Comparison of Pharmacological Activity of Macitentan and Bosentan in Preclinical Models of Systemic and Pulmonary Hypertension

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 article info abstract

Article history: Aims: The endothelin (ET) system is a tissular system, as the production of ET isoforms is mostly autocrine or

Received 29 October 2013 paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and Accepted 12 February 2014 sustained receptor binding properties designed to achieve a more efficacious ET receptor blockade. To determine Available online xxxx 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 ET /ET receptor antagonist, bosentan, which does not display sustained receptor occupan- Endothelin A B Pharmacology cy and shows less tissue distribution. – Blood pressure Main methods: After establishing dose response curves of both compounds in conscious, hypertensive Dahl salt- Pulmonary hypertension sensitive and pulmonary hypertensive bleomycin-treated rats, macitentan was administered on top of the max- Rat imal effective dose of bosentan. 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/).

Introduction receptor antagonist with increased tissue distribution and slow receptor dissociation, resulting in sustained receptor occupancy time. In the Pulmonary arterial hypertension (PAH) is a severe disease caused by monocrotaline rat model of pulmonary hypertension, macitentan constriction and remodeling of pulmonary vessels, progressing to right prevented development of right ventricular hypertrophy and improved heart failure and death (Voelkel et al., 2012). Until now, short-term survival (Bolli et al., 2012; Gatfield et al., 2012; Iglarz et al., 2008). Slow clinical trials in patients with PAH showed that endothelin (ET) receptor receptor dissociation minimizes competition between the ligand and antagonists (ERAs) can improve exercise capacity, symptoms, cardio- the drug at the target binding site and thus is a property that can im- pulmonary hemodynamic variables, and delay time to clinical worsen- prove efficacy of drugs (Swinney, 2006). Such an increased ability of ing (McLaughlin, 2011). A search for new ERAs with superior ET macitentan to block the ET receptors would be expected to achieve su- receptor blockade – and better safety – was critical in order to maximize perior efficacy vs. ERAs with fast receptor dissociation kinetics, particu- the potential to delay remodeling and disease progression. larly in pathological conditions in which the ET system is upregulated.

Recently, the novel ERA macitentan (10 mg/day) showed a highly Macitentan also has an active metabolite, a dual ETA/ETB receptor antag- signiﬁcant 45% reduction in the combined endpoint of morbidity and onist with a long half-life, ACT-132577, which likely contributes to the mortality in a long-term Phase III clinical trial (SERAPHIN) in patients pharmacological activity of macitentan (Iglarz et al., 2008). suffering from PAH (Pulido et al., 2013). Macitentan is a dual ETA/ETB 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 ⁎ Corresponding author at: Drug Discovery Department, Actelion Pharmaceuticals Ltd, of the maximal effective dose of bosentan, another dual ET /ET ERA. To Gewerbestrasse 16, CH-4123 Allschwil, Switzerland. Tel.: +41 61 565 63 41; fax: +41 A B 61 565 89 03. do so, the hemodynamic effects of these ERAs and their combination E-mail address: [email protected] (M. Iglarz). 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 2 M. Iglarz et al. / Life Sciences xxx (2014) xxx–xxx

Dahl salt-sensitive (DSS) rats and pulmonary hypertensive, bleomycin- instilled 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 Fig. 1. Scheme describing the ‘add-on’ protocol. the guidelines of the Swiss Animal Protection Law under licenses 164 and 185. on top of the maximal effective dose of macitentan. The maximal effec- Transmitter implantation in DSS rats tive dose of the second compound was administered at Tmax of the first tested compound. 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. Pharmacokinetics Paul, MN, USA) was implanted under anesthesia. In brief, a blood pressure-sensing catheter was placed in the descending aorta below In order to rule out any confounding drug–drug interaction on the the renal arteries, pointing upstream and implanted into the peritoneal pharmacological effect measured, the exposure of macitentan and its cavity to measure systemic arterial pressure (Brockway et al., 1991). active metabolite ACT-132577 was measured in the presence or absence of bosentan in similar conditions to the add-on protocol. Vehicle Transmitter implantation in bleomycin rats or bosentan 300 mg/kg was administered to Wistar rats (n =6/ group) 6 h prior to macitentan 30 mg/kg. Plasma samples were collect- Under sterile conditions, ventilated rats were instrumented micro- ed at 1, 2, 3, 4, 6, 8 and 24 h after oral administration of macitentan, surgically with a telemetry pressure transmitter implanted in the peri- and quantification of macitentan and ACT-132577 was determined by toneal cavity. The right ventricle was exposed using a right lateral liquid chromatography coupled to mass spectrometry (API4000, AB thoracotomy at the 6–7th intercostal space. A trench was made with a SCIEX, Concord, Ontario, Canada). 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 Test compounds allow measurement of pulmonary pressure. The chest was closed and the transmitter sutured to the abdominal musculature. Twenty-one Macitentan and bosentan were supplied by Actelion Pharmaceuti- days after transmitter implantation, a single dose of bleomycin sulphate cals Ltd. Gelatin 7.5%, administered at 5 mL/kg, was used as vehicle for (1.5 mg/kg/mL, Baxter AG, Volketswil, Switzerland followed by 1 mL/kg oral administration of the compounds by gavage. of air, adapted from Williams et al., 1992) was intratracheally instilled in each rat to induce mild pulmonary arterial hypertension. Statistics Data acquisition by telemetry All results are presented as mean ± SEM. Area between the mean ar- Blood pressure was collected continuously using the Dataquest ART terial pressure vs. time curves (ABC) is expressed in mm Hg h, MAP or Gold acquisition system (version 3.01). Systolic, mean and diastolic ar- MPAP in mm Hg, and HR in bpm. Differences between groups were an- terial pressures and heart rate (HR) were collected at 5-min intervals alyzed using an unpaired Student t-test. Statistical significance was de- during the entire experiment, resulting in a series of 1152 data points fined as P 0.05. 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 Results mean arterial pressure (MAP) or mean pulmonary arterial pressure (MPAP) vs. time curves of the control and treatment periods (integrated Determination of maximal effective doses in DSS rats and observation that differences between pre- and post-dose curves over time). the maximal efficacy of macitentan is greater than that of bosentan

Dose–response curves and add-on protocol DSS rats developed an increase in MAP up to approximately 180 mm Hg. MAP and HR baseline values were similar between the dif- First, dose–response curves of each ERA were constructed in both ferent experimental groups. After acute oral administration, macitentan systemic hypertensive DSS rats and bleomycin-induced pulmonary hy- dose-dependently decreased MAP in DSS rats. The maximal effective pertensive Wistar rats to determine the maximal effective dose and dose of macitentan was 30 mg/kg; this dose decreased MAP by 30 ± Tmax (time of observed maximal effect) of each ERA. Pharmacological 5mmHg(Table 1). The maximal effect was reached at about 24 h effects on MAP or MPAP and HR were measured up to 120 h after a sin- after administration (Tmax). Acute oral administration of bosentan gle gavage at doses ranging from 0.1 to 100 mg/kg (macitentan) or 3 to dose-dependently decreased MAP in DSS rats. At the maximal effective 600 mg/kg (bosentan). To determine whether macitentan could pro- dose of 100 mg/kg, bosentan decreased MAP by 15 ± 2 mm Hg vide superior pharmacological activity vs. bosentan, we designed a (Table 1), with a Tmax at 6 h. No effect on HR was observed. Based on study in which: 1) macitentan was administered on top of the maximal these data, the maximal effective doses of 30 mg/kg of macitentan and effective dose of bosentan established by the dose–response curve as 100 mg/kg of bosentan were selected for the add-on study. Tmax was shown in Figs. 1 and 2) the same dose of bosentan was administered 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 M. Iglarz et al. / Life Sciences xxx (2014) xxx–xxx 3

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MAP (mmHg) Vehicle, 5 ml/kg MAP (mmHg) 120 120 Vehicle, 5 ml/kg Control, n=5 Control, n=9 Vehicle, 5 ml/kg p.o. n=5 Bosentan, 100 mg/kg p.o. n=9 100 100 024487296024487296 Time (hour) Time (hour)

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MAP (mmHg) Bosentan, 100 mg/kg MAP (mmHg) Macitentan, 30 mg/kg 120 Control, n=5 120 Control, n=9 Bosentan, 100 mg/kg p.o. n=5 Bosentan, 100 mg/kg p.o. n=9 100 100 024487296024487296 Time (hour) Time (hour)

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Vehi + Vehi, n=5Bos + Vehi, n=9 Bos + Bos, n=5 Bos + Maci, n=9

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 was 33 ± 4 mm Hg (Fig. 2). Conversely, bosentan, administered orally bosentan using add-on protocol in DSS rats 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 The use of maximal effective doses of macitentan and bosentan was vehicle administered on top of macitentan 30 mg/kg (Fig. 3). confirmed using the add-on protocol: macitentan 30 mg/kg administered on top of macitentan 30 mg/kg did not cause an additional MAP Determination of maximal effective doses in bleomycin-treated rats and decrease as compared to vehicle on top of macitentan (−29 ± 2 and observation that the maximal efficacy of macitentan is greater than that −28 ± 5 mm Hg). Bosentan 100 mg/kg, administered when the maxi- of bosentan mal effect of bosentan 100 mg/kg was reached, did not induce an additional MAP decrease compared to vehicle on top of bosentan (−13 ± 2 Bleomycin-treated rats developed an increase in MPAP of approxi- and −14 ± 3 mm Hg, respectively; Fig. 2). mately 13 mm Hg vs. saline-instilled rats. MPAP and HR baseline values were similar between the different experimental groups. Acute oral ad- Comparison between macitentan and bosentan using add-on protocol in ministration of macitentan and bosentan dose-dependently decreased DSS rats MPAP in bleomycin rats (Table 1) without affecting HR (data not shown). At a dose of 30 mg/kg macitentan, the maximal decrease in Oral administration of macitentan 30 mg/kg, when the maximal ef- MPAP was 12 ± 3 mm Hg, about 24 h after administration (Tmax). At fect of bosentan 100 mg/kg had been reached, decreased MAP by an ad- the maximal effective dose of 300 mg/kg, bosentan decreased MPAP ditional 19 mm Hg compared to vehicle (p b 0.01) administered on top by7±2mmHg(Table 1), with a Tmax around 6 h. Based on these of bosentan 100 mg/kg. The maximal decrease induced by macitentan 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 4 M. Iglarz et al. / Life Sciences xxx (2014) xxx–xxx

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 10 30 100 300 600

Dahl salt-sensitive hypertensive rats Macitentan ΔMAP (mm Hg) −7±5 −12 ± 4 −15 ± 2 −19 ± 3 −25 ± 3 −30 ± 5 −33 ± 6 –– ΔABC (mm Hg h) −43 ± 37 −141 ± 38 −290 ± 44 −539 ± 98 −880 ± 160 −988 ± 159 −1714 ± 291 –– Bosentan ΔMAP (mm Hg) –– – −6±3 −8±2 −8±3 −15 ± 2 −13 ± 2 – ΔABC (mm Hg h) –– – −21 ± 19 −68 ± 20 −44 ± 27 −209 ± 9 −285 ± 61 –

Bleomycin-induced pulmonary hypertensive Wistar rats Macitentan ΔMPAP (mm Hg) – −5±1 −7±2 −8±0 −12 ± 4 −12 ± 3 −10 ± 1 –– ΔABC (mm Hg h) – −37 ± 14 −76 ± 18 −184 ± 35 −244 ± 58 −447 ± 104 −492 ± 60 –– Bosentan ΔMPAP (mm Hg) –– – −5±1 −6±1 −8±1 −5±1 −7±2 −6±1 ΔABC (mm Hg h) –– – −31 ± 21 −58 ± 40 −77 ± 12 −64 ± 12 −97 ± 20 −80 ± 7

300 mg/kg of bosentan were selected for the add-on study. Tmax was on top of bosentan 300 mg/kg. The maximal decrease induced by determined to be 6 h for bosentan and 24 h for macitentan. 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, Confirmation of selection of maximal effective dose of bosentan using when the maximal effect of macitentan 30 mg/kg had been reached, add-on protocol in bleomycin-treated rats did not induce an additional MPAP decrease compared to vehicle administered on top of macitentan 30 mg/kg (Fig. 5). The use of maximal effective doses of macitentan and bosentan was confirmed using the add-on protocol: macitentan 30 mg/kg adminis- Absence of drug–drug interaction tered on top of macitentan 30 mg/kg did not cause an additional MPAP decrease compared to vehicle on top of macitentan (−18 ± 2 and As shown in Fig. 6, administration of bosentan did not modify the −13 ± 2 mm Hg, p = 0.112). Similarly, as shown in Fig. 4, bosentan plasma concentrations of macitentan and its active metabolite ACT- 300 mg/kg administered when the maximal effect of bosentan 132577, ruling out a modification of macitentan pharmacokinetics by 300 mg/kg was reached did not cause an additional MPAP decrease bosentan during the add-on protocol. compared to vehicle on top of bosentan (−8±1and−7±1mmHg, respectively). Discussion

Comparison between macitentan and bosentan using add-on protocol in Comparison of efﬁcacy between compounds is rarely assessed fairly bleomycin-treated rats as it requires generating full dose–response curves in order to appropri- ately compare maximal effects. In our study, dose–response curves Oral administration of macitentan 30 mg/kg to bleomycin rats, were established in conscious rats with either systemic or pulmonary when the maximal effect of bosentan 300 mg/kg had been reached, in- hypertension. We compared the maximal efﬁcacy of macitentan and duced an additional MPAP decrease compared to vehicle administered bosentan both in DSS rats and in the bleomycin rat model of PH

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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 M. Iglarz et al. / Life Sciences xxx (2014) xxx–xxx 5

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40 g) 40 H m mHg) m m ( ( P P

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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 reaches higher plasma concentrations (Fig. 6) and has a prolonged develops rapidly progressing PH leading to death (Ryan et al., 2011; half-life compared to macitentan despite a lower potency than its Rey et al., 2012), bleomycin-treated rats develop a moderate but con- parent compound (Iglarz et al., 2008). The superior pharmacological stant and sustained increase in pulmonary pressure, which allows the activity of macitentan cannot be explained by a change in selectivity study of hemodynamics over a long period of time without dramatic profile towards ETA and ETB receptors as macitentan, ACT-132577 changes in MPAP (Williams et al., 1992). Macitentan achieved a superi- and bosentan, are all dual ERAs characterized by comparable ETA/ETB se- or blood pressure reduction compared to bosentan in both animal lectivity ratios as determined in isolated organs (50:1, 16:1 and 22:1 for models: −30 ± 5 mm Hg vs. −15 ± 2 mm Hg on MAP, respectively, macitentan, ACT-132577 and bosentan, respectively; Iglarz et al., 2008; in DSS rats, and −12 ± 3 mm Hg vs. −7 ± 2 mm Hg on MPAP, respec- Clozel et al., 1994). Pharmacological efficacy of a receptor antagonist de- tively, in bleomycin rats. In addition to these results, we used an ‘add- pends on its binding mode and affinity to the receptor and the number on’ protocol to confirm and demonstrate the superior pharmacological of receptors it can block in the target tissue (tissue distribution). Com- activity of macitentan vs. bosentan. bined optimization of these characteristics can also explain the superior In both models, macitentan was able to induce a further blood efficacy of macitentan relative to bosentan. pressure reduction when given on top of the maximal efficacious dose of bosentan, whereas the opposite was not true, suggesting that Receptor binding mode differentiates macitentan from bosentan macitentan is able to block more efficiently ET receptors. In addition ACT-132577, the active metabolite of macitentan, may contribute to Macitentan displays a slow receptor dissociation rate, as in pul- the superior activity of macitentan over bosentan. Indeed ACT-132577 monary 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 6 M. Iglarz et al. / Life Sciences xxx (2014) xxx–xxx

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Control, n=7 Macitentan, 30 mg/kg p.o. n=7 0 024487296 Time (hour) Maci + Vehi, n=6 Maci + Bos, n=7

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 D) favors partitioning to tissues and its pKa value corresponds to a (Gatfield et al., 2012). The slower dissociation kinetics of macitentan high fraction of the non-ionized form, able to cross cell membranes. translated into an insurmountable antagonism in studies of second With a pKa of 6.2, 6% of macitentan is non-ionized in an aqueous envi- messenger signaling, whereas bosentan showed surmountable compet- ronment at physiological pH—a relatively high percentage compared itive antagonism. As a result, macitentan inhibited the ET-1-induced with 1% of bosentan. Macitentan shows a distribution of 800 to 1 be- sustained elevation in intracellular calcium across the whole range of tween octanol and aqueous buffer, which predicts a good distribution ET-1 concentrations tested, which was not observed with bosentan. to lipids and tissues. In comparison, bosentan has a lower affinity for DSS rats present high ET-1 plasma concentrations compared to normal the lipophilic phase as shown by a distribution of 20 to 1 (Iglarz et al., animals (Quaschning et al., 2001). High ET-1 plasma concentrations, as 2008). We recently reported that, using microautoradiography analysis, a result of spillover of locally produced ET-1, reflect a saturation of tissue lung distribution of macitentan was greater than that of bosentan in receptors (Frelin and Guedin, 1994). Indeed, DSS and bleomycin-treated bleomycin-instilled rats (Iglarz et al., 2011). Thus, the improved physi- rats show, respectively, increased cardiac and lung tissue production of cochemical properties of macitentan should favor its penetration in ET-1 (Mutsaers et al., 1998; Emoto et al., 2005). Therefore, in such con- the media and allow a more complete blockade of ET receptors than ditions of high local ET-1 concentrations, the slow receptor dissociation bosentan. It might explain the additional effect of macitentan on top rate of macitentan might confer a more efficient receptor blockade, con- of bosentan, as macitentan could engage receptors left unblocked after tributing to a more effective blood pressure reduction. dosing of bosentan.

Tissue distribution differentiates macitentan from bosentan Conclusion Drug distribution in the target tissue is important to maximize the In conclusion, our data demonstrate, using different approaches and number of receptors blocked. Macitentan's distribution coefﬁcient (log animal models, that macitentan is more efﬁcacious than bosentan

12000 in vivo. Although bosentan has a similar mode of action (ETA and ETB Gelatin 5 ml/kg + macitentan 30 mg/kg, n = 6 blockade), macitentan displays improved ability to achieve a more ef- Bosentan 300 mg/kg + macitentan 30 mg/kg, n = 6 fective blockade of ET receptors via increased tissue distribution and better receptor binding properties. This improved efﬁcacy may explain 8000 the unique efﬁcacy of macitentan in reducing disease progression in patients with PAH as demonstrated in the SERAPHIN trial.

4000 ACT-132577 Conﬂict of interest statement Plasma conc. (ng/ml) All authors are employees of Actelion Pharmaceuticals Ltd. Macitentan 0 0 13 6 12 18 24 Acknowledgments Time (hours)

The authors are grateful to Stéphane Delahaye, Rolf Wuest and 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 Fabienne Drouet for DMPK analysis. This research is entirely supported prior to macitentan 30 mg/kg. n = 6/group. Data are expressed as mean ± SD. 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 7

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