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Characterization of Angiotensin II Antagonism Displayed by SK-1080, a Novel Nonpeptide AT1-Receptor Antagonist

Lee, Sung Ho; Jung, Yi Sook; Lee, Byung Ho; Yun, Sun Il; Yoo, Sung Eun*; Shin, Hwa Sup

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Journal of Cardiovascular Pharmacology: March 1999 - Volume 33 - Issue 3 - p 367-374
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Abstract

The pivotal role of the renin-angiotensin system in the regulation of blood pressure has been well established (1). Many attempts have been made to reduce the contribution of angiotensin II to the development of high blood pressure by inhibiting the synthesis of angiotensin II by renin or angiotensin-converting enzyme (ACE). Renin inhibitors still have not been well developed as a new type of therapeutic agent because of their low bioavailability and fast degradation in the liver (2). On the other hand, several ACE inhibitors have proven to be clinically effective in the treatment of hypertension and congestive heart failure, with good oral activity and long duration of action (3,4). However, some evidence suggested that their unwanted side effects, such as dry cough and angioedema, result from the lack of specificity of ACE for angiotensin I (5-7). Furthermore, it was reported that in humans, angiotensin II can also be formed by a chymotrypsin-like proteinase, which is not affected by ACE inhibitors (8,9). Thus these problems prompted many studies to be conducted for the development of more useful drugs that block the action of angiotensin II directly and more completely without the undesirable side effects of ACE inhibitors. The discovery of losartan, a competitive AT1-receptor antagonist (10,11) without partial agonistic activity (12) opened a new era for the development of new selective, orally active angiotensin II-receptor antagonists as novel antihypertensive agents with the property of direct and more specific interruption of the angiotensin II receptor itself. Currently losartan is on the market as the first angiotensin II-receptor antagonist that was launched as a novel antihypertensive agent since proven to be orally active in animals (12-14) and humans (15-17).

SK-1080 (KR-31080; 2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) belongs to a novel class of nonpeptide angiotensin II-receptor antagonists with a high affinity for AT1 receptor (Fig. 1). SK-1080 is structurally different from losartan and its series of imidazole analogues in having pyridylimidazole and pyridine N-oxide moieties. In this study, we characterized the pharmacologic properties of SK-1080 as an angiotensin II-receptor antagonist in comparison with losartan by examining its antagonistic effects on the binding of [125I]-[Sar1, Ile8]-angiotensin II and [125I] CGP 42112A to human recombinant AT1- and AT2-receptor subtypes, on the angiotensin II-induced contraction of rabbit and rat aortic segments, and on the angiotensin II-induced pressor response in pithed rats.

FIG. 1
FIG. 1:
Chemical structure of SK-1080.

METHODS

Radioligand binding assay

For the competition and equilibrium binding studies on AT1 and AT2 receptors, human recombinant AT1 and AT2 receptors (BioSignal Inc., Montreal, Canada) were used to exclude the possible interaction between drugs and other receptors in various tissues. Binding assays were performed in 96-well plates by incubating aliquots of the human recombinant AT1 and AT2 receptor (BioSignal Inc.) with 0.21 nM [125I]-[Sar1, Ile8]-angiotensin II, and 0.5 nM [125I]-CGP 42112A, respectively. From the preliminary experiments with those concentrations of radioligands, the binding parameters were well fit to the control data provided by the receptor vendor (data not shown). Test compounds were dissolved at 2.5 mM in dimethyl sulfoxide and serially diluted to 10 concentrations for the evaluation of activity in the total assay volume of 250 μl. The assay buffer contained 50 mM Tris, 5 mM MgCl2, 1 mM EDTA, and 0.1% bovine serum albumin (pH 7.4). Specific [125I]-[Sar1, Ile8]-angiotensin II and [125I]-CGP 42112A binding was determined experimentally from the difference between counts in the absence and the presence of unlabeled angiotensin II and [Sar1, Ile8]-angiotensin II at the concentration of 10 μM, respectively. After incubation at 37°C for 60 min (or 180 min for AT2 receptor), the incubation mixtures were filtered through GF/C glass-fiber filters (Wallac, Turku, Finland), which were presoaked in 0.3% polyethylenimine and rapidly washed 9 times with 200 μl of ice-cold 50 mM Tris buffer (pH 7.4) by using the Inotech harvester (Inotech, Dottikoh, Switzerland). The filters were covered with MeltiLex (melted on scintillator; Wallac), sealed in sample bag, followed by drying in the microwave oven, and counted by MicroBeta (Wallac). The assays were performed in three separate experiments run in quadruplicate.

The ability of antagonists to inhibit specific [125I]-[Sar1, Ile8]-angiotensin II and [125I]-CGP 42112A binding was estimated by IC50 values, which are the molar concentrations of unlabeled drugs necessary to displace 50% of specific binding. The Ki value was calculated from the equation Ki = IC50/(1 + L/Kd), where L equals the concentration of [125I]-[Sar1, Ile8]-angiotensin II or [125I]-CGP 42112A (18). The data from binding experiments were analyzed by nonlinear regression, by using the PRISM computer program (GraphPad Software Inc., San Diego, CA, U.S.A.).

In vitro potency in rat and rabbit aorta

This study conformed with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health. The descending thoracic aorta was isolated from male Sprague-Dawley rats (350-450 g; Korea Research Institute of Chemical Technology, KRICT, Taejon, Korea) and male New Zealand White rabbits (2-3 kg; Samyook Experimental Animal Co., Suwon, Korea), respectively. The endothelial layer of aorta was destroyed by gentle rubbing of the luminal surface with a cotton swab moistened with Krebs solution. The aorta was cut into ring segments of 3-4 mm in width, and the vascular rings were mounted in 20-ml organ baths containing Krebs bicarbonate buffer of the following composition (in mM): NaCl, 118; KCl, 4.7; CaCl2, 2.5; NaHCO3, 25; MgSO4, 1.2; KH2PO4, 1.2; and glucose, 11.0. The Krebs buffer was kept at pH 7.4 by continuous bubbling with a gas mixture (95% O2/5% CO2) at 37°C. The isometric contraction was recorded with force-displacement transducers (Grass FT03; Grass Instruments, Quincy, MA, U.S.A.) and displayed on a chart recorder (Multicorder MC 6625; Hugo Sachs Electronic, March, Freiburg, Germany).

In experiments with rat aorta, a resting tension of 2 g was applied, and the rings were allowed to equilibrate for 60 min. After each ring was treated for 30 min with a single dose of SK-1080 (10−9, 3 × 10−9, 10−8M), losartan (10−7, 3 × 10−7, 10−6M), or vehicle (0.1% dimethyl sulfoxide), a cumulative concentration-contractile response curve for angiotensin II (10−10-10−5M) was determined in the same ring. In each ring, only one full dose-response curve was obtained to avoid the tachyphylactic response to angiotensin II. The aortic rings were rinsed several times and allowed to return to baseline tension. Then the rings were contracted with high-K+ solution (an equimolar concentration of NaCl was substituted with KCl) to obtain a reference contractile response, as a percentage of which all the responses from rat aorta were expressed.

In experiments with rabbit aorta, the rings were allowed to equilibrate for 90 min under a resting tension of 2 g. The first control cumulative concentration-contractile response curve for angiotensin II (10−10-10−5M) was determined to ensure stable reactivity to subsequently added angiotensin II. Then the tissue was washed 3 times until baseline tension was recovered. After each ring was treated for 30 min with a single dose of SK-1080 (3 × 10−9, 10−8, 3 × 10−8M), losartan (10−7, 3 × 10−7, 10−6M), or vehicle (0.1% dimethyl sulfoxide), the second cumulative concentration-contractile response curve for angiotensin II was established. To exclude any influence of multiple dosing with SK-1080 and losartan on the concentration-contractile response curve, each tissue was incubated only with one concentration of the antagonist. Responses from rabbit aorta were expressed as percentage of the maximal angiotensin II response obtained from the first cumulative concentration-response curve. The pA2 values were determined according to the Schild equation (19) with pKB values being calculated from the equation of (antagonist)/(dose ratio - 1).

To test the specificity of SK-1080 as an angiotensin II-receptor antagonist, the concentration-contractile responses to norepinephrine, KCl, serotonin, and histamine also were examined in the endothelium-removed rabbit aorta in the presence and the absence of SK-1080 at 10 μM. Responses in this study were expressed as percentage of the maximal response obtained from the first cumulative concentration-response curve.

In vivo potency and specificity as angiotensin II antagonist in pithed rats

Male Sprague-Dawley rats (350-450 g; KRICT) were anesthetized with sodium pentobarbital (35 mg/kg, i.p.). After a tracheotomy was performed, artificial ventilation with room air was initiated with rodent ventilator (model 7025; Ugo Basile, Varese, Italy; frequency, 60 cycles/min; stroke volume, 1 ml/100 g body weight). Two polyethylene (PE-50) catheters connected to PE-10 catheters that were filled with heparinized saline solution (20 IU/ml) were inserted into the left femoral artery and vein for recording arterial blood pressure and drug administration, respectively. The arterial catheter was connected to an Isotec pressure transducer (Healthdyne, Marietta, GA, U.S.A.) coupled to a Graphtec Linearcorder (model 3310; Graphtec Corp., Tokyo, Japan). Heart rate was derived from the arterial pulse pressure via a cardiotachometer (type 576; Graphtec). Subsequently the animals were pithed by inserting and driving a steel rod (2 mm in diameter) via the orbit and the foramen magnum down into the whole length of the spinal canal (20). The animals were kept warm at 37°C by means of a thermostat-controlled heating pad. Arterial blood pressure and heart rate were continuously recorded through the whole experiment.

Forty minutes after surgery, when consistent control values for blood pressure and heart rate were possible to obtain, the experiment was commenced. To construct the dose-pressor response curve for angiotensin II, angiotensin II (0.01-1,000 μg/kg/0.1 ml, i.v.) was injected cumulatively, with each successive injection given immediately after the maximal effect of the preceding dose was reached (10-20 s). After each injection, the catheter was flushed with 0.2 ml of saline. Only one full dose-response curve was obtained in each rat. Fifteen minutes before injection of angiotensin II, the animal was pretreated with a single i.v. dose of SK-1080 (0.03, 0.1, and 0.3 mg/kg), losartan (1, 3, and 10 mg/kg), and vehicle (0.05N KOH, 1 ml/kg). A similar protocol also was carried out with norepinephrine, vasopressin, and isoproterenol to determine specificity of SK-1080. Full dose-pressor response curves for norepinephrine (0.01-100 μg/kg, i.v.) and vasopressin (0.01-100 IU/kg, i.v.) and a full dose-heart rate response curve for isoproterenol (0.001-1 μg/kg, i.v.) were determined in pithed rats pretreated with SK-1080 (0.3 mg/kg, i.v.) or vehicle (0.05N KOH, 1 ml/kg). The results were expressed as mm Hg of diastolic arterial blood pressure. The doses (ID50) of compounds that inhibited by 50% the pressor response to angiotensin II (10 μg/kg, i.v.) were calculated by linear regression as an indirect measure of antagonism.

Chemicals

SK-1080, L-158809, PD-123177, and losartan were synthesized at the Bio-Organic Science Division, KRICT. Sodium pentobarbital was purchased from Hanlim Pharm. Co. (Seoul, Korea), and ketamine hydrochloride from Yuhan Co. (Seoul, Korea). [Sar1, Ile8]-angiotensin II, angiotensin II acetate, arterenol bitartrate, vasopressin acetate, and isoproterenol hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [125I]-[Sar1, Ile8]-angiotensin II and [125I]-CGP 42112A (2,200 Ci/mmol) were obtained from NEN Life Science Products (Boston, MA, U.S.A.). SK-1080 and losartan were dissolved in dimethyl sulfoxide and further diluted in the buffer for angiotensin II-binding assay and for isolated tissue experiments, and dissolved in 0.05N KOH in saline for intravenous administration in rats. All chemicals were prepared just before use.

Statistical analysis

All values are expressed as mean ± SEM. Data were analyzed by Student's t test or one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons (Sigma Stat; Jandel Co., San Rafael, CA, U.S.A.). In all comparisons, the difference was considered to be statistically significant at p < 0.05.

RESULTS

Radioligand binding assay

Against the human recombinant AT1 receptor, [125I]-[Sar1, Ile8]-angiotensin II interacted with a single population of binding sites with the dissociation constant (Kd) of 0.24 ± 0.01 nM. The corresponding number of binding sites labeled by the radioligand was 46.3 ± 0.7 fmol/mg of protein. SK-1080, losartan, L-158809, and PD-123177 competed dose-dependently with 0.21 nM [125I]-[Sar1, Ile8]-angiotensin II against the binding sites of the human recombinant AT1, where they appeared to exhibit monophasic inhibition curves (Fig. 2). SK-1080 displayed high specific affinity for the human recombinant AT1 receptor (IC50, 1.01 ± 0.12 nM) without any binding affinity for the human recombinant AT2 subtype, against which PD-123177 showed moderate activity (IC50, 4.3 ± 1.4 μM). SK-1080 was ∼12 times more potent than losartan (IC50, 12.30 ± 1.42 nM) and equipotent with L-158809 (IC50, 1.44 ± 0.34 nM) in displacing labeled angiotensin II from the human recombinant AT1 receptor. The Hill coefficients for the inhibition by SK-1080, losartan, and L-158809 were 0.96, 0.86, and 0.99, respectively, which were not significantly different from unity. The results from saturation-binding assay using [125I]-[Sar1, Ile8]-angiotensin II conducted in the presence of SK-1080 (1 nM) and losartan (10 nM) were depicted in Fig. 3. The Scatchard transformations of [125I]-[Sar1, Ile8]-angiotensin II saturation curves revealed that these two antagonists did not affect the total number of binding sites labeled by [125I]-[Sar1, Ile8]-angiotensin II, but increased the dissociation constant of the radioligand by a factor of 1.74 ± 0.20 with SK-1080 and 1.37 ± 0.15 with losartan. Saturation-binding assay also showed their competitive interaction with the receptor.

FIG. 2
FIG. 2:
Inhibition of specific [125I]-[Sar1, Ile8]-angiotensin II binding to human recombinant AT1 (A) and of specific [125I]-CGP 42112A binding to human recombinant AT2 receptor (B) by SK-1080 (open circles), losartan (solid circles), L-158809 (open triangles), and PD-123177 (solid triangles), respectively. The dose-response curve for the inhibition of specific binding by these compounds was determined by incubating the radioligand with 10 concentrations of each compound in the medium of receptor source. The data points represent the mean ± SEM of three separate experiments run in quadruplicate.
FIG. 3
FIG. 3:
Scatchard transformations of saturation-binding data for specific [125I]-[Sar1, Ile8]-angiotensin II binding to human recombinant AT1 receptor in the absence (open circles) or presence of SK-1080 (1 nM, solid circles) and losartan (10 nM, open triangles). The data points represent the mean of three separate experiments run in quadruplicate.

In vitro potency in rat and rabbit aorta

SK-1080 and losartan inhibited the angiotensin II-induced contractions of the rat aorta in a concentration-dependent manner (Fig. 4), but with dissimilar types of antagonism. SK-1080 (10−9, 3 × 10−9, and 10−8M) produced a rightward shift in the concentration-contractile response curve to angiotensin II with a significant reduction in the maximal contractile response by 28.3, 38.8, and 31.0% at each concentration, respectively (the calculated pKB, 9.97; Fig. 4A). In contrast, losartan (10−7, 3 × 10−7, and 10−6M) produced a parallel rightward shift in the concentration-response curve without any changes in the maximal contractile response (pA2 value, 8.02; slope of the Schild plot, 1.03; Fig. 4C).

FIG. 4
FIG. 4:
Effects of SK-1080 (top) and losartan (bottom) on the concentration-contractile response curve to angiotensin II in isolated rat aorta (A, C) and rabbit aorta (B, D). SK-1080: Vehicle (open circles), 10−9 M (solid circles), 3 × 10−9 M (open triangles), 10−8 M (solid triangles), and 3 × 10−8 M (open squares). Losartan: Vehicle (open circles), 10−7 M (solid circles), 3 × 10−7 M (open triangles), and 10−6 M (solid triangles). The data points represent the mean percentage of the maximal response ± SEM (n = 4-8).

The antagonistic pattern of SK-1080 and losartan with the rabbit aorta were similar to those with rat aorta. Thus SK-1080 (3 × 10−9, 10−8, and 3 × 10−8M) produced a rightward shift in the concentration-contractile response curve to angiotensin II with a significant reduction in the maximal contractile response by 19.1, 15.4, and 25.3% at each concentration, respectively (the calculated pKB, 9.51; Fig. 4B). In contrast, losartan (10−7, 3 × 10−7, and 10−6M) produced a parallel rightward shift in the concentration-response curve without any changes in the maximal contractile response (pA2 value, 7.59; slope of the Schild plot, 1.34; Fig. 4D). At the higher concentration (10 μM), SK-1080 did not change the concentration-response curve to norepinephrine, KCl, serotonin, and histamine in rabbit aortic preparations (Fig. 5).

FIG. 5
FIG. 5:
Effects of vehicle (open circles) and SK-1080 (10−5 M, solid circles) on the concentration-contractile response curve to norepinephrine (A), potassium chloride (B), serotonin (C), and histamine (D) in isolated rabbit aorta. The data points represent the mean percentage of the maximal response ± SEM (n = 4-5).

In vivo potency and specificity of angiotensin II antagonist in pithed rats

In pithed rats treated with vehicle (control group) under the experimental conditions used, the mean diastolic arterial pressure and heart rate were 30.9 ± 4.8 mm Hg and 302.1 ± 8.8 beats/min, respectively. The baseline values for diastolic arterial pressure and heart rate were similar in all groups of pithed rats. Cumulatively administered angiotensin II induced a gradual increase in diastolic arterial pressure with dose (Emax, 112.0 ± 7.5 mm Hg; ED50, 0.68 ± 0.05 μg/kg; Fig. 6A). The pretreatment with SK-1080 (0.03, 0.1, and 0.3 mg/kg, i.v.) did not significantly affect diastolic arterial pressure (32.0 ± 4.1, 30.5 ± 2.0, and 27.5 ± 2.1 mm Hg, respectively). However, SK-1080 not only caused a dose-dependent rightward shift in the dose-pressor response curve to angiotensin II with an ID50 value of 0.07 mg/kg, but also significantly decreased the maximal pressor response to angiotensin II (Emax, 86.5 ± 8.5, 56.7 ± 4.4, and 26.3 ± 6.7 mm Hg at 0.03, 0.1, and 0.3 mg/kg, respectively). Losartan (1.0, 3.0, and 10.0 mg/kg, i.v.) slightly reduced diastolic arterial pressure (33.9 ± 3.2, 30.4 ± 1.3, and 23.0 ± 1.4 mm Hg, respectively). Losartan dose-dependently shifted to the right the dose-pressor response curve to angiotensin II in a parallel manner, with ID50 value of 1.74 mg/kg, but without any change in the maximal response to angiotensin II, unlike SK-1080 (Fig. 6B). At a dose of 0.3 mg/kg, i.v., SK-1080 did not alter the dose-response curves to norepinephrine, vasopressin, and isoproterenol (Fig. 7).

FIG. 6
FIG. 6:
Effects of intravenously administered SK-1080 (A) and losartan (B) on the log dose-pressor response curve to angiotensin II in anesthetized pithed rat. SK-1080: Vehicle (open circles), 0.03 (solid circles), 0.1 (open triangles), and 0.3 mg/kg (solid triangles). Losartan: Vehicle (open circles), 1.0 (solid circles), 3.0 (open triangles), and 10.0 mg/kg (solid triangles). The data points represent the mean ± SEM (n = 6-9).
FIG. 7
FIG. 7:
Effects of intravenously administered vehicle (open circles) and SK-1080 at 0.3 mg/kg (solid circles) on the log dose-pressor response curve to norepinephrine (A) and vasopressin (B) and on the log dose-tachycardic response curve to isoproterenol (C) in anesthetized pithed rat. The data points represent the mean ± SEM (n = 5-6).

DISCUSSION

The results of this study with various experimental models showed that SK-1080 containing pyridylimidazole and pyridine N-oxide moieties is a potent AT1-selective antagonist in vitro and in vivo. In radioligand-binding studies, SK-1080 totally displaced specifically bound [125I]-[Sar1, Ile8]-angiotensin II from human recombinant angiotensin AT1 receptor with 12 times greater potency than losartan, but without interaction with human recombinant angiotensin AT2 receptor, from which PD 123177, a AT2-selective antagonist, displaced specifically bound [125I]-CGP 42112A. The analysis of the competition curve showing characteristics of monophasic inhibition indicated binding of SK-1080 to a single class of AT1 receptors with a Hill coefficient of 0.96. In further studies with radioligand-saturation experiments, SK-1080 caused an increase in dissociation constant of [125I]-[Sar1, Ile8]-angiotensin II without reduction in the maximal binding capacity (Bmax) of human recombinant AT1 receptor. All these data provide strong evidence that SK-1080 competitively interacts with AT1 receptors, as for losartan.

Several functional in vitro and in vivo studies were performed to characterize the mode of interaction of SK-1080 with AT1 and AT2 receptors. Unlike losartan, which exerted a parallel rightward shift in the concentration-response curve without any changes in the maximal contractile response in rat and rabbit aorta, SK-1080 caused a rightward shift in the concentration-response curve to angiotensin II with a reduction of maximal contractile response by 20-25%, suggesting an insurmountable antagonism of angiotensin II-induced contraction. This unusual pharmacologic behavior exerted by SK-1080 was also reported previously for other nonpeptide AT1 antagonists such as BIBR 277, GR 138950, or EXP3174 (21,22). This phenomenon of insurmountable antagonism by SK-1080 also was reproduced in the anesthetized pithed rat. That is, SK-1080 caused a rightward shift in the dose-pressor response curve to angiotensin II with a dose-dependent reduction in the maximal pressor response to angiotensin II. By contrast, losartan produced a rightward parallel shift in dose-pressor response curve to angiotensin II without reduction in the maximal response in pithed rat, as reported by others (12,23). Several hypothetical mechanisms proposed to explain insurmountable antagonism exhibited by AT1-receptor antagonists include the action on multiple receptors, a slow dissociation of the receptor-antagonist complex, and allosteric modification of receptors (10,24). At present, however, it remains unclear how an insurmountable antagonism can be displayed in isolated vessels and in vivo by nonpeptide AT1-receptor antagonists, including SK-1080, which showed competitive antagonism in the binding study. The insurmountable antagonism may not be limited to a single mechanism and may be influenced by factors such as the agonist/antagonist used, tissues, species, and experimental conditions, as suggested by Bond et al. (25).

As in the binding assay, SK-1080 was shown to be significantly more potent than losartan in other experiments: >50-fold in blocking the contractile effect of angiotensin II in rat and rabbit aorta (on the basis of calculated pKB) and ∼25-fold in blocking the pressor effect of angiotensin II in the pithed rat. The selective and specific interaction of SK-1080 with angiotensin II receptors was further substantiated by the results from the functional experiments demonstrating no effects of SK-1080 on the pressor response to norepinephrine and vasopressin, and on the heart-rate response to isoproterenol in pithed rat, and its minimal effects on the contractile response to norepinephrine, KCl, and 5-HT in the isolated rabbit aorta.

In summary, the results from this study with various binding and functional experiments demonstrate that SK-1080 is a highly potent selective nonpeptide AT1-receptor antagonist with a mode of insurmountable antagonism. Thus further studies are needed to evaluate the therapeutic potential of this compound.

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Keywords:

SK-1080; KR-31080; Angiotensin; AT1-receptor antagonist

© 1999 Lippincott Williams & Wilkins, Inc.