LFS-13929; No of Pages 7 Life Sciences xxx (2014) xxx–xxx
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Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension Marc Iglarz ⁎, Alexandre Bossu, Daniel Wanner, Céline Bortolamiol, Markus Rey, Patrick Hess, Martine Clozel Drug Discovery Department, Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, 4123 Allschwil, Switzerland
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Aims: The endothelin (ET) system is a tissular system, as the production of ET isoforms is mostly autocrine or paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and sustained receptor binding properties designed to achieve a more efficacious ET receptor blockade. To determine if these features translate into improved efficacy in vivo, a study was designed in which rats with either systemic or pulmonary hypertension and equipped with telemetry were given macitentan on top of maximally effective Keywords: doses of another dual ETA/ETB receptor antagonist, bosentan, which does not display sustained receptor occupanEndothelin cy and shows less tissue distribution. Pharmacology Main methods: After establishing dose–response curves of both compounds in conscious, hypertensive Dahl saltBlood pressure sensitive and pulmonary hypertensive bleomycin-treated rats, macitentan was administered on top of the maxPulmonary hypertension imal effective dose of bosentan. Rat Key findings: In hypertensive rats, macitentan 30 mg/kg further decreased mean arterial blood pressure (MAP) by 19 mm Hg when given on top of bosentan 100 mg/kg (n = 9, p b 0.01 vs. vehicle). Conversely, bosentan given on top of macitentan failed to induce an additional MAP decrease. In pulmonary hypertensive rats, macitentan 30 mg/kg further decreased mean pulmonary artery pressure (MPAP) by 4 mm Hg on top of bosentan (n = 8, p b 0.01 vs. vehicle), whereas a maximal effective dose of bosentan given on top of macitentan did not cause any additional MPAP decrease. Significance: The add-on effect of macitentan on top of bosentan in two pathological models confirms that this novel compound can achieve a superior blockade of ET receptors and provides evidence for greater maximal efficacy. © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Article history: Received 29 October 2013 Accepted 12 February 2014 Available online xxxx
Introduction Pulmonary arterial hypertension (PAH) is a severe disease caused by constriction and remodeling of pulmonary vessels, progressing to right heart failure and death (Voelkel et al., 2012). Until now, short-term clinical trials in patients with PAH showed that endothelin (ET) receptor antagonists (ERAs) can improve exercise capacity, symptoms, cardiopulmonary hemodynamic variables, and delay time to clinical worsening (McLaughlin, 2011). A search for new ERAs with superior ET receptor blockade – and better safety – was critical in order to maximize the potential to delay remodeling and disease progression. Recently, the novel ERA macitentan (10 mg/day) showed a highly significant 45% reduction in the combined endpoint of morbidity and mortality in a long-term Phase III clinical trial (SERAPHIN) in patients suffering from PAH (Pulido et al., 2013). Macitentan is a dual ETA/ETB
⁎ Corresponding author at: Drug Discovery Department, Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, CH-4123 Allschwil, Switzerland. Tel.: +41 61 565 63 41; fax: +41 61 565 89 03. E-mail address:
[email protected] (M. Iglarz).
receptor antagonist with increased tissue distribution and slow receptor dissociation, resulting in sustained receptor occupancy time. In the monocrotaline rat model of pulmonary hypertension, macitentan prevented development of right ventricular hypertrophy and improved survival (Bolli et al., 2012; Gatfield et al., 2012; Iglarz et al., 2008). Slow receptor dissociation minimizes competition between the ligand and the drug at the target binding site and thus is a property that can improve efficacy of drugs (Swinney, 2006). Such an increased ability of macitentan to block the ET receptors would be expected to achieve superior efficacy vs. ERAs with fast receptor dissociation kinetics, particularly in pathological conditions in which the ET system is upregulated. Macitentan also has an active metabolite, a dual ETA/ETB receptor antagonist with a long half-life, ACT-132577, which likely contributes to the pharmacological activity of macitentan (Iglarz et al., 2008). To demonstrate that the improved physicochemical properties and binding of macitentan could translate into superior pharmacological activity, a study protocol was designed that tested macitentan's ability to induce an additional decrease in blood pressure when administered on top of the maximal effective dose of bosentan, another dual ETA/ETB ERA. To do so, the hemodynamic effects of these ERAs and their combination were monitored in conscious, freely moving, systemic hypertensive,
http://dx.doi.org/10.1016/j.lfs.2014.02.018 0024-3205/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
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Dahl salt-sensitive (DSS) rats and pulmonary hypertensive, bleomycininstilled Wistar rats, two pathological models associated with activated ET-1 production (Park et al., 1997; Schiffrin, 2001). Methods Animals Two-month-old DSS rats were purchased from Charles River (USA) and two-month-old Wistar rats from Harlan (The Netherlands). All rats were maintained under identical conditions in accordance with the guidelines of the Swiss Animal Protection Law under licenses 164 and 185. Transmitter implantation in DSS rats In DSS rats, hypertension was induced and maintained by administration of 1% NaCl in drinking water. Five to six weeks after starting salt administration, a telemetry system (PA-C40, Data Sciences, St. Paul, MN, USA) was implanted under anesthesia. In brief, a blood pressure-sensing catheter was placed in the descending aorta below the renal arteries, pointing upstream and implanted into the peritoneal cavity to measure systemic arterial pressure (Brockway et al., 1991). Transmitter implantation in bleomycin rats Under sterile conditions, ventilated rats were instrumented microsurgically with a telemetry pressure transmitter implanted in the peritoneal cavity. The right ventricle was exposed using a right lateral thoracotomy at the 6–7th intercostal space. A trench was made with a stainless steel trocar from the peritoneal cavity to the thorax. The pressure catheter was inserted into the ventricle to measure right ventricular pressure and further advanced through the pulmonary valve to allow measurement of pulmonary pressure. The chest was closed and the transmitter sutured to the abdominal musculature. Twenty-one days after transmitter implantation, a single dose of bleomycin sulphate (1.5 mg/kg/mL, Baxter AG, Volketswil, Switzerland followed by 1 mL/kg of air, adapted from Williams et al., 1992) was intratracheally instilled in each rat to induce mild pulmonary arterial hypertension.
Fig. 1. Scheme describing the ‘add-on’ protocol.
on top of the maximal effective dose of macitentan. The maximal effective dose of the second compound was administered at Tmax of the first tested compound.
Pharmacokinetics In order to rule out any confounding drug–drug interaction on the pharmacological effect measured, the exposure of macitentan and its active metabolite ACT-132577 was measured in the presence or absence of bosentan in similar conditions to the add-on protocol. Vehicle or bosentan 300 mg/kg was administered to Wistar rats (n = 6/ group) 6 h prior to macitentan 30 mg/kg. Plasma samples were collected at 1, 2, 3, 4, 6, 8 and 24 h after oral administration of macitentan, and quantification of macitentan and ACT-132577 was determined by liquid chromatography coupled to mass spectrometry (API4000, AB SCIEX, Concord, Ontario, Canada).
Test compounds Macitentan and bosentan were supplied by Actelion Pharmaceuticals Ltd. Gelatin 7.5%, administered at 5 mL/kg, was used as vehicle for oral administration of the compounds by gavage.
Statistics Data acquisition by telemetry Blood pressure was collected continuously using the Dataquest ART Gold acquisition system (version 3.01). Systolic, mean and diastolic arterial pressures and heart rate (HR) were collected at 5-min intervals during the entire experiment, resulting in a series of 1152 data points per day for each animal. For both blood pressure and HR, each rat served as its own control, by using the data of the last 24 h before drug or vehicle administration. An area between curves (ABC) was calculated for mean arterial pressure (MAP) or mean pulmonary arterial pressure (MPAP) vs. time curves of the control and treatment periods (integrated differences between pre- and post-dose curves over time). Dose–response curves and add-on protocol First, dose–response curves of each ERA were constructed in both systemic hypertensive DSS rats and bleomycin-induced pulmonary hypertensive Wistar rats to determine the maximal effective dose and Tmax (time of observed maximal effect) of each ERA. Pharmacological effects on MAP or MPAP and HR were measured up to 120 h after a single gavage at doses ranging from 0.1 to 100 mg/kg (macitentan) or 3 to 600 mg/kg (bosentan). To determine whether macitentan could provide superior pharmacological activity vs. bosentan, we designed a study in which: 1) macitentan was administered on top of the maximal effective dose of bosentan established by the dose–response curve as shown in Figs. 1 and 2) the same dose of bosentan was administered
All results are presented as mean ± SEM. Area between the mean arterial pressure vs. time curves (ABC) is expressed in mm Hg h, MAP or MPAP in mm Hg, and HR in bpm. Differences between groups were analyzed using an unpaired Student t-test. Statistical significance was defined as P 0.05.
Results Determination of maximal effective doses in DSS rats and observation that the maximal efficacy of macitentan is greater than that of bosentan DSS rats developed an increase in MAP up to approximately 180 mm Hg. MAP and HR baseline values were similar between the different experimental groups. After acute oral administration, macitentan dose-dependently decreased MAP in DSS rats. The maximal effective dose of macitentan was 30 mg/kg; this dose decreased MAP by 30 ± 5 mm Hg (Table 1). The maximal effect was reached at about 24 h after administration (Tmax). Acute oral administration of bosentan dose-dependently decreased MAP in DSS rats. At the maximal effective dose of 100 mg/kg, bosentan decreased MAP by 15 ± 2 mm Hg (Table 1), with a Tmax at 6 h. No effect on HR was observed. Based on these data, the maximal effective doses of 30 mg/kg of macitentan and 100 mg/kg of bosentan were selected for the add-on study. Tmax was determined to be 6 h for bosentan and 24 h for macitentan.
Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
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Fig. 2. Superior pharmacological activity of macitentan vs. bosentan in Dahl-salt sensitive rats. Whereas addition of bosentan (Bos) 100 mg/kg on top of bosentan 100 mg/kg did not induce further blood pressure reduction, addition of macitentan (Maci) 30 mg/kg on top of bosentan 100 mg/kg further decreased blood pressure by 19 mm Hg, **p = 0.004 vs. vehicle (Vehi). Vehicle, second dose of macitentan 30 mg/kg or bosentan 100 mg/kg (arrow) was administered 6 h after the first dose. n = 5–9/group. Data are expressed as mean ± SEM.
Confirmation of selection of maximal effective doses of macitentan and bosentan using add-on protocol in DSS rats The use of maximal effective doses of macitentan and bosentan was confirmed using the add-on protocol: macitentan 30 mg/kg administered on top of macitentan 30 mg/kg did not cause an additional MAP decrease as compared to vehicle on top of macitentan (− 29 ± 2 and −28 ± 5 mm Hg). Bosentan 100 mg/kg, administered when the maximal effect of bosentan 100 mg/kg was reached, did not induce an additional MAP decrease compared to vehicle on top of bosentan (−13 ± 2 and −14 ± 3 mm Hg, respectively; Fig. 2). Comparison between macitentan and bosentan using add-on protocol in DSS rats Oral administration of macitentan 30 mg/kg, when the maximal effect of bosentan 100 mg/kg had been reached, decreased MAP by an additional 19 mm Hg compared to vehicle (p b 0.01) administered on top of bosentan 100 mg/kg. The maximal decrease induced by macitentan
was 33 ± 4 mm Hg (Fig. 2). Conversely, bosentan, administered orally at 100 mg/kg, when the maximal effect of macitentan 30 mg/kg had been reached, did not induce an additional MAP decrease compared to vehicle administered on top of macitentan 30 mg/kg (Fig. 3). Determination of maximal effective doses in bleomycin-treated rats and observation that the maximal efficacy of macitentan is greater than that of bosentan Bleomycin-treated rats developed an increase in MPAP of approximately 13 mm Hg vs. saline-instilled rats. MPAP and HR baseline values were similar between the different experimental groups. Acute oral administration of macitentan and bosentan dose-dependently decreased MPAP in bleomycin rats (Table 1) without affecting HR (data not shown). At a dose of 30 mg/kg macitentan, the maximal decrease in MPAP was 12 ± 3 mm Hg, about 24 h after administration (Tmax). At the maximal effective dose of 300 mg/kg, bosentan decreased MPAP by 7 ± 2 mm Hg (Table 1), with a Tmax around 6 h. Based on these data, the maximal effective doses of 30 mg/kg of macitentan and
Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
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Table 1 Dose–response of macitentan and bosentan on blood pressure in DSS and bleomycin rats: maximal decreases and area between curves. MAP: mean arterial pressure, MPAP: mean pulmonary arterial pressure, ABC: area between curves. Data are expressed as mean ± SEM. Dose (mg/kg)
0.1
0.3
1
3
Dahl salt-sensitive hypertensive rats Macitentan ΔMAP (mm Hg) ΔABC (mm Hg h) Bosentan ΔMAP (mm Hg) ΔABC (mm Hg h)
−7 ± 5 −43 ± 37 – –
−12 ± 4 −141 ± 38 – –
−15 ± 2 −290 ± 44 – –
−19 −539 −6 −21
± ± ± ±
3 98 3 19
−25 −880 −8 −68
± ± ± ±
3 160 2 20
−30 −988 −8 −44
± ± ± ±
5 159 3 27
−33 −1714 −15 −209
± ± ± ±
−7 ± 2 −76 ± 18 – –
−8 −184 −5 −31
± ± ± ±
0 35 1 21
−12 −244 −6 −58
± ± ± ±
4 58 1 40
−12 −447 −8 −77
± ± ± ±
3 104 1 12
−10 −492 −5 −64
± ± ± ±
Bleomycin-induced pulmonary hypertensive Wistar rats Macitentan ΔMPAP (mm Hg) – −5 ± 1 ΔABC (mm Hg h) – −37 ± 14 Bosentan ΔMPAP (mm Hg) – – ΔABC (mm Hg h) – –
10
30
100
300
600
6 291 2 9
– – −13 ± 2 −285 ± 61
– – – –
1 60 1 12
– – −7 ± 2 −97 ± 20
– – −6 ± 1 −80 ± 7
on top of bosentan 300 mg/kg. The maximal decrease induced by macitentan on top of bosentan was −11 ± 1 mm Hg (p b 0.01 vs. vehicle) (Fig. 4). Conversely, bosentan, administered orally at 300 mg/kg, when the maximal effect of macitentan 30 mg/kg had been reached, did not induce an additional MPAP decrease compared to vehicle administered on top of macitentan 30 mg/kg (Fig. 5).
300 mg/kg of bosentan were selected for the add-on study. Tmax was determined to be 6 h for bosentan and 24 h for macitentan. Confirmation of selection of maximal effective dose of bosentan using add-on protocol in bleomycin-treated rats The use of maximal effective doses of macitentan and bosentan was confirmed using the add-on protocol: macitentan 30 mg/kg administered on top of macitentan 30 mg/kg did not cause an additional MPAP decrease compared to vehicle on top of macitentan (− 18 ± 2 and − 13 ± 2 mm Hg, p = 0.112). Similarly, as shown in Fig. 4, bosentan 300 mg/kg administered when the maximal effect of bosentan 300 mg/kg was reached did not cause an additional MPAP decrease compared to vehicle on top of bosentan (−8 ± 1 and −7 ± 1 mm Hg, respectively).
Absence of drug–drug interaction As shown in Fig. 6, administration of bosentan did not modify the plasma concentrations of macitentan and its active metabolite ACT132577, ruling out a modification of macitentan pharmacokinetics by bosentan during the add-on protocol. Discussion Comparison of efficacy between compounds is rarely assessed fairly as it requires generating full dose–response curves in order to appropriately compare maximal effects. In our study, dose–response curves were established in conscious rats with either systemic or pulmonary hypertension. We compared the maximal efficacy of macitentan and bosentan both in DSS rats and in the bleomycin rat model of PH
Comparison between macitentan and bosentan using add-on protocol in bleomycin-treated rats Oral administration of macitentan 30 mg/kg to bleomycin rats, when the maximal effect of bosentan 300 mg/kg had been reached, induced an additional MPAP decrease compared to vehicle administered 200
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Time (hour) Fig. 3. Absence of pharmacological activity of bosentan on top of macitentan in conscious Dahl-salt sensitive rats. Addition of bosentan (Bos) 100 mg/kg on top of macitentan (Maci) 30 mg/kg did not induce further blood pressure reduction. Vehicle (Vehi) or bosentan dose (arrow) was administered 24 h after macitentan. n = 4–5/group. Data are expressed as mean ± SEM.
Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
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Fig. 4. Superior pharmacological effect of macitentan on top of bosentan in conscious bleomycin-induced pulmonary hypertensive rats. While addition of bosentan (Bos) 300 mg/kg on top of bosentan 300 mg/kg did not induce further mean pulmonary arterial pressure (MPAP) reduction, addition of macitentan (Maci) 30 mg/kg on top of bosentan 300 mg/kg further decreased blood pressure by 4 mm Hg, **p b 0.01 vs. vehicle (Vehi). Vehicle, second dose of bosentan 300 mg/kg or macitentan 30 mg/kg (arrow) was administered 6 h after the first dose of bosentan 300 mg/kg. n = 8/group. Data are expressed as mean ± SEM.
associated with lung fibrosis. Unlike the monocrotaline rat model that develops rapidly progressing PH leading to death (Ryan et al., 2011; Rey et al., 2012), bleomycin-treated rats develop a moderate but constant and sustained increase in pulmonary pressure, which allows the study of hemodynamics over a long period of time without dramatic changes in MPAP (Williams et al., 1992). Macitentan achieved a superior blood pressure reduction compared to bosentan in both animal models: −30 ± 5 mm Hg vs. − 15 ± 2 mm Hg on MAP, respectively, in DSS rats, and −12 ± 3 mm Hg vs. −7 ± 2 mm Hg on MPAP, respectively, in bleomycin rats. In addition to these results, we used an ‘addon’ protocol to confirm and demonstrate the superior pharmacological activity of macitentan vs. bosentan. In both models, macitentan was able to induce a further blood pressure reduction when given on top of the maximal efficacious dose of bosentan, whereas the opposite was not true, suggesting that macitentan is able to block more efficiently ET receptors. In addition ACT-132577, the active metabolite of macitentan, may contribute to the superior activity of macitentan over bosentan. Indeed ACT-132577
reaches higher plasma concentrations (Fig. 6) and has a prolonged half-life compared to macitentan despite a lower potency than its parent compound (Iglarz et al., 2008). The superior pharmacological activity of macitentan cannot be explained by a change in selectivity profile towards ETA and ETB receptors as macitentan, ACT-132577 and bosentan, are all dual ERAs characterized by comparable ETA/ETB selectivity ratios as determined in isolated organs (50:1, 16:1 and 22:1 for macitentan, ACT-132577 and bosentan, respectively; Iglarz et al., 2008; Clozel et al., 1994). Pharmacological efficacy of a receptor antagonist depends on its binding mode and affinity to the receptor and the number of receptors it can block in the target tissue (tissue distribution). Combined optimization of these characteristics can also explain the superior efficacy of macitentan relative to bosentan. Receptor binding mode differentiates macitentan from bosentan Macitentan displays a slow receptor dissociation rate, as in pulmonary arterial smooth muscle cells, the receptor occupancy half-life
Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
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Fig. 5. Absence of pharmacological activity of bosentan on top of macitentan in conscious bleomycin rats. Addition of bosentan (Bos) 300 mg/kg on top of macitentan (Maci) 30 mg/kg did not induce further blood pressure reduction. Vehicle (Vehi) or bosentan second dose (arrow) was administered 24 h after macitentan. n = 6–7/group. Data are expressed as mean ± SEM.
of macitentan (17 min) was 15-fold longer than that of bosentan (Gatfield et al., 2012). The slower dissociation kinetics of macitentan translated into an insurmountable antagonism in studies of second messenger signaling, whereas bosentan showed surmountable competitive antagonism. As a result, macitentan inhibited the ET-1-induced sustained elevation in intracellular calcium across the whole range of ET-1 concentrations tested, which was not observed with bosentan. DSS rats present high ET-1 plasma concentrations compared to normal animals (Quaschning et al., 2001). High ET-1 plasma concentrations, as a result of spillover of locally produced ET-1, reflect a saturation of tissue receptors (Frelin and Guedin, 1994). Indeed, DSS and bleomycin-treated rats show, respectively, increased cardiac and lung tissue production of ET-1 (Mutsaers et al., 1998; Emoto et al., 2005). Therefore, in such conditions of high local ET-1 concentrations, the slow receptor dissociation rate of macitentan might confer a more efficient receptor blockade, contributing to a more effective blood pressure reduction. Tissue distribution differentiates macitentan from bosentan
Conclusion
Drug distribution in the target tissue is important to maximize the number of receptors blocked. Macitentan's distribution coefficient (log 12000
Gelatin 5 ml/kg + macitentan 30 mg/kg, n = 6 Bosentan 300 mg/kg + macitentan 30 mg/kg, n = 6
Plasma conc. (ng/ml)
D) favors partitioning to tissues and its pKa value corresponds to a high fraction of the non-ionized form, able to cross cell membranes. With a pKa of 6.2, 6% of macitentan is non-ionized in an aqueous environment at physiological pH—a relatively high percentage compared with 1% of bosentan. Macitentan shows a distribution of 800 to 1 between octanol and aqueous buffer, which predicts a good distribution to lipids and tissues. In comparison, bosentan has a lower affinity for the lipophilic phase as shown by a distribution of 20 to 1 (Iglarz et al., 2008). We recently reported that, using microautoradiography analysis, lung distribution of macitentan was greater than that of bosentan in bleomycin-instilled rats (Iglarz et al., 2011). Thus, the improved physicochemical properties of macitentan should favor its penetration in the media and allow a more complete blockade of ET receptors than bosentan. It might explain the additional effect of macitentan on top of bosentan, as macitentan could engage receptors left unblocked after dosing of bosentan.
8000
In conclusion, our data demonstrate, using different approaches and animal models, that macitentan is more efficacious than bosentan in vivo. Although bosentan has a similar mode of action (ETA and ETB blockade), macitentan displays improved ability to achieve a more effective blockade of ET receptors via increased tissue distribution and better receptor binding properties. This improved efficacy may explain the unique efficacy of macitentan in reducing disease progression in patients with PAH as demonstrated in the SERAPHIN trial.
ACT-132577
4000
Conflict of interest statement All authors are employees of Actelion Pharmaceuticals Ltd. Macitentan
0 0 1
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Time (hours) Fig. 6. Plasma concentrations of macitentan and its active metabolite ACT-132577 in the presence of bosentan 300 mg/kg or vehicle. Vehicle or bosentan was administered 6 h prior to macitentan 30 mg/kg. n = 6/group. Data are expressed as mean ± SD.
Acknowledgments The authors are grateful to Stéphane Delahaye, Rolf Wuest and Fabienne Drouet for DMPK analysis. This research is entirely supported by Actelion Pharmaceuticals Ltd.
Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
M. Iglarz et al. / Life Sciences xxx (2014) xxx–xxx
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Please cite this article as: Iglarz M, et al, Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.02.018
