480
Original Contributions
DuP 753 Is a Specific Antagonist for the
Angiotensin Receptor
Nour-Eddine Rhaleb, Noureddine Rouissi, Francois Nantel,
Pedro D'Orl6ans-Juste, and Domenico Regoli
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2-n-Butyl-4-chloro-5-hydroxy-methyl-l-[(2'-(lff)-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol
potassium salt (DuP 753) is a nonpeptide angiotensin II receptor antagonist that inhibits the
contractile effects of angiotensin II competitively and shows pA1 values of 8.27 on the rabbit
aorta and jugular vein, 8.66 on the rat portal vein and stomach, 8.19 on the rat urinary bladder,
and 836 on human colon, ileum, and urinary bladder. This agent (more than 1 0 * M) exhibits
no agonistic activity and does not affect the contractile effects of norepinephrine, acetylcholine,
bradykinin, desArg'-bradykinin, substance P, neurokinin A, neurokinin B, or bombesin in the
various tissues. The present results demonstrate that DuP 753 is a potent nonpeptide
antagonist with high affinity, specificity, and selectivity for the angiotensin receptor. (Hypertension 1991;17:480-484)
D
uP 753 (2-rc-butyl-4-chloro-5-hydroxymethyl-l-[(2'-(l//)-tetrazol-5-yl)biphenyl4-yl)methyl]imidazol potassium salt) is a
nonpeptide antagonist of angiotensin II (Ang II) that
is orally active and fairly specific for Ang II. DuP 753
shows a similar pharmacological spectrum as EXP
6803 and EXP 6155, two other antagonists previously
reported.1 In fact, DuP 753 does not inhibit either
the pressor effects of norepinephrine or vasopressin
or the depressor effect of bradykinin in anesthetized
rats.2 The compound has also been shown to reduce
blood pressure in furosemide-treated normotensive
rats2 or in animals affected by various forms of
experimental or spontaneous hypertension.3 In the
present study, DuP 753 was tested in vitro on selective peptide monoreceptor systems to determine the
extent of its specificity for the angiotensin receptor
and exclude all possible interference with receptors
for kinins (B! and B2), neurokinins (NK-1, NK-2,
NK-3), bombesin (BB] and BB2), and the a-adrenergic and the muscarinic receptors.
From the Department of Pharmacology, Medical School University of Sherbrooke, Sherbrooke, Quebec, Canada.
The work was performed with the financial support of the
Medical Research Council of Canada (M.R.C.C). N.E.R. and
P.DJ. are, respectively, student and scholar of the Heart and
Stroke Foundation of Canada. F.N. is a student of the Georges
Phe'nix Foundation and D.R. is a Career Investigator of the
M.R.C.C.
Address for correspondence: Dr. Domenico Regoli, Department of Pharmacology, Medical School, University of Sherbrooke,
Sherbrooke, Quebec, Canada J1H 5N4.
Received October 1, 1990; accepted November 16, 1990.
Methods
Male Sprague-Dawley rats (150-200 g) and Albino
New Zealand rabbits (1-1.5 kg) were killed by stunning and exsanguination. The aorta, jugular vein, and
pulmonary artery of the rabbits, the stomach, urinary
bladder, and the portal vein of the rats were rapidly
removed from the animals and kept in oxygenated
95% O2-5% CO2) Tyrode's (for the stomach and
urinary bladder) or Krebs' (for the isolated vessels)
solutions. Strips of the rabbit veins were prepared
according to Gaudreau et al4; the rat portal vein was
prepared according to Mastrangelo et al5; strips of
the rat stomach as well as strips of the rat urinary
bladder were prepared according to Vane.6 The
human tissues were taken from different donors of
both sexes (age range, 40-65 years). The human
urinary bladder was prepared according to Dion et
al.7 The longitudinal strips of human ileum and colon
were prepared by the method described by Crema et
al8 and modified by Couture et al.9 The strips were
suspended in 10-ml organ baths filled with oxygenated Krebs' (at 37°C) or Tyrode's (at 32°C) solutions
under a tension of 4.0 g (human urinary bladder), 2.0
g (rabbit aorta, rat stomach, human ileum, human
colon), 1.0 g (rabbit pulmonary artery, rat urinary
bladder), or 0.5 g (rabbit jugular and rat portalmesenteric vein). Changes of tension produced by
various agents were recorded with isometric transducers (model FT 03C, Grass Instrument Co.,
Quincy, Mass.) calibrated as to 1 g=30 mm on
polychannel recorders (model 7D, Grass). After an
equilibration period of 60-120 minutes, during which
the tissues were washed and the tension adjusted
Rhaleb et al
Anglotensin Receptor Antagonist
481
TABLE 1. Antagonist Effects of DuP 753 Against Anglotensin II in Six Isolated Organs and Absence of Antagonistic Effect by DuP 753
Against Other Peptide or Nonpeptide Agonists
Response
Control
+DuP 753
(21.7 /xM)
DesArg'-BK (27.7)
17.2±2.8
21.3±3.1
NE (12.2)
46.8±4.3
48.0±2.1
6.6±0.7
Tissue
pA 2
pAio
A
Agonist
(nM)
Rabbit aorta
8.27
7.19
1.08
Rabbit jugular vein
8.27
7.33
0.94
SP (6.5)
6.8±0.7
BK (2.4)
NKA (4.4)
9.0±1.0
8.5±0.9
26.3±5.5
23.6±5.3
24.4±4.5
NKB (20.7)
NE (486.4)
15.3±1.7
13.3±1.9
10.8±1.7
BB (6.2)
Ach (138.0)
BB (6.2)
16.0±1.5
45.5±4.4
Rabbit pulmonary artery
8.27
7.33
0.94
Rat portal vein
8.66
7.66
1.00
NE (72.9)
Rat stomach
8.66
7.63
1.03
Rat urinary bladder
8.19
7.27
0.92
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Human urinary bladder
Human colon
8.36
Human ileum
8.36
7.49
7.49
0.87
0.87
8.36
7.41
0.95
Ach (55.0)
Ach (550.4)
NKA (88.2)
Ach (550.4)
15.4±3.1
26.8±5.1
42.0±7.3
351.3±57
56.0±1.2
BK (8.1)
14.4±2.9
78.3±13.2
Ach (550.4)
92.5±11.6
23.7±5.5
14.8±1.9
54.7±5.9
31.5*3.4
40.3±5.5
350.0±60
54.0±6.1
13.2±2.9
80.8±12.3
90.0±12.7
pA2 and pA10 are log of the molar concentration of antagonist that reduces the effect of a double (pA2) or 10 times (pA10) higher
concentration of agonist (angiotensin II) to that of a single one. Values in parentheses are the nanomolar concentration of the agonists. A,
Difference between pA2 and pA,0. Values of response are mean±SEM of at least five determinations and indicate contraction in millimeters
(30 mm = l g). BK, bradykinin; NE, norepinephrine; SP, substance P; NKA, neurokinin A; BB, bombesin; Ach, acetylcholine.
every 15 minutes, the contractile agents were applied
in increasing concentrations to build complete concentration-response curves. Thereafter, average concentrations (producing approximately 70% of the
maximal response) of each agent were tested in the
absence and in the presence of DuP 753, which had
been applied 10 minutes before. In this way, pAi and
pA,0 values were measured. In the rabbit aorta,
cumulative concentration-response curves were recorded for Ang II in the absence and in the presence
of increasing concentrations of the antagonist (from
10.8 nM to 54.2 fiM) to build a Schild plot.
Results obtained with DuP 753 against Ang II are
presented in terms of pA2 and pA10, whereas for the
other agonists the mean±SEM of the contractions
recorded in the absence and in the presence of the
antagonist are compared. Significance of differences
between controls and experimental (recorded in the
presence of DuP 753) values are evaluated with
Student's t test for paired samples. Probability values
lower than 0.05 were considered significant.
Bradykinin (BK), desArg'-bradykinin (desArg9BK), substance P, neurokinin A, and neurokinin B
were prepared in our laboratory by the solid-phase
method10; Ang II and bombesin were purchased from
Bachem, acetylcholine and norepinephrine from
Sigma Chemical Co., St. Louis, Mo., ascorbic acid
and sulfolane from Anachemia, Lachine, Quebec,
Canada. DuP 753 was a generous gift of Dr. R.D.
Smith, Du Pont Co., Wilmington, Del. Concentrated
solutions (1-5 mg/ml) of peptides, acetylcholine, and
DuP 753 were prepared in water and that of neurokinin
B in 10% sulfolane. Solutions of norepinephrine were
made in water containing 0.5% ascorbic acid.
Results
Specificity of DuP 753 for the Anglotensin Receptor
Under the experimental conditions described
above, DuP 753 exerted a potent antagonistic effect
against Ang II in the nine isolated organs without
influencing the effects of the other peptide or nonpeptide stimulants (Table 1). Thus, DuP 753 was
found to be inactive as stimulant and as antagonist 1)
in the rabbit aorta on the Bj receptor, which is
activated by desArg9-BK,11 and on the a-adrenergic
receptor, 2) in the rabbit jugular vein on the kinin
Bj11 and the substance P (NK-1) receptors, 3) on the
rabbit pulmonary artery on the neurokinin A (NK-2)
and the a-adrenergic receptors, 4) in the rat portal
vein on the neurokinin B (NK-3) and the a-adrenergic receptors, 5) in the rat stomach on the bombesin
(BB2)12 and the muscarinic receptors, 6) in the rat
urinary bladder on the bombesin (BB,)12 and the
muscarinic receptors, and 7) in human preparations,
namely human urinary bladder, colon, and ileum on
the muscarinic receptors. As shown in Table 1, no
significant differences were observed between the
effects of the various agents other than obtained in the
absence (control) and in the presence of DuP 753.
Activity of DuP 753 on Angiotensin Receptor in
Various Organs
The pA2 values measured with DuP 753 against
Ang II in the various organs were very similar, in fact
482
Hypertension
Vol 17, No 4 April 1991
RABBIT AORTA
10 Min.
1.0 g
w
i s % vr M
DDK
ATn
DDK
9
1.1 x 10" 7 M
&S x l O
9
M
DUP 753
I.I x 1<T7 M
DUP 753
IX) x
RABBIT JUGULAR VEIN
ww
w
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
0.5
BK
UT9 M
I<r*M
DUP 753
DUP 753
S
1.0 x 1<T M
DUP 753
S
LO x 1<T M
1.0 x W 5 M
FIGURE 1. Representative traces showing myotropic effects of 1) angiotensin II (8.5xlO~9 M) and desArg9 bradykinin (DBK)
(1.1 XlO-7 M) on rabbit aorta and 2) angiotensin II (8.5x10-* M), substance P (SP) (6.5xlO~9 M), and BK (8.0xl0-8 M) on
rabbit jugular vein in absence and in presence of DuP 753 (l.OxlO'5 M). Abscissa, time; ordinate, tension in grams; ATn,
angiotensin II.
almost identical (between 8.19 and 8.27) for four of
the nine preparations, slightly higher (8.66) for the
rat stomach and portal vein, where active metabolic
degradation of the peptide agonist might favor the
metabolically resistant nonpeptide antagonist (unpublished observations from our laboratory). Interestingly enough, the three human tissues investigated
in the present experiment showed pA2 values very
similar to those of the animal preparations. These
findings indicate that the receptor for angiotensin in
arterial and venous vessels as well as in the rat and
human gastrointestinal and urinary systems is the
same. The data shown in Table 1 also suggest that
DuP 753 exerts a competitive antagonism against
Ang II since the difference between pA2 and pA10
values is near to 1.0 in all preparations according to
Schild.13 It is also worthy of mention that no agonistic
effect was observed in any organ with high concentrations (10~5 M) of DuP 753 (Figure 1). Moreover,
such high concentrations of DuP 753 completely
blocked the myotropic effects of Ang II on the rabbit
aorta and jugular vein without interfering with the
responses of these tissues to desArg9-BK or BK and
substance P, respectively (Figure 1).
Competitivity of DuP 753 for Angiotensin
To demonstrate that DuP 753 is indeed a competitive antagonist for angiotensin, concentration-re-
sponse curves were measured with Ang II on the
rabbit aorta, in the absence and in the presence of
five concentrations (from 10.8 nM to 54.1 /iM) of
DuP 753. The results are illustrated in Figure 2 by
showing the curves on panel A and the Schild plot on
panel B. Thus, the curves of angiotensin are displaced to the right by the antagonist; the curves are
parallel to the control and the maximum effect of
angiotensin is obtained in the presence of antagonist
at the five concentrations used. Figure 2A illustrates
the data plotted according to Schild.14 Individual
values fit on a straight line with a slope not significantly different from a unity (0.96) and a pA2 value of
8.33 extrapolated at the intersection of the line with
the abscissa, which corresponds to the pA2 (8.27) of
DuP 753 measured experimentally.
The antagonistic effect of DuP 753 is completely
reversible in 20-30 minutes for the low (5.5 nM) and
60-90 minutes for the high (0.54 pm) concentration
of antagonist (results not shown).
Discussion
The results presented above demonstrate that the
nonpeptidic angiotensin antagonist developed by the
Du Pont Company is selective for the angiotensin
receptor and does not interact with the receptors for
Rhaleb et al
Angiotensin Receptor Antagonist
483
10
Al'n + Ifl.NnM AIM.
AT,, 4 54.1 IIM
,, + S.4l|iM
A AT,,
,, ^ S4.I pM
' ANT. DUP 753
! 50
c
o
O
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FIGURE 2. Panel A: Concentration-response curve of angiotensin II on rabbit aorta
in presence of various concentrations of DuP
753. Ordinate, concentration expressed as a
percentage of the response to angiotensin II;
abscissa, log of the molar concentration of the
agonist. Each point represents the average,
vertical bars represent SEM. Panel B: Schild
plot of DuP 753 against angiotensin II on
rabbit aorta. Ordinate, log (DR-1) of agonist;
abscissa, log of molar concentration of antagonist; A Tn, angiotensin II.
ATn.-LogIM]
4-
Q
B
10-
r= 0.96
/
8
7
6
5
DUP 753. -Log[M]
4
other peptides and biologically active amines. These
results confirm the findings obtained by Wong et al2
in anesthetized rats against BK and noradrenaline;
they extend the characterization of DuP 753 to other
receptor systems such as the Bj for the kinins, the
three receptors for the neurokinins, the two receptors for bombesin, and the cholinergic muscarinic
receptor. The present data have all been obtained in
vitro, using monoreceptor systems that have been
identified and characterized in recent years.12-15-17
The contractile responses of the various preparations
(the arteries, the veins, the stomach, and urinary
bladder) appear to be due to the direct activation of
receptors by the various agents, since the potential
contributions of a large number of other endogenous
stimulants have been excluded by systematic specificity studies.512-1718 It is therefore suggested that the
competition between Ang II and DuP 753 occurs, in
the various tissues, primarily at the level of the
smooth musclefibers,where the angiotensin receptor
appears to be localized.19 The majority of the preparations used in the present study are isolated vessels, which are stimulated by Ang II and by at least
one of the other peptides. One interesting example is
the rabbit jugular vein, whose contractions in response to very similar concentrations of Ang II,
bradykinin, and substance P17 appear to be due to the
activation of the same intracellular mechanism, that
is, phosphatidylinositol (PI3) turnover.17 Thus, the
selective antagonist exerted by DuP 753 against
angiotensin must occur at the receptor site and may
not involve any postreceptor mechanism, even if DuP
753 would enter into the cells.
Affinities of DuP 753 in the various preparations,
evaluated in terms of pA2, are very similar; pA2
values varied between 8.19 (the rat urinary bladder)
484
Hypertension
Vol 17, No 4 April 1991
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and 8.66 (the rat stomach and the rat portal vein),
were the same (8.27) in three of the nine preparations, and 8.36 in the three human tissues. There is
no doubt that the angiotensin receptor is the same in
large arteries, large veins, and in the gastrointestinal
and urinary systems from both humans and other
mammals. It is therefore concluded that DuP 753
reduces or blocks the actions of angiotensin in several peripheral organs; DuP 753 is, however, inactive
on one of the two angiotensin binding sites in the
adrenal glands.20 Despite the multiplicity of peripheral effects, the major target of DuP 753 is the
arterial resistance vessels, where angiotensin exerts
its major physiological role in the regulation of blood
pressure. Similarities of pA2 values in the various
organs suggest that diffusion barriers, metabolism,
and the local redistribution of DuP 753 may not play
any major role for the interaction of the antagonists
with the receptor. Apparent affinities for angiotensin
and its antagonist expressed by the pD2 and pA2
values, may therefore be closed to the real affinities.21
pD2 values of Ang II and pA2 of DuP 753, as
measured in two of the most sensitive preparations,
the rabbit jugular vein and the rabbit aorta (Table 1),
indicate that the affinity of the antagonist is very
similar to that of the naturally occurring peptide. It is
therefore suggested that angiotensin and DuP 753
interact in a similar way with the receptor site. The
analysis of the antagonism, performed on the rabbit
aorta by using a wide range of concentrations, indicates that DuP 753 interacts with angiotensin at the
receptor level in a competitive manner. Indeed, the
difference between pA2 and pA10 and the slope of the
Schild plot presented in Figure 2 are near to unity.
Concentration-response curves obtained with angiotensin in the presence of increasing concentrations of
DuP 753 are parallel to the control curve, and the
maximum effect is reached even in the presence of
very high concentrations of DuP 753. These data and
the slope of the Schild plot indicate that DuP 753
exerts a competitive antagonism against angiotensin
in vitro, despite the fact that a prolonged duration of
action has been demonstrated for DuP 753 in anesthetized animals.2 This difference in the duration of
antagonism of DuP 753 between in vitro and in vivo
experiments may be explained at least in part by the
metabolism, which is very active for Ang II but not
for the nonpeptidic antagonist.
Acknowledgments
We acknowledge the secretarial work of C. Theberge and the technical assistance of M. Boussougou.
References
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KEY WORDS • angiotensin II • vascular smooth muscle •
antihypertensive agents • renin-angiotensin system
DuP 753 is a specific antagonist for the angiotensin receptor.
N E Rhaleb, N Rouissi, F Nantel, P D'Orléans-Juste and D Regoli
Hypertension. 1991;17:480-484
doi: 10.1161/01.HYP.17.4.480
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