EFFECTS OF TIMEPIDIUM BROMIDE, HYOSCINE

N-BUTYLBROMIDE AND ATROPINE ON THE ISOLATED

GUINEA PIG GALLBLADDER AND SPHINCTER OF ODDI

Hideyuki TAKENAGA, Tetsuo MAGARIBUCHI

and Hiromichi NAKAJIMA

Pharmacological Research Laboratory, Tanabe Seiyaku Co., Ltd.,
Toda, Saitama 335, Japan

Accepted December 7, 1979

Abstract-Effects of timepidium bromide (TB), hyoscine-N-butylbromide (HB) and

atropine (Atr) were studied on the isolated guinea pig gallbladder and sphincter of
Oddi. In the isolated gallbladder, the contraction induced by methacholine was
competitively antagonized with TB, HB and Atr: pA2 values for TB, HB and Atr were
8.44, 7.55 and 9.11, respectively. Contraction of gallbladder after field stimulation
at 5 Hz was also inhibited by TB, HB and Atr as well as by tetrodotoxin (TTX). On
the other hand, TB had no significant influence on the electrically evoked (30 Hz)
contraction which remained after treatment with TTX. It was estimated that the
inhibitory activity of TB on the contraction of gallbladder was 1/5-1/6 that of Atr and
7 times as potent as HB. In the isolated sphincter of Oddi, TB, HB and Atr reduced the
perfusion pressure increased by acetylcholine (ACh), the activity of TB being 1/4 that
of Atr and 3 times of HB. The contractile responses to field stimulation (5 Hz) were
also inhibited by TB, HB and Atr. The inhibitory activity of TB was 1/5 that of Atr
and 12 times of HB. It was also demonstrated that TB produced no significant effect
on the noradrenaline (Nor)-induced contraction and isoproterenol (Iso)-induced
relaxation in the gallbladder or Nor-induced increase of the perfusion pressure in the
sphincter of Oddi. It is suggested that TB acts on the gallbladder and sphincter of
Oddi by virtue of its anticholinergic activity.

Timepidium bromide (TB) is a new anticholinergic agent which produces a potent

spasmolytic action on various visceral smooth muscles (1-3). The antisialogogue and
mydriatic activities of TB are, however, lower than the spasmolytic activity (2). In in vivo
experiments, it has been reported that TB inhibits the spontaneous or bethanechol-induced
motility of gallbladder and reduces the perfusion pressure in the sphincter of Oddi (3, 4).
The present experiments were carried out to investigate the effect of TB on the biliary system
in more detail. For this purpose, isolated guinea pig gallbladder and sphincter of Oddi
were used and the results were compared to those of other known anticholinergic agents
as hyoscine-N-butylbromide (HB) and atropine (Atr).

MATERIALS AND METHODS

Male guinea pigs, weighing 250-500 g, were stunned and bled. The entire gallbladder

and sphincter of Oddi were removed. The gallbladder was dissected, the bile was washed

out and longitudinal strips, 20 mm long and 5 mm wide, were prepared. The preparation

was vertically suspended in an organ bath with 30 ml of modified Locke solution. A tension
of approx. 500 mg was initially applied to the gallbladder preparation and isometric con
traction was recorded by means of a force-displacement transducer coupled to an ink
writing oscillograph. Experiments were carried out at 37±1'C.
In the sphincter of Oddi, the motility was estimated by measuring the change in perfusion
pressure. A polyethylene cannula (inner diameter, 2 mm) was passed into the pouch through
the common bile duct and ligated. The cannula was attached to a Y-tube, one arm of
which was connected to a pressure transducer, and the other was perfused with modified
Locke solution at a constant inflow rate of 0.6-1.2 ml/hr. The preparation was then vertically
placed in an organ bath with 30 ml of modified Locke solution and the perfusion pressure
was recorded on an ink-writing oscillograph. Since the sphincter of Oddi exhibited rather
irregular, spontaneous activity at 37'C, experiments were performed at room temperature
(26-28 °C) to reduce the spontaneous activity.
Field stimulation was applied via two Ag-AgCl loop electrodes (diameter, 5 mm) to the
gallbladder and two Ag-AgCI plate electrodes (7 x 4 mm) to the sphincter of Oddi. In the
gallbladder, the strips were stimulated at 10 min intervals with rectangular pulse of 1 msec
duration at 1-50 Hz for 20 sec, while the sphincter of Oddi was driven at 10 min intervals
with rectangular pulse of 1 cosec duration at 2-50 Hz for 5 sec.
The composition of modified Locke solution was as follows (mM) : NaCl 141.9, KCI
5.6, CaCI2 2.2, MgCI2 0.5, NaHCO3 11.9, NaH2PO4 0.4, and glucose 5.5. The solution
was aerated with 95%02 and 5 % CO2. The test compound dissolved in modified Locke
solution (0.3 ml) was added directly to the organ bath and the response of the isolated
preparation was measured 30 min after the addition of the test compound.
Drugs used in the present experiments were: timepidium bromide (Tanabe Seiyaku Co.,
Ltd.), atropine sulfate (Wako Pure Chem. Ind., Ltd.), tetrodotoxin (Sankyo Co., Ltd.),
phentolamine mesylate (CIBA-Geigy Ltd.), propranolol hydrochloride (Sumitomo Chem.
Ind., Ltd.), (-)-noradrenaline bitartrate hydrate (Calbiochem) acetylcholine chloride,
methacholine chloride and (±)-isoproterenol hydrochloride (Nakarai Chem. Ltd.). Hyoscine
N-butylbromide was synthetized for the present work in our Organic Chemistry Research
Laboratory.

RESULTS

1. Isolated gallbladder
1-1. Antagonistic effects of TB, HB and Atr on methacholine (MeCh)-induced contraction:
Figure 1 shows the effects of TB, HB and Atr on the contraction induced by MeCh, which
was added cumulatively to the organ bath. The MeCh-induced contraction was compe
titively antagonized by these compounds and pA2 values (5) of TB, HB and Atr were 8.44,
7.55 and 9.11, respectively. Thus, the potency of TB was about 1/5 that of Atr and 7 times
that of HB.
1-2. Inhibitory effects of TB, HB and Atr on the contraction induced by field stimulation:
Stimulation at 1 Hz initiated the contraction and its amplitude increased with increasing
frequencies of stimulation until a peak reached at 30 Hz or more. Figure 2 shows that
 FIG. 1. Effects of TB, HB and Atr on the contraction induced by methacholine (MeCh)
in the isolated gallbladder. Each point is the mean of 5 experiments and the
vertical bars represent SE of the mean.

TTX (10-s g/ml) and Atr (10-6 g/ml) completely abolished the contraction after stimulation
with 5 Hz but did not inhibit completely the contraction caused by stimulation at 30 Hz.
As demonstrated in Figs. 2 and 3, TB as well as HB and Atr inhibited dose-dependently
the contraction evoked by stimulation at 5 Hz. The mean doses producing a 50 % decrease
in the electrically induced contraction (ID50 values) were 2.4 x 10-s g/ml, 1.7 x 10-' g/ml
and 4.1 x 10-9 g/ml for TB, HB and Atr, respectively. This means that the inhibitory activity
of TB was 1/6 that of Atr, whereas it was 7 times as potent as HB. TB (10-6 g/ml)
also inhibited, for the most part, the contraction induced by stimulation at 30 Hz (Fig. 2).
However, as shown in Fig. 2, TB had no significant influence on the contraction remained
after treatment with TTX (10-6 g/ml).
1-3. Effects of TB on the noradrenaline (Nor)-induced contraction and isoproterenol
(Iso)-induced relaxation : TB at the concentration of 10-6 g/ml was used, the concentration
which exhibted a strong inhibitory action on the electrically induced contraction as described
above. Nor (10-'-10-5 g/ml) produced a dose-dependent increase in the contraction of

FIG. 3. Dose-response curves for the effects

of TB, HB and Atr on the contraction
induced by field stimulation at 5 Hz
FIG. 2. Effects of tetrodotoxin (TTX), Atr in the isolated gallbladder. Each point
and TB on the contraction induced by is the mean of 5 experiments and the
field stimulation at 5 Hz and 30 Hz in vertical bars represent SE of the mean.
the isolated gallbladder. -0 TB , -/ HB, -F-1 Atr.
 FIG. 4. Effects of TB on the noradrenaline (Nor)-induced contraction and isoproterenol
(Iso)-induced relaxation in the isolated gallbladder. A: Effects of TB, phentol
amine (Phen) and propranolol (Prop) on the Nor-induced contraction.
B: Effects of TB and Prop on the Iso-induced relaxation.

the isolated gallbladder: the amplitude of the contraction induced by Nor (10-5 g/ml) was
approx. 1/10 that induced by ACh (10-5 g/ml). As shown in Fig. 4 A, TB had no signi
ficant influence on the contraction induced by Nor (10-6 g/ml). A similar effect was also
observed in preparations pretreated with propranolol (10-6 g/ml). On the contrary, the
a-adrenergic blocking agent, phentolamine (10-6 g/ml), inhibited the Nor-induced con
traction and a slight relaxation was thus elicited. This relaxation was prevented by the
8-adrenergic blocking agent, propranolol (10-6 g/ml). TB also produced no significant
influence on the Iso-induced relaxation, which was inhibited by propranolol (Fig. 4B).

2. Isolated sphincter of Oddi

2-1. Effects of TB, HB and Atr on the perfusion pressure increased by acetylcholine
(ACh) : When the preparation was treated with ACh, the perfusion pressure once increased
and then fell either rapidly or slowly. In some preparations, a higher concentration of
ACh (at or more than 10-5 g/ml) produced spontaneous oscillatory changes in the slowly
fall phase of perfusion pressure. In any case, the initial response to ACh (10-6-10-4 g/ml)
was increased in a dose-related manner. As illustrated in Fig. 5, TB, HB and Atr dose
dependently reduced the peak perfusion pressure increased by 10-6 g/ml ACh, the concen
tration of which exhibited a 50% increase in the pressure. ID50 values of TB, HB and Atr
were 4.9 x 10-9 g/ml, 5.6 x 10-8 g/ml and 1.2 x 10-9 g/ml, respectively. Thus, the potency
of TB was approx. 1/4 that of Atr and 12 times that of HB.
2-2. Effects of TB, HB and A tr on the perfusion pressure increased by field stimulation:
Field stimulation of the sphincter of Oddi caused a transient increase in the perfusion
pressure. Response to field stimulation increased with increasing frequencies of stimulation
until it became maximum at approx. 40 Hz. It was found that the increased perfusion
pressure after stimulation at 5 Hz was completely inhibited by TTX (10-6 g/ml) or Atr
(10-6 g/ml), while it was dose-dependently suppressed by TB, HB and Atr (Fig. 6). ID50
values were 7.1 x 10-9 g/ml, 2.3 x 10-$ g/ml and 1.5 x 10-9 g/ml for TB, HB and Atr, re
spectively. Thus, the inhibitory activity of TB was estimated to be 1/5 that of Atr and 3
times that of HB. On the other hand, as shown in Fig. 6, increase in the perfusion pressure
induced by field stimulation at 5 Hz was usually followed by a decrease below the control
level. Such a decrease in the perfusion pressure was generally inhibited by TTX, Atr, TB
and HB.
2-3. Effect of TB on the perfusion pressure increased by Nor: Increase in the perfusion
pressure caused by 10-5 g/ml Nor was not affected by TB (10-6 g/ml), whereas it was strongly
inhibited by phentolamine (10-6 g/ml). Unlike the effect on the isolated gallbladder, Iso
produced no significant change in the perfusion pressure in the sphincter of Oddi.

FIG. 5. Effects of TB, HB and Atr on the perfusion pressure increased by acetylcholine
(ACh) in the isolated sphincter of Oddi. A: Experimental records. B: Dose
response curves for the effects of TB, HB and Atr. Each point is the mean of
5 experiments and the vertical bars represent SE of the mean. -0 TB, -/
HB, -Fl Atr.

FIG. 6. Effects of TB, HB and Atr on the perfusion pressure increased by field stimu
lation at 5 Hz in the isolated sphincter of Oddi. A: Experimental records. B:
Dose-response curves for the effects of TB, HB and Atr. Each point is the mean
of 5 experiments and the vertical bars represent SE of the mean. -0 TB,
-/ HB , -L Atr.
 DISCUSSION
It has been reported that the gallbladder and sphincter of Oddi contract through the
cholinergic mechanism (6-9). a-Adrenergic mechanism is also responsible for the con
traction, whereas relaxation can be elicited by a-adrenergic mechanism (10-14). Similar
results were obtained in the present experiments: ACh, MeCh or Nor induced contraction
of the isolated gallbladder or perfused preparation of the sphincter of Oddi. The con
tractions induced by ACh or MeCh and Nor were blocked by Atr and phentolamine, re
spectively. On the other hand, Iso produced a relaxing effect on the gallbladder preparation
and this effect was inhibited by propranolol. In perfusion experiments with the sphincter
of Oddi, however, little significant effect was produced by Iso. In relation to the perfusion
experiments, Persson (11) has reported that in about one-third of the preparations Iso caused
a small decrease in the basal tension.
As shown in the present experiments, field stimulation evoked contraction in the isolated
gallbladder and sphincter of Oddi. The contractile responses to field stimulation at a
frequency of 5 Hz were completely abolished by TTX and Atr, suggesting that cholinergic
mechanisms are involved in the responses. On the contrary, the contraction induced by
stimulation at 30 Hz was not completely suppressed by TTX and Atr. Thus, it seems likely
that a small part of contraction evoked at 30 Hz is myogenic in nature and cholinergic activity
is not involved. In experiments with the sphincter of Oddi, it was observed that the increase
in perfusion pressure (positive response) after stimulation at 5 Hz was followed by a negative
response. Mechanism by which such a negative response is evoked is unknown, although
in general the response was inhibited by TTX, Atr, TB and HB. Contractile properties
under transmural stimulation have also been reported in the gallbladder (15, 16) and the
sphincter of Oddi (17-19).
As demonstrated in the present experiments, TB and HB competitively antagonized
the contraction induced by MeCh in the gallbladder and reduced dose-dependently the
perfusion pressure increased by ACh in the sphincter of Oddi. TB and HB also produced
a dose-dependent inhibitory effect on the contraction evoked by field stimulation at 5 Hz
in both the gallbladder and sphincter of Oddi. The results suggest that TB and HB exhibit
anticholinergic activity on the biliary system, as in the case with gastrointestinal tract (1-4).
On the other hand, TB produced no significant influence on the Nor-induced contraction
in the gallbladder and sphincter of Oddi and on the Iso-induced relaxation in the gallbladder
preparation. Therefore, a and (3-adrenergic mechanisms may not be involved in the in
hibitory action of TB on the guinea pig biliary system. Direct action of TB on the smooth
muscle of the gallbladder may also be excluded, since TB had no significant influence on
the contraction induced by field stimulation at 30 Hz in the presence of TTX.
From these lines of evidences, it is most probable that TB exhibits spasmolytic action
on the isolated guinea pig gallbladder and sphincter of Oddi in virtue of its anticholinergic
activity. The anticholinergic activity of TB on the biliary system was found to be 1/4-1/6
that of Atr, the activity of which was similar to its spasmolytic activity in cat and rat stomach
(2, 3).
 Acknowledgements: We should like to express our gratitude to Professor Emeritus

H. Kumagai, University of Tokyo and Dr. A. Kiyomoto, Director of Pharmacological

Research Laboratory, Tanabe Seiyaku Co., Ltd., for their constant encouragement and

pertinent suggestions.

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