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Vol. 290, Issue 3, 1085-1091, September 1999
Zeneca Pharmaceuticals, Alderley Park, Macclesfield, United Kingdom
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The endothelins (ETs), potent vasoconstrictor peptides, have been implicated in the pathogenesis of various cardiovascular disorders. In the present study, we describe the novel, potent, orally active, selective ETA receptor antagonist ZD1611 [3-{4-[3-(3-methoxy-5-methylpyrazin-2-ylsulfamoyl)-2-pyridyl]phenyl}-2,2-dimethylpropanoic acid]. ZD1611 competitively inhibited 125I-labeled ET-1 binding at human cloned ETA and ETB receptors with pIC50 values of 8.6 ± 0.1 and 5.6 ± 0.1, respectively, showing 1000-fold selectivity for the ETA receptor. ZD1611 caused a parallel rightward shift of the concentration response curve to ET-1 in the rat isolated aorta yielding a concentration of antagonist that caused a 2-fold rightward shift in the ET-1-response curve (pA2) of 7.5 ± 0.3. When administered i.v. to anesthetized rats and dogs, ZD1611 caused dose-related rightward shifts of partial dose-response curves to the precursor of ET-1, big ET-1. Threshold doses for significant antagonist activity were determined as 0.1 mg/kg and 0.3 mg/kg in the rat and dog, respectively. Importantly, ZD1611 was able to reverse an established big ET-1-induced pressor response in pithed rats in the presence of continuous big ET-1 infusion. Failure of ZD1611 to inhibit the BQ3020 (ETB-selective)-induced depressor response in pithed rats indicated a lack of activity at the endothelial ETB receptor. ZD1611 was orally active in the rat at 0.3 mg/kg and had a duration of action of more than 7 h, and, in the dog, a dose of 0.6 mg/kg p.o. was active for at least 6 h. In conclusion, these data demonstrate that ZD1611 is a potent and orally active, selective ETA receptor antagonist with a long duration of action which may be of therapeutic use.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Since
its discovery in 1988, endothelin (ET) 1 remains one of the most potent
vasoconstrictors yet described (Yanagisawa et al., 1988
). Originally
isolated from endothelial cells (Yanagisawa et al., 1988), ET-1 belongs
to a family of three isopeptides termed ET-1, ET-2, and ET-3 (Inoue et
al., 1989). ET-1 is the predominant isoform in the vasculature
and elicits a variety of actions including constriction, dilatation
(via the release of nitric oxide or prostacyclin), and cell
proliferation (Ohlstein and Douglas, 1993; see Masaki, 1995). The ETs
mediate their effects via two known receptor subtypes, the
ETA and the ETB receptor.
Both receptor subtypes have been sequenced and cloned in animals and
humans and belong to the seven- transmembrane, G protein-coupled
receptor superfamily (Arai et al., 1990; Sakurai et al., 1990). The
receptors may be distinguished pharmacologically, based on the rank
order of potency of the ET isoforms, at the ETA
receptor ET-1 
ET-2 
ET-3; and at the
ETB receptor, all three isopeptides are
equipotent (Sakurai et al., 1992).
Within the vasculature, ETA receptors have been
localized to smooth muscle cells where they mediate the constrictor and
mitogenic actions of ET (Sakurai et al., 1992; Ohlstein and Douglas,
1993). Constrictor ETB receptors have also been
identified on vascular smooth muscle of a number of animal species
(Ihara et al., 1991; Moreland et al., 1992). However, whereas mRNA for
ETB receptors has been identified in human
vascular smooth muscle (Davenport et al., 1995), the functional
relevance of this receptor remains unclear, and the
ETA receptor appears to predominate (Godfraind, 1993; Maguire and Davenport, 1995). ETB
receptors are also located on endothelial cells where they mediate
vasodilatation, via the release of nitric oxide and/or prostacyclin
(Warner et al., 1989).
There is now substantial evidence supporting a role for ET in the
pathogenesis of various acute and chronic cardiovascular disorders.
Raised ET plasma levels have been described in patients with
atherosclerosis (Lerman et al., 1991), pulmonary hypertension (Stewart
et al., 1991), chronic heart failure (Love and McMurray, 1997),
acute and chronic renal failure (Tomita et al., 1989; Stockenhuber et
al., 1992), and after myocardial infarction (Miyauchi et al., 1989) and subarachnoid hemorrhage (Suzuki et al., 1992). In
addition, ET receptor antagonists have beneficial effects in various
experimental models of these conditions (Miyauchi et al., 1993;
Patel et al., 1996).
If ET receptor antagonists are to be of therapeutic use in the
treatment of chronic disease, the development of orally active compounds would be advantageous. Ideally, such antagonists should block
the constrictor actions of ET while preserving the dilator effects;
i.e., be selective for the ETA receptor. In
addition, antagonist selectivity for the ETA
receptor is important given that the ETB receptor
has been implicated in the clearance of plasma ET-1 (Gasic et al.,
1992; Fukuroda et al., 1994). Here, we describe the pharmacological
profile of ZD1611
[3-{4-[3-(3-methoxy-5-methylpyrazin-2-ylsulfamoyl)-2-pyridyl]phenyl}-2,2-dimethylpropanoic acid] (Fig. 1), a novel, orally active,
selective ETA receptor antagonist. By using
established in vitro and in vivo methods, we have evaluated both the
potency and the duration of action of ZD1611 and demonstrated a lack of
activity at the ETB receptor.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Preparation of Cell Membranes.
The cDNA for human
ETA and ETB receptors were
obtained by polymerase chain reaction of a human kidney cDNA library
and a human placental cDNA template, respectively. The cDNAs were
subcloned and stably expressed in mouse erythroleukemic (MEL)-C88 cells according to previously described methods (Needham et al., 1992).
Radioligand Binding in Cell Membranes. MEL-C88 cell membranes containing either cloned human ETA or ETB receptors (1.5 µg of protein per assay well) were incubated with 30 pM 125I-labeled ET-1 in the presence of increasing concentrations of ZD1611 (100 pM to 10 µM), in a final incubation volume of 225 µl. Samples were incubated for 180 min at 30°C followed by filtration through Whatman (Maidstone, Kent, UK) GF/B filters with a Brandel (Bethesda, MD) cell harvester. 125I-labeled ET-1 binding was quantified by gamma counting. Nonspecific binding was defined by 125I-labeled ET-1 binding in the presence of a 3000-fold excess of unlabeled ET-1.
In Vitro Potency in Isolated Rat Aorta. Male Sprague-Dawley rats (300-350 g) were sacrificed by decapitation and exsanguination. The aorta was quickly removed and placed in oxygenated (95% O2/5% CO2) physiological salt solution (PSS) composed of 119 mM NaCl, 4.7 mM KCl, 1.6 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 22.6 mM NaHCO3, 5 mM dextrose, and 0.03 mM EDTA maintained at pH 7.4. The aortae were stripped of adherent fat and connective tissue. Subsequently, ring segments (2-mm long, 2-3 mm i.d.) were cut and denuded of endothelium by gentle abrasion of the intimal surface.
Isometric tension studies were carried out as described previously (Bialecki and Tulenko, 1989). Briefly, each aortic ring was mounted on
two stainless steel pins (130-µm radius) and submerged in a 5-ml
organ chamber containing PSS gassed continuously with 95%
O2/5% CO2 and maintained
at 37°C. The PSS was supplemented with 1 µM propranolol to prevent
potential activation of smooth muscle 
adrenoceptors by ET-induced
norepinephrine release from intramural neurons (Takimoto et al., 1993).
Vascular rings were placed under their predetermined optimal passive
load (approximately 3 g) and equilibrated for 30 min. Isometric
tension measurements were made with linear tension displacement transducers (Grass FT03) and recorded on a Grass model 7D polygraph.
After the equilibration period, tissues were exposed to 80 mM KCl to
assess viability. During the plateau phase of this contraction, denudation of vascular endothelium was confirmed by the lack of relaxation to 1 µM acetylcholine. The tissues were then washed until
the tension returned to baseline levels and equilibrated for another 60 min. Cumulative concentration-response curves (CRCs) to ET-1 were then
constructed in the absence or presence of increasing concentrations of
ZD1611. ZD1611 was added 60 min before the construction of the CRC to
ET-1. One CRC to ET-1 was constructed per preparation and paired
segments of rat aorta were studied for comparison. Each experiment was
terminated by the addition of 30 mM BaCl2, and
responses to ET-1 are expressed as a percentage of this reference contraction. Changes in isometric tension development, determined from
developed wall stress (g/cm2), were calculated
and analyzed with Bioreport software (Modular Instruments, Malvern, PA).
The concentration of ET-1 required for half the maximal contraction
(EC50) was determined from log-logit plots of
individual CRCs and expressed as the negative log of the molar
concentration (pD2). The concentration of
antagonist that caused a 2-fold rightward shift in the ET-1-response
curve (pA2) was used as an index of potency for a
competitive antagonist. Schild plots were constructed and
pA2 values were calculated by linear regression
analysis, if the slope did not differ from unity (Arunlakshana and
Schild, 1959). Data are presented as the mean ± S.E.
In Vivo Potency of i.v. ZD1611 in Pithed Rats. Male Alderley Park rats (280-330 g) were anesthetized with 2% halothane and artificially respired through a tracheal cannula. Rats were pithed by passing a needle (2 mm diameter) through the orbit, the foramen magnum, and down the spinal cord. The left femoral vein and right carotid artery were isolated, and heparin-filled catheters were implanted for the administration of compounds and the measurement of mean arterial pressure (MAP), respectively. Body temperature, measured rectally, was maintained at 38°C via a heated pad.
MAP and heart rate (derived from pulse pressure) were allowed to stabilize over a 10-min period, and a baseline reading was taken. Rats with an initial baseline MAP of <55 mm Hg or >70 mm Hg were excluded from the study. The precursor of ET-1, big ET-1, was used for in vivo analysis of the effects of ZD1611. Exogenously administered big ET-1 is converted to the biologically active peptide ET-1 in vivo (Hemsen et al., 1991) via a phosphoramidon-sensitive ET-converting enzyme (Fukuroda et
al., 1990). In the present study, the use of big ET-1 in vivo was
preferred because this compound fails to elicit the initial depressor
response associated with i.v. administered ET-1 (Yanagisawa et al.,
1988) and yielded a greater maximum response than that to ET-1 itself.
A partial cumulative dose-response curve to i.v. big ET-1 (starting at
0.3 nmol/kg) was constructed until pressor responses >30 mm Hg
were achieved. After a 55-min recovery period, ZD1611 (0.03-0.3 mg/kg)
or vehicle was administered, and the big ET-1-response curve was
repeated 5 min later. The activity of ZD1611 was calculated as a ratio
of the dose of big ET-1 required to give a 30-mm Hg rise in MAP in the
absence and then the presence of the compound.
Reversal of an Established Big ET-1 Pressor Response by i.v. ZD1611. To study the ability of ZD1611 to reverse big ET-1-induced pressor responses, big ET-1 was infused i.v. in pithed rats (as previously described), at 0.065 nmol/kg/min. Infusion was continued until a sustained increase in MAP of approximately 100 mm Hg was obtained. ZD1611 (0.03-1 mg/kg) or vehicle was then administered i.v. in a cumulative manner, in the presence of the big ET-1 infusion.
Activity of i.v. ZD1611 against BQ3020-Induced Depressor Responses. The selectivity of ZD1611 for the ETA-mediated pressor response over the ETB-mediated depressor response was investigated in pithed rats. Rats were pithed as previously described. In addition to left femoral vein and right carotid artery cannulation for compound administration and blood pressure/heart rate measurements, respectively, the right jugular vein was cannulated for noradrenaline infusion. Noradrenaline (3 µg/ml) was infused at an initial infusion rate of 30 µl/min, which was increased until MAP was stabilized at 140 to160 mm Hg. Two partial dose-response curves to the selective ETB receptor agonist BQ3020, separated by a 30-min recovery period, were constructed in the presence of the noradrenaline-induced pressor response. ZD1611 (1.0 mg/kg) or vehicle was administered i.v. 5 min before the second response curve.
In Vivo Potency of i.v. ZD1611 in Anesthetized Dogs. To investigate potency in more than one species, ZD1611 was also administered to dogs. Female beagle dogs (9-14 kg) were fasted overnight and predosed with acetylpromazine (ACP injection, 2 mg/ml) at a dose of 1 mg/dog administered s.c. The animals were subsequently anesthetized with i.v. sodium pentobarbitone (Sagatal, 60 mg/ml) at a dose of 45 mg/kg and anesthesia maintained with a constant infusion of 6 mg/kg/h. Animals were intubated using a 9- mm tracheal tube and artificially respired. The right jugular vein was cannulated with a dual lumen catheter for the infusion of anesthetic and administration of compounds, and the femoral artery for blood pressure measurements. Atenolol (3 mg/kg i.v.) was administered 10 min before vagotomization. After a 30-min stabilization period, a partial cumulative dose-response curve to i.v. big ET-1, with doses that elicited a pressor response >20 mm Hg (0.1-1 nmol/kg), was constructed. Following a 2-h recovery period, ZD1611 (0.1; 0.3 mg/kg) or vehicle was administered, and the big ET-1-response curve was repeated 20 min later. As before, the activity of ZD1611 was calculated as a ratio of the dose of big ET-1 required to give a 20-mm Hg rise in MAP in the absence and then the presence of the compound.
In Vivo Potency of p.o. Administered ZD1611 in Conscious Rats. Male Alderley Park rats (280-330 g) were anesthetized with i.v. Saffan (alfaxalone, 0.9%, w/v; alfadolone, 0.3%, w/v; 1:2.5 distilled water) and fitted with in-dwelling cannulas in the carotid artery and jugular vein. The catheters were externalized at the back of the neck and filled with heparin (5000 U/ml). During the recovery period, rats were housed individually with free access to food and water. Subsequently, the rats were fasted overnight with unlimited water available.
On the day of the experiment, the rats were placed in perspex housing and restrained. The arterial catheter was drained, refilled with heparinized saline (50 U/ml), and connected to a pressure transducer for measurement of MAP. Heart rate was derived from pulse pressure. After a 10-min stabilization period, a partial cumulative dose-response curve to i.v. big ET-1 was constructed until an increase in MAP of 30 mm Hg was achieved. The animals were then returned to their cages and allowed to recover for 2 h. ZD1611 (0.1-1.0 mg/kg p.o.) or vehicle was then administered p.o. during the recovery period, and the big ET-1 dose-response curve was repeated at time intervals of 0.5, 2, and 4 h after dosing. In separate studies, a dose of 1.5 mg/kg p.o. was administered and the big ET-1 response was measured at 0.5-, 7-, and 10-h time intervals. As before, the activity of ZD1611 was calculated as a ratio of the dose of big ET-1 required to give a 30-mm Hg rise in MAP in the absence and then the presence of the compound.In Vivo Potency of p.o. Administered ZD1611 in Dogs. In preliminary experiments with conscious dogs, reproducible pressor responses to big ET-1 were difficult to obtain. To investigate the oral activity of ZD1611 in dogs, it was necessary to administer ZD1611 to conscious dogs and then assess its activity under anesthesia. Female Alderley Park beagles (8-11 kg) were dosed p.o. with a 10-ml gelatin capsule containing either 0.6 mg/kg ZD1611 in solution or vehicle. After a rest period of 60 min, the dogs were anesthetized and prepared as described above. Partial dose-response curves to big ET-1 were constructed 3 and 6 h after oral dosing. Activity was assessed by comparing the big ET-1 ED20 values between drug- and vehicle-treated groups.
Materials. Big ET-1, ET-1, and BQ3020 were obtained from Cambridge Research Biochemicals (Cheshire, UK). Acetylcholine, atenolol, bacitracin, benzaminidine, BSA, noradrenaline, phenanthroline, and propranolol were purchased from Sigma Chemical Co. (Poole, UK or St. Louis, MO). Heparin was obtained from C.P. Pharmaceuticals (Wrexham, UK), and penicillin and streptomycin were purchased from Life Technologies (Paisley, UK). 125I-labeled ET-1 was purchased from Amersham International plc (Amersham Pharmacia Biotech, UK). Saffan was obtained from Pitman-Moore (Uxbridge, Middlesex, UK) Ltd. and Sagatal obtained from Rhone Merieux. Acetylpromazine was obtained from C-Vet (Leyland, Lancashire, UK). Stock solutions (100 µM) of ET-1 were made by dissolving in distilled H2O with 0.1% BSA (fraction V) and stored in aliquots at 4°C for <10 days. Working solutions were prepared daily as needed. ZD1611 was dissolved in dimethyl sulfoxide to give 100 µM stock solutions on the day required.
Statistical Analyses. In all cases n equals the number of individual animals studied. pIC50 and pD2 values (negative log of the IC50 and EC50, respectively) are given as arithmetic mean with S.E.M. Significant difference between the Schild regression slope and unity was tested using the Student's one-sample t test. Antagonist activity of ZD1611 in vivo was calculated as a ratio of the dose of big ET-1 required to give a 20- or 30-mm Hg rise in MAP in the absence and presence of the compound; ED20 and ED30, respectively. Mean dose ratios are expressed as geometric mean with 95% CLs. Differences between dose ratios were tested by using Students' t test for unpaired data. A p value < .05 was considered to be significant.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibition of 125I-Labeled ET-1 Binding by ZD1611 in
Human Cloned ET Receptors.
In competition-binding experiments with
human cloned ETA receptors, ZD1611 competed with
125I-labeled ET-1 in a monophasic manner with a
pIC50 value of 8.6 ± 0.1 (n = 5). ZD1611 was 1000-fold less potent at the human
ETB receptor with a pIC50
value of 5.6 ± 0.1 (n = 3) (Fig.
2). Slopes were not significantly
different from unity.
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Selectivity of ZD1611 for ET Receptors.
When tested at a
concentration of 10
4 M, with standard
ligand-binding assays, ZD1611 had no significant affinity for the
following receptors (derived from whole rat forebrain unless noted
otherwise): 
adrenoceptors (1A,
2A), adrenoceptors
(1, rat cortex; 2, rat cerebellum), muscarinic (M1, rat cortex;
M2, rat cardiac atrium), nicotinic,
-aminobutyric acid (GABAA),
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate,
N-methyl-D-aspartate, histamine
(H1), and 5-hydroxytryptamine (serotonin)
(5-HT1A, 5-HT2) receptors
(data not shown).
Effects of ZD1611 on Responses to ET-1 in Rat Aorta.
ET-1
potently contracted rat aorta segments with a mean
pD2 value of 8.8 ± 0.2. ZD1611 caused a
parallel rightward shift of the CRC to ET-1 without suppression of the
maximal response (Fig. 3). Schild
analysis yielded a pA2 value for ZD1611 of
7.5 ± 0.3 and a slope of 0.80 ± 0.20 (n 11; p = not significant compared to unity),
indicating competitive antagonism (Fig. 3).
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In Vivo Potency of ZD1611 in Rats. ZD1611 caused a dose-related rightward shift of the partial dose-response curve to big ET-1 in pithed rats. The threshold dose for significant antagonist activity was established as 0.1 mg/kg (i.v.). An additional effect was observed with 0.3 mg/kg, but this failed to reach statistical significance due to variability within the group (Table 1).
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In Vivo Potency of ZD1611 in Dogs. ZD1611 caused a dose-related rightward shift of the partial dose-response curve to big ET-1 in anesthetized dogs at 0.1 and 0.3 mg/kg, i.v. The shift at 0.3 mg/kg was significantly different from control (Table 2).
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Reversal of an Established Big ET-1 Pressor Response by
ZD1611.
An increase in MAP from a mean resting value of 64 ± 1 mm Hg to approximately 100 mm Hg was achieved and maintained
following infusion of big ET-1 (0.065 nmol/kg/min) in pithed rats.
ZD1611 (0.03-1.0 mg/kg i.v.) caused a dose-related reversal of the big ET-1 pressor response in the presence of a continuous big ET-1 infusion
(Fig. 4). The dose of ZD1611 required to
produce a 50% reversal of the big ET-1 response was 0.2 (95%
CLs = 0.1-0.3) mg/kg.
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Activity of ZD1611 against BQ3020-Induced Depressor Responses. The effect of ZD1611 on the depressor response to the selective ETB agonist BQ3020 was assessed in pithed rats. A small attenuation of the BQ3020 depressor response was observed on administration of vehicle, with a mean dose ratio of 2.3 (95% CLs = 1.7-3.1; n = 4). However, no additional, significant effect (p > .05) was obtained after the infusion of ZD1611 (1.0 mg/kg; mean dose ratio, 3.0; 95% CLs = 1.4-6.1; n = 4), indicating that ZD1611 was without activity at the endothelial ETB receptor.
Oral Activity of ZD1611 in Conscious Rats. ZD1611 caused dose- and time-dependent antagonism of the pressor response to big ET-1 in conscious rats. At 0.3 mg/kg p.o., ZD1611 was active for >4 h (Table 3 and Fig. 5), whereas at 1.5 mg/kg p.o., ZD1611 remained active for 7 h after administration (Fig. 5).
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Oral Activity of ZD1611 in Dogs. The effect of ZD1611 on the pressor response to big ET-1 was investigated at a single dose of 0.6 mg/kg p.o. ZD1611 was active for at least 6 h (Table 4). The dose ratios (ratio of doses of big ET-1 required to give a 20-mm Hg increase in MAP in dogs treated with either vehicle or ZD1611) were 4 and 2.2 at 3 and 6 h, respectively.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present study, we describe the novel, nonpeptide
ETA receptor antagonist ZD1611. ZD1611 has high
affinity for human cloned ETA receptors
(pIC50 = 8.6) and is 1000-fold selective for
human ETA receptors compared with human
ETB receptors (pIC50 = 5.6). The potency and selectivity of ZD1611 is similar to that reported
for peptide, e.g., FR139317 (IC50 at porcine
ETA = 0.53 nM and ETB = 4650 nM; Sogabe et al., 1993) and nonpeptide ET receptor antagonists, including SB217242 (Ki at
human ETA = 1.1 nM and at human
ETB = 111 nM; Ohlstein et al., 1996), BMS-182874
(Ki at human ETA = 48 nM and at human ETB = >50 µM; Webb et
al., 1995), and bosentan (Ki at human
ETA = 4.7 nM and at human
ETB = 95 nM; Clozel et al., 1994).
The ability of ZD1611 to antagonize ET-induced constrictor responses in
vitro was investigated by using the rat isolated aorta, a preparation
in which ETA receptor-mediated contractions to
ET-1 have been well characterized (Panek et al., 1992; Ohlstein et al.,
1994). ZD1611 caused a parallel rightward shift of the CRC to ET-1
without affecting the maximal response, indicating competitive antagonism. Schild analysis of the data yielded a
pA2 value of 7.5 ± 0.3. Consistent with the
binding data, this pA2 value is comparable to
those described for other nonpeptide ETA receptor antagonists such as PD156707 (pA2 = 7.5; Reynolds
et al., 1995) and BMS 182874 (KB = 520 nM; Webb et al., 1995), and nonselective antagonists including SB217242
(KB = 4.4 nM; Ohlstein et al., 1996)
and bosentan (pA2 = 7.2; Clozel et al., 1994),
although other compounds have higher potency in vitro, e.g., A-127722
(pA2 = 9.2; Opgenorth et al., 1996) and SB209670
(pA2 = 9.4; Ohlstein et al., 1994).
The selectivity and potency of ZD1611 observed in vitro was
paralleled in vivo. The pressor response to big ET-1 (which is converted to ET-1 in vivo) appears to be mediated mainly via the ETA receptor, although a small
ETB component has been inferred by some studies
(McMurdo et al., 1993; Clozel et al., 1994). ZD1611 effectively blocked
the pressor response to big ET-1 in pithed rats. Importantly, ZD1611
exhibited high in vivo potency in two animal species, the rat and the
dog, suggesting a nonspecies-dependent action. The threshold i.v. dose
of ZD1611 required to block the big ET-1 pressor response was
determined as 0.1 and 0.3 mg/kg in the rat and dog, respectively. In
contrast, the compound had no significant effect on the depressor
response induced by the selective ETB receptor
agonist BQ3020, which acts on endothelial ETB
receptors to release nitric oxide and/or prostacyclin (Warner et al.,
1989). In humans, ET-induced vasoconstriction is mediated predominantly
via the ETA receptor subtype (Godfraind, 1993;
Maguire and Davenport, 1995), and the mitogenic properties of ET-1 also appear to be mediated via the ETA receptor (see
Ohlstein and Douglas, 1993). Under conditions associated with raised ET
levels, the pharmacological profile of ZD1611 would allow for blockade
of the deleterious constrictor and mitogenic effects of ET without affecting its beneficial dilator action.
In many clinical situations ET receptor antagonists will be required to
reverse established vasoconstriction. Cumulative i.v. administration of
ZD1611 produced dose-dependent reversal of an established big
ET-1-induced pressor response in the pithed rat with a dose of 0.2 mg/kg being required to reverse the response by 50%. Given the
apparently slow dissociation of ET-1 from its binding sites (Hirata et
al., 1988; Devadason and Henry, 1997), the mechanism(s) involved in
this response is unclear. However, ET receptor internalization,
recycling, and subsequent externalization (Marsault et al., 1993) may
allow ZD1611 to compete with ET-1 and reverse established and sustained contractions.
An essential characteristic of any compound used for the treatment of chronic disease is good oral potency and duration of action. When administered p.o., ZD1611 caused concentration-dependent blockade of the pressor response to big ET-1 with effective threshold doses of 0.3 and 0.6 mg/kg in conscious rats and dogs, respectively. These doses were only slightly higher than those required for activity via the i.v. route, indicating good oral bioavailability. ZD1611 was active for more than 4 h at a dose of 0.3 mg/kg p.o. and more than 7 h at 1.5 mg/kg p.o. in conscious rats. In dogs, ZD1611 was active at a dose of 0.6 mg/kg p.o. for at least 6 h. ZD1611 therefore has a long duration of action when administered p.o., which may allow an infrequent dosing regimen in humans.
In summary, ZD1611 is a selective and potent ETA receptor antagonist with functional activity. ZD1611 potently inhibits ETA-mediated, ET-1-induced vasoconstriction both in vitro and in vivo but has no action at the dilator ETB receptor. In addition, ZD1611 is effective in reversing established ET-1-induced constriction, a profile which may be of clinical importance. ZD1611 appears to be highly bioavailable when administered p.o. and has a long duration of action. These data suggest that ZD1611 may be of therapeutic utility in the treatment of acute or chronic conditions associated with raised levels of ET.
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Acknowledgments |
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We acknowledge the technical expertise of Kevin Rogers in the synthesis of ZD1611 and the valuable advice provided by Dr. Les Hughes.
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Footnotes |
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Accepted for publication April 30, 1999.
Received for publication December 18, 1998.
1 From Zeneca Pharmaceuticals, Wilmington, Delaware.
Send reprint requests to: Dr. C. Wilson, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, SK10 4TG, UK.
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Abbreviations |
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ET, endothelin; CRC, concentration-response curve; ED20, effective dose required to produce an increase in mean arterial pressure of 20 mm Hg; ED30, effective dose required to produce an increase in mean arterial pressure of 30 mm Hg; MAP, mean arterial pressure; MEL, mouse erythroleukemic; PSS, physiological salt solution.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A promising therapeutic target?
Heart
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93-94.
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), 0.3 (
), 1 (
),
and 3 (
) µM on concentration-response curves to ET-1 (
) in rat
isolated aorta. Values are arithmetic means with S.E.M. and are
expressed as a percentage of the maximum response to 30 mM barium
chloride (n 
