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Circulation. 1996;93:1860-1870

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(Circulation. 1996;93:1860-1870.)
© 1996 American Heart Association, Inc.

Articles

Systemic Endothelin Receptor Blockade Decreases Peripheral Vascular Resistance and Blood Pressure in Humans

William G. Haynes, BSc, MRCP, MD; Charles J. Ferro, BSc, MRCP; Kevin P. J. O'Kane, BSc, MRCP; David Somerville, RGN; Charmaine C. Lomax, RGN; David J. Webb, MD, FRCP, FFPM

From the Department of Medicine, University of Edinburgh, Western General Hospital, UK.

Correspondence to Professor David J. Webb, University Department of Medicine, Western General Hospital, Edinburgh EH4 2XU, UK. E-mail d.j.webb@ed.ac.uk.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
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Background Although local inhibition of the generation or actions of endothelin-1 has been shown to cause forearm vasodilatation, the systemic effects of endothelin receptor blockade in healthy humans are unknown. We therefore investigated the cardiovascular effects of a potent peptide endothelin ETA/B receptor antagonist, TAK-044, in healthy men.

Methods and Results Two randomized, placebo-controlled, crossover studies were performed. In nine subjects, TAK-044 (10 to 1000 mg IV over a 15-minute period) caused sustained dose-dependent peripheral vasodilatation and hypotension. Four hours after infusion of the highest dose (1000 mg), there were decreases in mean arterial pressure of 18 mm Hg and total peripheral resistance of 665 AU and increases in heart rate of 8 bpm and cardiac index of 0.9 L·min-1·m-2 compared with placebo. TAK-044 caused a rapid, dose-dependent increase in plasma immunoreactive endothelin (from 3.3 to 35.7 pg/mL within 30 minutes after 1000 mg). In a second study in eight subjects, intravenous administration of TAK-044 at doses of 30, 250, and 750 mg also caused peripheral vasodilatation, and all three doses abolished local forearm vasoconstriction to brachial artery infusion of endothelin-1. Brachial artery infusion of TAK-044 caused local forearm vasodilatation.

Conclusions The endothelin ETA/B receptor antagonist TAK-044 decreases peripheral vascular resistance and, to a lesser extent, blood pressure; increases circulating endothelin concentrations; and blocks forearm vasoconstriction to exogenous endothelin-1. These results suggest that endogenous generation of endothelin-1 plays a fundamental physiological role in maintenance of peripheral vascular tone and blood pressure. The vasodilator properties of endothelin receptor antagonists may prove valuable therapeutically.


Key Words: endothelin • receptors • vasculature • blood pressure • drugs • hemodynamics


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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The endothelins are a family of three isopeptides with extremely potent and characteristically sustained vasoconstrictor and pressor actions.1 They also have mitogenic and neuroendocrine properties that act to increase blood pressure.2 Endothelin-1 is the predominant isopeptide in the vascular endothelium3 and is therefore likely to be the most physiologically relevant of the three isopeptides in regulation of vascular tone. Endothelin-1 is generated from a precursor, big endothelin-1, through the action of a unique neutral metalloprotease, ECE.4 Two distinct endothelin receptors have been identified and characterized.5 6 The ETA receptor has a high affinity for endothelin-1 and is selectively expressed in vascular smooth muscle cells.5 The ETB receptor has equal affinity for all three endothelins. Although vascular expression of the ETB receptor has been thought to be limited to vascular endothelial cells,6 recent evidence suggests that ETB receptors are expressed in vascular smooth muscle cells7 and are functionally active.8 9

The physiological relevance of endogenous generation of endothelin-1 in control of blood pressure has been unclear. If basal generation of endothelin-1 contributes to resistance-vessel tone, then drugs that inhibit the generation or actions of endothelin would be expected to cause vasodilatation and decrease blood pressure and might have potential therapeutic value in diseases associated with sustained peripheral vasoconstriction, such as hypertension and chronic heart failure. However, results of animal studies using ECE inhibitors and endothelin receptor antagonists have been contradictory. Some have shown no apparent effect of antiendothelin therapy on blood pressure in normotensive animals.10 11 12 13 14 15 However, most of these studies had not been designed primarily to test this hypothesis, with the result that they may have lacked statistical power to confidently exclude a hypotensive effect. In addition, in some, blood pressure was not measured for sufficient time after dosing to detect the expected slow-onset hypotensive effect of antiendothelin therapy. Other studies have shown that endothelin blockade apparently reduces blood pressure significantly only in hypertensive animals,16 17 leading to suggestions that endothelin-1 has a pathological rather than a physiological role. However, there were similar percentage decreases in blood pressure in normotensive and hypertensive animals in these and other studies,16 17 18 suggesting that endothelin plays a similar role in hypertensive and normotensive animals. The lack of a significant effect of antiendothelin therapy on blood pressure in normotensive animals may be due to the relative imprecision of measurement of small changes in blood pressure. Other studies have shown that ECE inhibitors and endothelin receptor antagonists do decrease blood pressure in normotensive animals.18 19 20 21 22 These positive studies have usually examined hemodynamic responses for several hours after drug administration, thereby taking into account the known slow reversal of endothelin-1–induced vasoconstriction by antiendothelin therapy.23

In the first such study in humans, we recently demonstrated that brachial artery infusion of the ECE inhibitor phosphoramidon and the ETA receptor antagonist BQ-123 causes progressive forearm vasodilatation.24 These effects of phosphoramidon and BQ-123 on forearm blood flow indicate a physiological role for basal generation of endothelin-1 in maintenance of vascular tone. However, homeostatic mechanisms often obscure the blood pressure effects of quite large changes in resistance vessel tone, with the result that changes in blood pressure may be quite small in relation to the effects of a drug on peripheral resistance.25 Thus, the magnitude of any potential effect of an endothelin antagonist on blood pressure is difficult to predict without systemic administration. As far as we are aware, the effects of systemic endothelin receptor blockade on hemodynamics in healthy human subjects have not previously been reported.

Therefore, we examined the hemodynamic effects of systemic administration of a potent combined ETA and ETB receptor peptide antagonist, TAK-044,26 27 28 29 in healthy male subjects. Because animal data show that endothelin receptor antagonists increase circulating endothelin concentrations,30 31 we also measured plasma immunoreactive endothelin concentrations. In addition, in a second study, we investigated whether systemic pretreatment with TAK-044 blocked forearm vasoconstriction to brachial artery infusion of endothelin-1 and whether local administration of TAK-044 caused direct vasodilatation of forearm resistance vessels.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Subjects
Eighteen healthy male subjects between 21 and 60 years of age and within 15% of ideal body weight were recruited. Studies were conducted with the approval of the Lothian Healthy Volunteer Research Ethics Subcommittee and the Inveresk Clinical Research Ethics Committee and with the written, informed consent of each subject. No subject received vasoactive or nonsteroidal medication in the week before or during the study. In addition, subjects abstained from alcohol for 48 hours, from caffeine-containing drinks and cigarettes for at least 24 hours, and from food for at least 10 hours before any measurements were made.

Drugs
TAK-044 is a cyclic hexapeptide (cyclo[D-{alpha}-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]-L-alanyl-L-{alpha}-aspartyl-D-2-(2-thienyl)glycyl-L-leucyl-D-tryptophyl] disodium salt; molecular weight, 972) that potently antagonizes 125I–endothelin-1 binding at both ETA (IC50=0.08 nmol/L) and ETB (IC50=120 nmol/L) receptors in vitro.26 TAK-044 also blocks constriction of isolated coronary vessels to endothelin-1 (ETA and ETB receptor agonist) and sarafotoxin S6c (ETB receptor agonist).26 Specificity of TAK-044 for endothelin receptors has been shown in vitro in porcine coronary arteries, in which it does not affect vasoconstriction to histamine, serotonin, acetylcholine, U-46619, and potassium and in which it is without direct vasoactive effects even at concentrations of 100 µmol/L.29 Specificity has also been shown in vivo, where systemic pretreatment of rats with TAK-044 at 10 mg/kg does not alter pressor or depressor responses to phenylephrine, angiotensin II, nitroglycerin, and acetylcholine.28 In contrast, TAK-044 dose-dependently blocks the pressor response to bolus doses of endothelin-1 and sarafotoxin S6c in rats, with 90% blockade apparent at a dose of 10 mg/kg, the effects of which persist for 3 hours.28 29 For the clinical studies in humans, we chose to use doses of TAK-044 ranging from 10 to 1000 mg. This dose range was based on the animal evidence of specificity and efficacy at doses from 0.1 to 10 mg/kg and also on the safety profile of TAK-044 in toxicological studies at higher doses (information on file, Takeda Euro R&D Centre GmbH). Pharmaceutical grade TAK-044 for parenteral use was obtained from Takeda Euro R&D Centre GmbH and was dissolved in physiological saline (0.9%; Baxter Healthcare Ltd). The placebo was dextrose (50 mg), also dissolved in physiological saline.

Endothelin-1 was administered intra-arterially at a dose of 5 pmol/min, based on previous work showing that this dose of endothelin-1 causes slow-onset vasoconstriction of human forearm resistance vessels in vivo.9 24 Pharmaceutical grade endothelin-1 was obtained from Clinalfa AG (NovaBiochem) and dissolved in physiological saline (0.9%; Baxter Healthcare Ltd) to a final concentration of 5 pmol/mL.

Drug Administration
For intravenous administration of TAK-044, an antecubital vein was cannulated at least 1 hour before dosing. TAK-044 or placebo was dissolved in physiological saline and infused at 200 mL/h over a period of 15 minutes (total volume, 50 mL). This cannula was not used for blood sampling. For intra-arterial infusion of endothelin-1 or TAK-044, the left brachial artery was cannulated under local anesthesia (1% lidocaine; Astra Pharmaceuticals Ltd) with a 27–standard wire gauge steel needle attached to a 16-gauge epidural catheter (Portex Ltd). Patency was maintained by infusion of 0.9% physiological saline via a Welmed P1000 syringe pump (Welmed Clinical Care Systems). The total rate of intra-arterial infusion was maintained constant throughout all intra-arterial studies at 1 mL/min.

Measurements
Systemic hemodynamics. Blood pressure and heart rate were measured with semiautomated oscillometric monitors (study 1, Hewlett-Packard M1165A, Hewlett-Packard GmbH; study 2, Takeda UA 751, Takeda Medical Inc). Cardiac function (stroke volume, cardiac output, and heart rate) was measured with a noninvasive bioimpedance methodology (BoMed NCCOM3, BoMed Medical Manufacturer Ltd). Absolute cardiac output measured by bioimpedance has been validated against thermodilution measurements with correlation coefficients ranging from 0.83 to 0.90 and mean differences ranging from 2% to 12%.32 33 34 35 In addition, bioimpedance measures of changes in cardiac output after drug intervention are in close agreement with simultaneous thermodilution measurements.35 Furthermore, within-subject coefficient of variation is lower with bioimpedance than with thermodilution (4.7% versus 7.8%).33 The safety and reproducibility of the bioimpedance technique confers specific advantages in studies of drug action in healthy subjects.25

Side-effect assessments. The following assessments were performed to detect potential adverse effects: 12-lead ECGs, visual analogue scale (for sedation), urinalysis, clinical chemistry screen (liver enzymes, electrolytes, creatinine, blood urea, protein), and hematology screen (full blood cell count, white blood cell differential count).

Forearm blood flow. Blood flow was measured simultaneously in both forearms by venous occlusion plethysmography using indium/gallium-in-Silastic strain gauges as previously described,9 24 except that single-channel Hokanson EC 4 plethysmographs (DE Hokanson Inc) were used. Recordings of forearm blood flow were made repeatedly over 3-minute periods.

Pharmacokinetic and endothelin assays. Fifteen-milliliter venous blood samples were obtained at intervals for assay of serum TAK-044 and plasma immunoreactive endothelin concentrations. TAK-044 was extracted from sodium acetate (pH 5) buffered serum by methanol/acetic acid–conditioned Varian Certify II cartridges and was measured by HPLC. Eluates were evaporated to dryness under vacuum at 40°C, and the dry residues were taken up in 200 µL of 39% acetonitrile. Chromatographic separation was achieved by a column-switching technique using two Alltech C18 HPLC columns with Gilson model 307 HPLC pumps. The first mobile phase comprised 40% acetonitrile and 60% 0.01 mol/L KH2PO4/0.005 mol/L tetrabutylammonium bromide, pH 3.8. The second mobile phase comprised 45% acetonitrile/1% acetic acid/54% water. Detection was achieved by fluorimetry (excitation, 286 nm; emission, 348 nm) with Hitachi F-1050 fluorescence detectors. The limit of quantification of the assay, defined as the lowest quantifiable amount of compound at which the loss of precision was <20% and the accuracy was between ±20%, was 5 ng/mL of TAK-044.

Plasma immunoreactive endothelin was measured by radioimmunoassay (ITS Production BV) as previously described.36 The sensitivity of this assay is 2 pg/mL immunoreactive endothelin. The assay does not cross-react with TAK-044. Cross-reactivity of the assay with endothelin-1, endothelin-2, endothelin-3, and big endothelin-1 is 100%, 52%, 96%, and 7%, respectively.

Study Design
Study 1: Dose-Ranging Hemodynamic Study
Two groups of five subjects were recruited to a double-blind, ascending-dose, crossover study with a randomized placebo phase. Group 1 subjects were studied on five occasions, receiving placebo and 10, 100, 500, and 1000 mg of TAK-044, with 7 days between phases. Group 2 subjects were studied on four occasions, receiving placebo and 30, 250, and 750 mg of TAK-044, with 7 days between phases. The ascending-dose design enabled us to evaluate safety and tolerability of TAK-044 at lower doses before proceeding to higher doses and entailed that the study days for groups 1 and 2 occur on different days of the same week for the first 4 weeks of dosing. For example, in the first week, subjects in group 1 received either placebo or 10 mg on Monday and group 2 subjects received placebo or 30 mg on Wednesday.

In each study phase, subjects were admitted to the research unit the day before dosing and fasted from 11 PM. At least 1 hour before dosing, an antecubital venous cannula was sited in each arm for administration of TAK-044 and blood sampling. Subjects received a 15-minute intravenous infusion of TAK-044 or placebo at {approx}9 AM and, apart from voiding, were not permitted to stand until 4 hours after dosing. Hemodynamic measurements were made and blood samples were obtained for TAK-044 and endothelin concentrations before and after dosing (see Figs 1 through 4DownDownDownDown). Sedation was assessed and 12-lead ECGs were recorded before and after dosing. Blood and urine samples were obtained for safety assessments before and 24 hours after dosing. Subjects were fasted until 4 hours after dosing, when they received a light meal. An evening meal was provided 10 hours after dosing. Subjects were discharged 24 hours after dosing.



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  		Figure 1. Time course of the effects of the highest dose of TAK-044 (1000 mg) on mean arterial pressure (MAP), heart rate (HR), stroke index (SI), cardiac index (CI), and total peripheral resistance index (TPRI) in group 1 from study 1. TAK-044 significantly decreased MAP (P<.001) and TPR (P<.001) and increased HR (P<.001), SI (P=.034), and CI (P<.001); these effects were maximal at 4 hours and sustained for at least 12 hours. Data shown represent placebo-corrected changes from predose (change from predose [active] minus mean change from predose [placebo]).



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  		Figure 2. Mean hemodynamic changes ({Delta}) over 24 hours after dosing with TAK-044 in study 1. For the placebo columns (open), mean change from predose is shown. For the active treatment columns (stippled), placebo-corrected changes from predose are shown (change from predose [active] minus mean change from predose [placebo]). *P<=.05 for comparison with predose; {dagger}P<=.05 for linear contrast test of trend with dose. SBP indicates systolic blood pressure; DBP, diastolic blood pressure; other abbreviations as in Fig 1Up.



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  		Figure 3. Effect of TAK-044 on plasma immunoreactive endothelin levels in study 1. A, Group 1 results after infusion of placebo ({diamondsuit}) and TAK-044 at 10 mg ({square}), 100 mg ({bullet}), 500 mg ({circ}),and 1000 mg ({blacksquare}). B, Group 2 results after infusion of placebo ({diamondsuit}) and TAK-044 at 30 mg ({square}), 250 mg ({bullet}), and 750 mg ({circ}). TAK-044 dose-dependently increased circulating endothelin concentrations.



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  		Figure 4. Graphs showing pharmacokinetic profiles of TAK-044 in study 1. A, Results after infusion of TAK-044 at 10 mg ({blacksquare}), 30 mg ({circ}), and 100 mg ({bullet}). B, Results after infusion of TAK-044 at 250 mg ({square}), 500 mg ({bullet}), 750 mg ({circ}), and 1000 mg ({blacksquare}). Results are shown here only for the first 4 hours after dosing, although pharmacokinetic calculations were based on all time points up to 24 hours (see Table 2Up). Plasma levels after 4 hours were <0.025 µg/mL for all doses.

Study 2: Effect of TAK-044 on Forearm Vasoconstriction to Endothelin-1
Eight subjects were recruited to a five-phase, double-blind, randomized, placebo-controlled crossover study, with at least 7 days between phases. These studies using forearm plethysmography were performed in a quiet clinical research ward maintained at a constant temperature between 22°C and 25°C. In each phase, subjects were admitted to the research unit at 7 AM, and blood and urine samples were obtained for safety assessments before dosing. At least 1 hour before dosing, an antecubital venous cannula was sited in each arm for administration of TAK-044 and blood sampling. In the first four phases, subjects received, in random order, placebo and 30, 250, and 750 mg of TAK-044 IV over 15 minutes, at {approx}9 AM. Brachial artery cannulation was performed after the infusion of TAK-044 or placebo had finished, and intra-arterial infusion of endothelin-1 (5 pmol/min) commenced 60 minutes after the start of TAK-044 dosing and continued for 120 minutes thereafter (ie, until 180 minutes after TAK-044 dosing). Measurements were made of forearm blood flow (see Fig 5Down) and blood pressure and cardiac output (-25, -15, -5, +15, +30, +45, +60, +90, +120, +150, and +180 minutes). Blood samples were obtained at -15, +15, +60, +120, and +180 minutes for assay of TAK-044 and endothelin concentrations. In the fifth phase, TAK-044 was infused intra-arterially via the brachial artery, with subjects receiving 10 mg over 1 hour followed by 100 mg over 1 hour. Measurements were made of forearm blood flow (see Fig 6Down), blood pressure, and cardiac function (-10 and +120 minutes), and blood samples were obtained for assay of endothelin (-15, +15, +60, and +120 minutes). In each phase, subjects remained supine until 3 hours after dosing and were fasted until 4 hours after dosing, when they received a light meal. Subjects were discharged 6 hours after dosing.



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  		Figure 5. Effect of TAK-044 on forearm vasoconstriction to locally administered endothelin-1 in study 2. On four separate occasions, subjects received a 15-minute intravenous infusion of placebo ({bullet}) and TAK-044 at 30 mg ({circ}), 250 mg ({blacksquare}), and 750 mg ({square}), followed 60 minutes later by brachial artery infusion of endothelin-1 (5 pmol/min for 120 minutes). Endothelin-1 caused a slow-onset forearm vasoconstriction after infusion of placebo. All three doses of TAK-044 significantly (P<.05) blocked forearm vasoconstriction to endothelin-1.



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  		Figure 6. Graph showing effect of brachial artery administration of TAK-044 on forearm blood flow in the last phase of study 2. TAK-044 was infused at 10 and 100 mg/h sequentially, each for 60 minutes. TAK-044 caused significant local vasodilatation of the cannulated arm during infusion at 10 mg/h (P=.0062). At 100 mg/h, local vasodilatation appears to diminish (P=.10); this is probably related to an increase in blood flow in the noninfused arm, which decreases the blood flow ratio between infused and noninfused arms (see text and Table 5).

Data Presentation and Statistical Analysis
Mean arterial pressure was calculated as diastolic blood pressure plus one third pulse pressure. Data for stroke volume and cardiac output were corrected for body surface area, calculated according to a standard nomogram, to provide measures of stroke and cardiac indexes. Total peripheral resistance index was calculated as mean arterial pressure divided by cardiac index and expressed in AU. For the systemic hemodynamic data, the change from the last measurement before dosing was calculated at each time point and corrected for the changes that occurred at the same time point after placebo. Plethysmographic data listings were extracted from computer data files and forearm blood flows (mL·dL forearm tissue-1·min-1) calculated for individual venous occlusion cuff inflations using a template spreadsheet (Excel 4.0; Microsoft Ltd). The last five flow recordings in each 3-minute measurement period were averaged. To reduce the variability of blood flow data, the ratio of flows in the infused and noninfused arms was calculated for each time point and expressed as a percentage change from the last baseline measurement (+55 minutes for phases A through D; -10 minutes for phase E), in effect using the noninfused arm as a contemporaneous control for the infused arm.37 38

Pharmacokinetics of TAK-044 were analyzed by use of SIPHAR software (version 4, SIMED). The following parameters were calculated: AUC, Cmax, and elimination half-life. Plasma immunoreactive endothelin concentrations were analyzed in a similar manner, with AUC and Cmax being calculated.

Absolute values are presented as mean±SEM. Placebo-corrected hemodynamic changes from baseline were arithmetically averaged over the relevant measurement period (0 to 24 hours for study 1; 0 to 3 hours for study 2), with uniform weighting given to each time point, and are shown in the tables with 95% CIs. Data were analyzed statistically by repeated-measures ANOVA. Factors included in the ANOVA were subject, dose of TAK-044, time point, and dose–time point interaction. In none of the analyses was there evidence of a statistically significant dose–time point interaction. Therefore, the adjusted dose group means from the ANOVA were compared with the null hypothesis, for all time points combined, by a two-sided t test. In addition, dose-response trends were assessed statistically by the technique of linear contrast. Linear contrast analyzes trends between groups of subjects that are categorized quantitatively, using variances derived from an ANOVA. Each linear contrast was calculated as the sum of the mean of each group multiplied by a coefficient that represented that group's dose (adjusted so that the sum of all coefficients equals zero).39 Statistical testing of a linear contrast involved calculation of its SEM using the pooled estimate of variance from the ANOVA, with a t statistic given by the linear contrast divided by this SEM. Simple regression analysis was used to explore whether there was a correlation between plasma endothelin and hemodynamic changes. Statistical analyses were performed by use of the software package SAS (version 6.07, SAS Institute Inc).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
General
In study 1, one subject withdrew from group 1 after the second phase for non–study-related reasons and was not replaced; he did not receive placebo and was therefore not included in the analysis. All other subjects completed the study protocols. TAK-044 was well tolerated, with no difference between placebo and TAK-044 phases in the prevalence of minor symptoms. There were no serious adverse events in either study, and no clinically significant abnormalities were detected on safety monitoring (urinalysis, hematology, clinical chemistry, ECG, and sedation scores).

Study 1: Dose-Ranging Hemodynamic Study
Baseline hemodynamic parameters did not differ between study days (Table 1Down). Compared with placebo, all doses of TAK-044 reduced blood pressure, with the hypotensive effect apparent within 30 minutes, maximal between 1 and 6 hours, and persisting to 24 hours at the higher doses (Figs 1Up and 2Up). For example, after the 30- and 1000-mg doses, mean arterial pressure was reduced at 4 hours by 8 and 18 mm Hg from baselines of 75 and 72 mm Hg, respectively. Diastolic and mean arterial pressures were reduced by all doses; systolic pressure was significantly decreased by all doses except 750 mg.


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  		Table 1. Baseline Hemodynamic Values in Groups 1 and 2 of Study 1

Heart rate was significantly increased by TAK-044 at all doses except 30 and 100 mg; this increase persisted to {approx}8 hours for doses >250 mg (Figs 1Up and 2Up). Most doses of TAK-044 significantly increased stroke and cardiac indexes (Fig 2Up). Total peripheral resistance index was significantly and substantially reduced at all doses, and this effect was sustained for up to 24 hours (Figs 1Up and 2Up). For example, after the 30- and 1000-mg doses, total peripheral resistance index was reduced at 4 hours by 378 and 665 AU from baselines of 1628 and 1605 AU, respectively. There were significant dose-related trends on linear contrast testing for heart rate, stroke index, cardiac index, and total peripheral resistance, although not for blood pressure (Fig 2Up).

TAK-044 increased plasma immunoreactive endothelin concentrations in a dose-dependent manner, with significant increases at all doses except for 10 mg (Table 2Down). For example, after 1000 mg, plasma endothelin concentrations increased from 3.3 to 35.7 pg/mL within 30 minutes. Compared with the sustained hemodynamic effects of TAK-044, increases in plasma endothelin were maximal within 30 minutes and waned rapidly, even at the highest doses (Fig 3Up). Even so, there was a significant correlation between the increase in plasma endothelin and the change in total peripheral resistance in both group 1 (r=-.15; P=.032) and group 2 (r=-.156; P=.021). In group 2 only, plasma endothelin was also correlated with change in systolic (r=-.189; P=.005) and mean arterial (r=-.160; P=.018) pressures. TAK-044 plasma concentrations increased dose-dependently (Fig 4Up); the terminal half-life was short (30 to 60 minutes) and tended to increase with dose (Table 2Down).


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  		Table 2. Summary Pharmacokinetic Parameters for Plasma Immunoreactive Endothelin Concentrations and TAK-044 Concentrations in Study 1

Study 2: Effect of TAK-044 on Forearm Vasoconstriction to Endothelin-1
As in the first study, all intravenous doses of TAK-044 significantly decreased diastolic blood pressure, increased heart rate and cardiac index, and caused peripheral vasodilatation (Table 3Down), with the effects sustained over the measurement period. There were significant dose-related trends for systolic blood pressure, mean arterial pressure, and total peripheral resistance (Table 3Down). As in study 1, plasma immunoreactive endothelin concentrations were increased in a dose-dependent manner, with significantly higher Cmax values after 250 mg (22.9 pg/mL; P<.0001) and 750 mg (37.2 pg/mL; P<.0001) compared with placebo (7.8 pg/mL). Similarly, there were significant correlations between plasma endothelin concentrations and changes in cardiac index (r=.226; P=.012), diastolic pressure (r=-.225; P=.011), mean arterial pressure (r=-.206; P=.021), and total peripheral resistance (r=-.249; P=.006).


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  		Table 3. Mean Hemodynamic Changes Over 24 Hours After Dosing With TAK-044 in Study 2

In the first four phases, forearm blood flow in the infused arm was not significantly different from that in the noninfused arm at baseline on any phase, and baseline blood flows were similar between the different treatment days (Table 4Down). Blood flow in the noninfused arm did not change significantly after placebo. Brachial artery infusion of endothelin-1 in the placebo phase caused a significant slow-onset local forearm vasoconstriction, reaching {approx}30% after 60 minutes (P=.0031 versus basal; Fig 5Up). Systemic administration of TAK-044 did not significantly change blood flow in the noninfused arm compared with placebo. However, forearm vasoconstriction to endothelin-1 was completely blocked by TAK-044 at doses of 30 mg (P=.34 versus basal; P=.02 versus placebo), 250 mg (P=.60 versus basal; P=.01 versus placebo), and 750 mg (P=.89 versus basal; P=.01 versus placebo), with no significant difference between doses (Fig 5Up, Table 4Down).


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  		Table 4. Forearm Blood Flows and Ratio of Blood Flows Between Infused and Noninfused Arms in Study 2

In the fifth phase, brachial artery infusion of TAK-044 at 10 mg caused significant local vasodilatation, with an increase in the ratio of blood flow between infused and noninfused arms of {approx}20% (P=.0062; Fig 6Up, Table 4Up). At the higher dose (100 mg/h), blood flow remained elevated in the infused arm but also increased in the noninfused arm, with the result that the percentage increase in the ratio of blood flow between the infused and noninfused arms fell toward baseline (P=.10; Fig 6Up, Table 4Up). A possible systemic effect of TAK-044 at the higher dose is supported by the fact that total peripheral resistance decreased by 347±113 AU at 2 hours, compared with an increase of 158±134 AU at 2 hours in the placebo phase. In addition, although circulating endothelin concentrations did not differ from placebo at baseline or 1 hour after the start of intra-arterial dosing, endothelin concentrations at 2 hours (17.3±1.6 pg/mL) were significantly greater than at 2 hours in the placebo phase (4.6±0.2 pg/mL; P=.01).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
These studies are the first report of the effects of systemic endothelin receptor blockade in healthy humans. We have shown that a 15-minute infusion of the endothelin ETA/B receptor antagonist TAK-044 decreased systolic blood pressure (by {approx}4%), diastolic blood pressure (by {approx}18%), and total peripheral resistance (by {approx}26%) over a 24-hour period. Systemic ETA/B receptor blockade also increased circulating immunoreactive endothelin (by up to 1000%) and blocked peripheral vasoconstriction to exogenous endothelin-1. In addition, local administration of TAK-044 caused forearm vasodilatation. These findings have implications for the physiological role of endothelin-1 generation, the pharmacology of endothelin receptor antagonists, and their ultimate therapeutic relevance.

Physiological Role of Endothelin-1 in Regulation of Blood Pressure
As noted earlier, animal data on the hemodynamic effects of systemic endothelin receptor antagonism are apparently contradictory. We have previously shown that brachial artery administration of an ECE inhibitor or ETA antagonist causes local forearm vasodilation, suggesting that basal vascular generation of endothelin-1 contributes to vascular tone.24 Our demonstration here that systemic administration of an ETA/B antagonist causes peripheral vasodilation and hypotension confirms that endogenous generation of endothelin plays a fundamental physiological role in the maintenance of blood pressure in humans.

Pharmacology of Endothelin Receptor Antagonists
TAK-044 decreased mean arterial pressure and increased heart rate and cardiac index, resulting in a substantial decrease in calculated peripheral resistance. The greater reduction in diastolic as opposed to systolic pressure is consistent with a primary action of TAK-044 on peripheral resistance. TAK-044 also increased forearm blood flow when administered via the brachial artery, although this local action was obscured at the higher dose by systemic vasodilatation. Taken together, these effects indicate that the resistance vessels are the major site of action after endothelin ETA/B receptor blockade with TAK-044. In spontaneously hypertensive rats, a 6-hour infusion of an ETA/B endothelin receptor antagonist (SB 209670) also decreases blood pressure through an effect on total peripheral resistance.40 However, heart rate tends to decrease in these animals, suggesting other sites of action for endothelin receptor antagonists. The difference between our results and these animal data may reflect differences in species, resting blood pressure, or mode of administration of the antagonist.

Vasodilatation and hypotension caused by TAK-044 occurred within 15 minutes and persisted for 12 to 24 hours. In contrast to its sustained hemodynamic actions, the marked increase in plasma endothelin concentrations caused by TAK-044 was relatively short in duration, and TAK-044 itself appeared to have a short half-life. In animals, the blood pressure–lowering effects of ECE inhibition or endothelin ETA receptor blockade usually take several hours to reach maximum,16 17 18 19 20 21 22 and forearm vasodilatation to these agents is also slow in onset.24 This gradual effect is thought to be related to the slow dissociation of endothelin-1 from its receptor, resulting in persistent vasoconstriction even after new receptor binding is inhibited. There are two speculative explanations for the rapid onset of vasodilatation observed here. First, the rapid effects of TAK-044 may be related to its potency as an endothelin receptor antagonist, with plasma concentrations being achieved that were sufficient to reverse, rather than prevent, endothelin-1 receptor binding. Second, TAK-044 is active at ETB as well as ETA receptors26 27 28 29 ; there is some evidence that vasoconstrictor ETB receptors may have a more rapid onset of action than ETA receptors.41

Endothelin-1 has a slow onset of action, and this may partially explain the sustained vasodilatation caused by ETA/B receptor blockade with TAK-044. In addition, although TAK-044 had a short half-life (30 to 60 minutes), TAK-044 concentrations were substantially greater than the IC50 for binding to ETA receptors (0.08 nmol/L or 0.08 ng/mL) for at least 12 hours at doses >500 mg. Furthermore, it is possible that TAK-044 concentrations were above this level for longer periods or at lower doses; however, the limit of quantification for the TAK-044 assay was 5 ng/mL, {approx}50-fold higher than the IC50 at ETA receptors. Finally, the dissociation between pharmacokinetic and pharmacodynamic parameters may reflect entry into and activity of TAK-044 in another tissue compartment. This might be within the vasculature or in the central or peripheral nervous system. Entry into and actions in other tissue compartments appear to explain the similar dissociation between actions and plasma concentrations observed for inhibitors of the renin-angiotensin system.42 43

The trend analysis shows that vasodilatation to TAK-044 was dose dependent. However, given that vasodilatation occurred at almost all doses, including the lowest (10 mg), it is probable that doses <10 mg may be effective. Indeed, the pharmacokinetic results, together with the in vitro pharmacology data discussed earlier, suggest that the initial plasma levels were probably sufficiently high even after 10 mg to block endothelin ETA receptors for at least 2 hours. This is confirmed by the complete blockade of forearm vasoconstriction to endothelin-1 at 3 hours by doses as low as 30 mg in the second study. We did demonstrate a dose response for the elevation of circulating immunoreactive endothelin by TAK-044. In addition, peripheral vasodilatation was related to plasma endothelin concentrations, further supporting a dose-dependent effect on peripheral resistance.

The increase in plasma immunoreactive endothelin after TAK-044 may have several components. The radioimmunoassay we used detected both endothelin-1 and endothelin-3. Although it also cross-reacted with big endothelin-1, this was to a limited degree (7%) and therefore is unlikely to explain the substantial increases in circulating endothelin concentrations. The increase in circulating endothelin may have been due to increased generation or decreased receptor-mediated clearance of endothelin isopeptides. Decreased clearance of endothelin by ETB receptors appears to be the most likely explanation, for several reasons. First, in animals, blockade of endothelin receptors of the ETB subtype but not of the ETA subtype increases plasma endothelin-1 and endothelin-3 concentrations30 and prolongs the half-life of exogenous 125I–endothelin-1.31 Second, blockade of endothelin receptors increases plasma endothelin-1 within 15 minutes,30 whereas de novo generation is thought to take several hours.1 Third, endothelin receptor blockade does not increase big endothelin-1 concentrations.30 The substantial increase in total immunoreactive endothelin in this study, together with the animal findings above, suggests that ETB receptor binding is an important mechanism in clearance of endogenous endothelin peptides.

It would be useful for the clinical development of endothelin receptor antagonists to have a simple and reproducible index of endothelin receptor blockade. Although increases in plasma immunoreactive endothelin correlated with decreases in total peripheral resistance, this association was relatively weak, with correlation coefficients of {approx}0.2. Changes in circulating endothelin concentrations probably only reflect antagonism at the ETB receptor, which, in addition to its functional roles, appears to mediate clearance of circulating endothelin-1.30 31 For a drug with ETA receptor blocking properties, such as TAK-044, pharmacodynamic effects may be apparent at concentrations that do not substantially increase circulating endothelin concentrations, as was the case here. This may help to explain the different timings of changes in circulating endothelin and peripheral resistance, as well as the rather weak correlation between these parameters. In the second study, we used forearm vasoconstriction to endothelin-1 1 to 3 hours after dosing with TAK-044 to test endothelin receptor blockade. Vasoconstrictor responses to locally infused endothelin-1 were completely inhibited by all three doses, consistent with the similar hemodynamic responses to these doses. This model is safer than using intravenous infusion of systemic doses of endothelin-1 to increase blood pressure, particularly given the sustained and potent nature of vasoconstriction to endothelin-1. Given that both ETA and ETB receptors mediate vasoconstriction to endothelin-1 in the forearm,9 blockade of vasoconstriction to endothelin-1 is likely to reflect antagonism at both ETA and ETB receptors. Antagonism at ETB receptors could be tested by brachial artery administration of a selective ETB receptor agonist, such as sarafotoxin S6c.9

Potential Therapeutic Role of Endothelin Receptor Antagonists
Experimental evidence supports a pathophysiological role for endothelin-1 in several diseases thought to be associated with acute vasoconstriction or vasospasm. These include acute renal failure,21 coronary vasospasm,44 unstable angina,45 myocardial infarction,46 and cerebral vasospasm associated with subarachnoid hemorrhage.21 The potent inhibition of peripheral vasoconstriction to exogenous endothelin-1, as a model of vasospasm, by TAK-044 in this study suggests that it could be of benefit in such conditions. Indeed, in experimental animal models, TAK-044 has been shown to prevent postischemic acute renal failure27 and limit myocardial infarction size.29 The sustained vasodilator actions of TAK-044 in healthy subjects suggest that orally available endothelin receptor antagonists with a similar profile of action may have a valuable therapeutic role in diseases associated with chronic peripheral vasoconstriction, such as essential hypertension, chronic heart failure, and chronic renal failure.

In conclusion, we have shown that systemic endothelin ETA and ETB receptor blockade with the peptide TAK-044 causes sustained and substantial peripheral vasodilatation and, to a lesser extent, hypotension. This response suggests a fundamental physiological role for endogenously generated endothelin-1 in cardiovascular regulation. Circulating immunoreactive endothelin concentrations were increased dose-dependently, and TAK-044 blocked forearm vasoconstriction to intra-arterial endothelin-1. Blockade of forearm vasoconstriction to brachial artery infusion of endothelin-1 appears to be a sensitive model for detecting endothelin receptor antagonism in humans. These findings support the development of endothelin receptor antagonists as therapies for diseases associated with acute and sustained peripheral vasoconstriction.


*    Selected Abbreviations and Acronyms
 
AU = arbitrary units
AUC = area under the concentration-time curve
Cmax = maximum drug concentration
ECE = endothelin-converting enzyme
ETA, ETB = endothelin receptors
HPLC = high-performance liquid chromatography


*    Acknowledgments
 
This work was supported by a grant from Takeda Euro R&D Centre. Dr Haynes is the recipient of a Wellcome Trust Advanced Training Fellowship (042145/114). Dr Ferro is supported by the British Heart Foundation (PG/94183). We wish to thank E. Stanley and Dr N. Lannigan of the Pharmacy Department at the Western General Hospital for preparing ampoules of endothelin-1 for parenteral use. We thank Dr Allyn L. Mark for his helpful comments regarding the manuscript.

Received December 8, 1995; revision received January 22, 1996; accepted January 22, 1996.


*    References
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