the absorption
of otilonium bromide following oral administration in man. Drugs Exp Clin Res 10:273–276.
Sutton JA, Kilminster SG and Mould GP (1997) The clinical pharmacology of single doses of
otilonium bromide in healthy volunteers. Eur J Clin Pharmacol 52:365–369.
Ullberg S and Larsson B (1981) Whole-body autoradiography. Methods Enzymol 77:64 –70.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017
Acknowledgments. We thank Prof. A. Giachetti, Dr. A. Crea, and
Dr. S. Manzini for their helpful advice and suggestions, and F. Parenti
and C. Azzurrini for their secretarial assistance.
647