The metabolism of the dual endothelin receptor antagonist macitentan in rat and dog

Article abstract of DOI:10.3109/00498254.2015.1070302

1. The metabolism of the endothelin receptor antagonist macitentan has been characterized in bile duct-cannulated rats and dogs.2. In both species, macitentan was metabolized along five primary pathways, i.e. conjugation with glucose (M9), oxidative depropylation (M6), aliphatic hydroxylation (M7), oxidative cleavage of the ethylene glycol linker (M4) and hydrolysis of the sulfamide moiety (M3). Most of the primary metabolites underwent subsequent biotransformation including conjugation with glucuronic acid or glucose, hydrolysis of the sulfamide group or secondary oxidation of the ethylene glycol moiety.3. Though there were species differences in their relative importance, all metabolic pathways were present in rat and dog. The depropylated M6 was the only metabolite present in plasma of both species.4. Metabolism was a prerequisite for macitentan excretion as relevant amounts of parent drug were neither detected in bile nor urine. Biliary excretion was the major elimination pathway, while renal elimination was of little importance.

Full text of DOI:10.3109/00498254.2015.1070302

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
The metabolism of the dual endothelin receptor
antagonist macitentan in rat and dog
Alexander Treiber, Tommaso Miraval, Martin H. Bolli, Jacques-Alexis Funel,
Jerome Segrestaa & Swen Seeland
To cite this article: Alexander Treiber, Tommaso Miraval, Martin H. Bolli, Jacques-
Alexis Funel, Jerome Segrestaa & Swen Seeland (2016) The metabolism of the dual
endothelin receptor antagonist macitentan in rat and dog, Xenobiotica, 46:3, 253-267, DOI:
10.3109/00498254.2015.1070302
To link to this article: http://dx.doi.org/10.3109/00498254.2015.1070302
Published online: 17 Aug 2015.
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ISSN: 0049-8254 (print), 1366-5928 (electronic)
Xenobiotica, 2016; 46(3): 253–267
2015 Taylor & Francis. DOI: 10.3109/00498254.2015.1070302
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RESEARCH ARTICLE
The metabolism of the dual endothelin receptor antagonist macitentan
in rat and dog
Alexander Treiber1, Tommaso Miraval1, Martin H. Bolli2, Jacques-Alexis Funel2, Jerome Segrestaa1, and
Swen Seeland1
2
1Department of Non-Clinical Drug Metabolism and Pharmacokinetics and Department of Medicinal Chemistry, Actelion Pharmaceuticals Ltd,
Allschwil, Switzerland
Abstract
Keywords
1. The metabolism of the endothelin receptor antagonist macitentan has been characterized in
bile duct-cannulated rats and dogs.
Endothelin receptor antagonist, macitentan,
metabolism in rat and dog
2. In both species, macitentan was metabolized along five primary pathways, i.e. conjugation
with glucose (M9), oxidative depropylation (M6), aliphatic hydroxylation (M7), oxidative
cleavage of the ethylene glycol linker (M4) and hydrolysis of the sulfamide moiety (M3). Most
of the primary metabolites underwent subsequent biotransformation including conjugation
with glucuronic acid or glucose, hydrolysis of the sulfamide group or secondary oxidation of
the ethylene glycol moiety.
3. Though there were species differences in their relative importance, all metabolic pathways
were present in rat and dog. The depropylated M6 was the only metabolite present in
plasma of both species.
History
Received 15 June 2015
Revised 3 July 2015
Accepted 4 July 2015
Published online 17 August 2015
4. Metabolism was a prerequisite for macitentan excretion as relevant amounts of parent drug
were neither detected in bile nor urine. Biliary excretion was the major elimination pathway,
while renal elimination was of little importance.
Introduction
endothelin receptor binding (Gatfield et al., 2012, 2014;
Iglarz et al., 2008), pharmacokinetic profile (Sidharta et al.,
2011) and liver safety (Treiber et al., 2014). The recently
completed SERAPHIN (Study with an Endothelin Receptor
Antagonist in Pulmonary arterial Hypertension to Improve
Clinical outcome) trial has demonstrated that macitentan
alone or in combination with phosphodiesterase-5 inhibitors
or prostanoid receptor agonists reduces the risk of a compos-
ite endpoint of morbidity and mortality in pulmonary arterial
hypertension patients (Pulido et al., 2013).
Pulmonary arterial hypertension is a progressive and severe
hemodynamic disease ultimately leading to right heart failure
and death (Galie et al., 2009b). Several drug classes have
shown beneficial effects in the treatment of pulmonary
arterial hypertension, including endothelin receptor antagon-
ists (Channick et al., 2001; Galie et al., 2005a; Langleben
et al., 2004; Pulido et al., 2013), phosphodiesterase-5
inhibitors (Galie et al., 2005b, 2009a), epoprostenol (Rubin
et al., 1990) and prostanoid analogues (Olschewski et al.,
2002; Simonneau et al., 2002). Endothelin receptor antagon-
ists were the first oral treatment option with bosentan
(TracleerÕ) being approved in 2001, followed by ambrisentan
(LetairisÕ/VolibrisÕ) in 2007. Macitentan (OpsumitÕ, N-[5-
(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-
4-pyrimidinyl-N0-propyl-sulfamide) has been developed as a
novel endothelin receptor antagonist with optimized
The approved dose of macitentan in pulmonary arterial
hypertension is 10 mg. Macitentan is slowly absorbed in man
after oral dosing and reaches peak plasma concentrations at
8 h after administration. Plasma levels decline thereafter with
a half-life of 16 h (Sidharta et al., 2011). Its oral bioavail-
ability is unknown but estimated as about 70% based on
pharmacokinetic modeling (de Kanter et al., 2015).
Macitentan is lipophilic with a log D of 2.9, negatively
charged at physiological pH and highly bound to plasma
proteins. It partitions well into tissues with an apparent
volume of distribution (Vss/F) of 50 l. Macitentan clearance
largely depends on cytochrome P450-mediated metabolism,
mostly CYP3A4 and CYP2Cs. Renal and biliary excretion
both contribute to overall drug elimination. Oxidative
Address for correspondence: Alexander Treiber, Department of Non-
Clinical Drug Metabolism and Pharmacokinetics, Actelion
Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, CH-4123,
Switzerland. Tel: +41 61 565 65 92. Fax: +41 61 565 89 03. E-mail:
alexander.treiber@actelion.com

254 A. Treiber et al.
Xenobiotica, 2016; 46(3): 253–267
Br
depropylation of its sulfamide moiety yields a major metab-
olite (ACT–132577, called M6 in this publication) which
retains pharmacological activity on endothelin receptors and
likely contributes to overall efficacy (Iglarz et al., 2008).
This report describes the metabolism of macitentan in rat
and dog, the two major species used for the non-clinical safety
assessment. Radiolabeled macitentan was given orally and
intravenously to bile duct-cannulated animals and excreta
were collected for up to 4 days. Metabolic patterns were
recorded using high performance liquid chromatography
(HPLC) coupled to radiodetection and metabolites structur-
ally identified using MS technology and authentic reference
compounds.
Br
N
H
N
H
N
O
S
O
N
O
2
N
N
*
macitentan
asterisk denotes position of radiolabel
Figure 1. Chemical structure of 14C-macitentan.
constants are given in Hz. Liquid chromatography coupled
to high resolution mass spectrometry (LC–HRMS): analytical
pump: Waters Acquity Binary, Solvent Manager, MS:
SYNAPT G2 MS, source temperature: 150 ꢀC, desolvatation
temperature: 400 ꢀC, desolvatation gas flow: 400 l/h; cone gas
flow: 10 l/h, extraction cone: 4 RF; lens: 0.1 V; sampling
cone: 30; capillary:1.5 kV; high resolution mode; gain: 1.0,
MS function: 0.2 s per scan, 120–1000 amu in full scan,
centroid mode. Lock spray: Leucine enkephalin 2 ng/ml
(556.2771 Da), scan time 0.2 s with interval of 10 s and
average of five scans; DAD: Acquity UPLC PDA detector.
Column: Acquity UPLC BEH C18 1.7 mm, 2.1 ꢁ 50 mm ID
from Waters, kept at 60 ꢀC in an Acquity UPLC Column
Manager. Eluent A: water with 0.05% formic acid; eluent B:
acetonitrile with 0.05% formic acid. Gradient: 2–98% B over
3.0 min with a flow of 0.6 ml/min. UV detection at 214 nm
and MS. Retention times are given in min.
Materials and methods
Chemicals and reagents
Macitentan (batch no. 600083/Batch 1) was manufactured
at Lonza AG (Visp, Switzerland) with a purity of 98.6%.
14C-labeled macitentan (lot nos. 031215 and 030514)
(Figure 1) was obtained from American Radiolabeled
Chemicals Inc. (St. Louis, MO) with a specific activity of
50 mCi/mmol as a stock solution in acetonitrile, with
chemical and radiochemical purities in excess of 97%. All
other chemicals and solvents used throughout this study were
of highest commercially available quality and obtained from
Sigma-Aldrich or Fluka (Buchs, Switzerland). Deuterated
water (NMR grade) was purchased from Merck (Darmstadt,
Germany). Liquid scintillation cocktails, Irga Safe Plus and
Flow Safe 2, were from Perkin Elmer (Zu¨rich, Switzerland)
and Berthold Technologies (Regensdorf, Switzerland). Pooled
rat and dog liver microsomes were purchased from TOTAM
Biologicals Ltd. (Northampton, United Kingdom).
6-Amino-5-(4-bromophenyl)-4-(2-((5-bromopyrimidin-2-yl)
oxy)ethoxy)pyrimidine 1-oxide (M2)
To a solution of 6-amino-5-(4-bromophenyl)-4-(2-((5-bromo-
pyrimidin-2-yl)oxy)ethoxy)-pyrimidine
(M3)
(50 mg,
Metabolite syntheses
0.11 mmol) in dichloromethane (2 ml), meta-chloroperben-
zoic acid (74 mg, 0.43 mmol) was added. The mixture was
stirred at room temperature for 16 h before it was concen-
trated. The crude product was purified on prep. TLC plates
using dichloromethane:methanol (10:1) to give M2 (36 g,
All reagents and solvents were used as purchased from
commercial sources (Sigma-Aldrich Switzerland, Lancaster
Synthesis GmbH, Germany, Acros Organics, NJ). Moisture-
sensitive reactions were carried out under an argon atmos-
phere. Progress of the reactions was followed either by
thin-layer chromatography (TLC) analysis (Merck, 0.2 mm
silica gel 60 F254 on glass plates) or by liquid chromatography
coupled to mass spectrometry (LC–MS). Preparative HPLC
was accomplished using Gilson 333/334 pumps, Gilson
LH215 autosampler, Dionex ISO-3100A make-up pump,
Dionex DAD-3000 detector, Thermo Finnigan MSQ Plus
mass spectrometer and Polymer Laboratories PL-ELS1000
ELSD detector; for column and gradient see below. Melting
points were determined by differential scanning calorimetry
using a DSC822e/400 instrument and the HSS7 sensor from
Mettler Toledo, Switzerland. For nuclear magnetic resonance
1
69%) as a beige foam. H NMR (500 MHz, CDCl3): 8.52
(s, 1 H), 8.51 (s, 2 H), 7.56–7.61 (m, 2 H), 7.22–7.28 (m, 2 H),
6.26 (s br, 2 H), 4.67–4.72 (m, 2 H), 4.61–4.66 (m, 2 H). 13
C
NMR (125 MHz, CDCl3): 163.4, 159.6, 157.9, 153.5, 144.5,
132.6, 131.4, 128.2, 123.2, 112.2, 100.7, 65.7, 65.1; 13C
DEPT NMR (500 MHz, CDCl3): CH: 159.6, 144.5, 132.6,
131.4, CH2: 65.7, 65.1; 1H 13C HSQC NMR (500 MHz,
CDCl3): 8.52(s, 1 H) ! 144.5; 8.51 (s, 2 H) ! 159.6; 7.56–
7.61 (m, 2 H) ! 132.5; 7.22–7.28 (m, 2 H) ! 131.5, 4.67–
1
4.72 (m, 2 H) ! 65.1; 4.61–4.66 (m, 2 H) ! 65.7; H 13C
HMBC NMR (500 MHz, CDCl3): 8.52 (s, 1 H) ! 157.9,
153.4; 8.51 (s, 2 H) ! 163.4, 159.6, 112.2; 7.56–7.61
(m, 2 H) ! 132.6, 128.2, 123.2, 100.7 (weak); 7.22–7.28
(m, 2 H) ! 131.4, 128.2, 100.7; 4.67–4.72 (m, 2 H) ! 157.9,
65.7; 4.61–4.66 (m, 2 H) ! 163.4, 65.1.
(NMR) spectroscopy,
a Bruker Avance II, 400 MHz
UltraShieldTM, 400 MHz (1H), 100 MHz (13C); or Bruker
Avance III HD, Ascend II 500 MHz (1H), 125 MHz (13C)
magnet equipped with a cryoprobe was used. Chemical shifts
are reported in parts per million (ppm) relative to tetra-
methylsilane, and multiplicities are given as s (singlet), d
(doublet), t (triplet), q (quartet), quint (quintuplet), h (hextet),
hept (heptuplet) or m (multiplet); br ¼ broad; coupling
6-Amino-5-(4-bromophenyl)-4-(2-((5-bromopyrimidin-2-yl)
oxy)ethoxy)pyrimidine (M3)
A solution of N-[5-(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimi-
dinyl)oxy]ethoxy]-4-pyrimidinyl]-N0-propylsulfamide

DOI: 10.3109/00498254.2015.1070302
Macitentan metabolism in rat and dog 255
(macitentan, 25 g, 42.5 mmol) in acetonitrile (500 ml) and
water (150 ml) was stirred at 60 ꢀC for 7 days. The acetonitrile
was removed by distillation on a rotavap and the precipitate
filtered off and washed with water. The solid material was
slurred in boiling methanol (500 ml), filtered off and washed
with methanol. The solid was dissolved in dichloromethane
(100 ml) and methanol (500 ml) and allowed to crystallize by
slowly removing the dichloromethane. The crystalline mater-
ial was collected, washed with methanol and dried under high
vacuum to give M3 (14.4 g, 73%) as a pale beige-grey powder.
m.p. 178 ꢀC. Optimized conditions use aqueous polar solvents
at higher temperature (e.g. water/dioxane at about 80–90 ꢀC)
and allow for isolation of crystalline M3 directly from the
CDCl3): 8.24 (s, 1 H), 7.60–7.65 (m, 2 H), 7.23–7.28 (m, 2
H), 4.98 (s br, 2 H), 4.43–4.49 (m, 2 H), 3.82–3.89 (m, 2 H),
2.95 (s br, 1 H).
6-Amino-5-(4-bromophenyl)pyrimidin-4-ol (M19)
(a) A solution of 5-(4-bromophenyl)-4,6-dichloropyrimidine
(3.0 g, 9.87 mmol) and 7 N NH3 in a mixture of 20 ml
methanol and 10 ml dioxane was stirred at room temperature
for 18 h before it was concentrated. The crude product was
purified by column chromatography on silica gel eluting with
dichloromethane:methanol (9:1) to give 5-(4-bromophenyl)-
6-chloropyrimidin-4-amine (1.48 g, 53%) as a white solid.
1H NMR (500 MHz, D6-DMSO): 8.22 (s, 1 H), 7.67–7.71 (m,
2 H), 7.24–7.28 (m, 2 H), 6.43 (s br, 2 H). 13C NMR
(125 MHz, D6-DMSO): 163.2, 157.6, 156.9, 132.7, 132.6,
132.5, 122.5, 114.7. (b) A solution of 5-(4-bromophenyl)-6-
chloropyrimidin-4-amine (1.48 g, 5.2 mmol) in 10 ml of 1 M
aqueous NaOH and 20 ml dioxane was stirred at 90 ꢀC for
72 h. The mixture was cooled to room temperature,
neutralized with 2 M aqueous HCl and the precipitate was
collected. The crude product was purified by preparative
1
reaction mixture. H NMR (300 MHz, CDCl3): 8.47 (s, 2 H),
8.21 (s, 1 H), 7.48–7.54 (m, 2 H), 7.16–7.23 (m, 2 H), 4.80
(s br, 2 H), 4.63–4.69 (m, 2 H), 4.56–4.63 (m, 2 H). 13C NMR
(75 MHz, CDCl3): 165.7, 163.8, 161.9, 159.7, 156.8, 132.4,
131.9, 130.8, 122.2, 112.2, 100.9, 66.1, 64.5. Elemental
analysis: calc. C 41.14%; H 2.81%; Br 34.21%; N 14.99%;
O 6.85%; found: C 40.86%, H 2.77%, Br 34.29%, N 14.90%,
O 7.00%.
1
HPLC to give M19 (200 mg, 14%) as a white solid. H NMR
2-((5-(4-Bromophenyl)-6-((N-propylsulfamoyl)amino)
pyrimidin-4-yl)oxy)acetic acid (M5)
(500 MHz, D6-DMSO): 11.71 (s br, 1 H), 7.82 (s, 1 H), 7.52–
7.57 (m, 2 H), 7.24–7.29 (m, 2 H), 6.18 (s br, 2 H). 13C NMR
(125 MHz, D6-DMSO): 160.7, 160.5, 148.7, 133.7, 133.2,
131.6, 119.8, 98.0.
To a solution of N-5-(4-bromophenyl)-6-(2-hydroxyethoxy)-
4-pyrimidinyl-N0-propyl-sulfamide (M4) (5.42 g, 12.6 mmol)
in acetone (80 ml), Jones reagent (20 ml, 10 g CrO3
dissolved in 17 g H2SO4 and filled up to 50 ml with
water) was added and the mixture was stirred at room
temperature for 18 h before it was diluted with water and
extracted with ethyl acetate. The organic extract was dried
over MgSO4, filtered and concentrated. The crude product
was purified by preparative HPLC (Waters XBridgeTM C18,
10 mm, 30 ꢁ 75 mm ID, gradient of acetonitrile in water
containing 0.5% of formic acid, flow: 75 ml/min) to give M5
(2.52 g, 45%) as a white solid. 1H NMR (500 MHz, D6-
DMSO): 11.8 (s br, 1 H), 8.39 (s, 1 H), 7.61–7.65 (m, 2 H),
7.27–7.31 (m, 2 H), 7.04 (s br, 1 H), 4.79 (s, 2 H), 2.77 (t,
J ¼ 7.1 Hz, 2 H), 1.42 (h, J ¼ 7.4 Hz, 2 H), 0.81 (t,
J ¼ 7.4 Hz, 3 H). 13C NMR (125 MHz, D6-DMSO): 172.5,
170.3, 165.5, 155.7, 133.2, 131.7, 130.8, 121.5, 105.0, 63.0,
45.1, 22.6, 11.8.
N-[5-(4-bromophenyl)-6-[2-hydroxyethoxy]-4-pyrimidinyl]-
sulfamide (M21)
(a) A solution of 5-(4-bromophenyl)-4,6-dichloropyrimidine
(5 g, 16.4 mmol) in 20 ml ethylene glycol was cooled to 4 ꢀC
before potassium tert. butylate (2.12 g, 18.9 mmol) was added.
The mixture was stirred at room temperature for 1 h before it
was again cooled to 4 ꢀC, diluted with 50 ml water and
extracted with 50 ml dichloromethane. The organic extract
was separated, concentrated and dried to give 2-((5-(4-
bromophenyl)-6-chloropyrimidin-4-yl)oxy)ethan-1-ol (5.3 g,
98%) as an off-white powder. 1H NMR (400 MHz, D6-
DMSO): 8.67 (s, 1 H), 7.68 (d, J ¼ 8.4 Hz, 2 H), 7.39 (d,
J ¼ 8.4 Hz, 2 H), 4.78 (t, J ¼ 5.4 Hz, 1 H), 4.40 (t, J ¼ 5.0 Hz,
2 H), 3.62 (q, J ¼ 5.2 Hz, 2 H). (b) A solution of 2-((5-(4-
bromophenyl)-6-chloropyrimidin-4-yl)oxy)ethan-1-ol (3.75 g,
11.1 mmol), K2CO3 (3.93 g, 26.4 mmol), sulfamide (1.20 g,
12.5 mmol) and tetrabutylammonium fluoride (8.98 g,
28.5 mmol) in 20 ml DMSO was stirred at 70 ꢀC for 2 h.
The mixture was cooled to room temperature, diluted with
aqueous CaCl2 and extracted with ethyl acetate. The organic
extract was washed twice with brine/water (1:1) and then
concentrated. The crude product was purified by preparative
HPLC (Waters XBridgeTM C18, 10 mm, 30 ꢁ 75 mm ID,
gradient of acetonitrile in water containing 0.5% aq. ammo-
nium hydroxide (25%), flow: 75 ml/min) to give M21 (1.05 g,
24%). 1H NMR (500 MHz, D6-DMSO): 9.73 (s br, 1 H), 8.47
(s, 1 H), 7.62 (d, J ¼ 8.4 Hz, 2 H), 7.26 (d, J ¼ 8.4 Hz, 2 H),
7.14 (s br, 2 H), 4.73 (t, J ¼ 5.4 Hz, 1 H), 4.32 (t, J ¼ 4.9 Hz,
2 H), 3.59 (q, J ¼ 5.3 Hz, 2 H). 13C NMR (125 MHz,
D6-DMSO): 166.4, 157.3, 156.0, 133.2, 131.9, 130.5, 121.6,
104.9, 68.6, 59.6.
2-((6-Amino-5-(4-bromophenyl)pyrimidin-4-yl)oxy)ethan-1-ol
(M18)
To a solution of ethylene glycol (2.9 ml, 51.3 mmol) in
dimethoxy ethane (10 ml), potassium tert. butylate (864 mg,
7.70 mmol) was added. The mixture was stirred at room
temperature for 15 min before 5-(4-bromophenyl)-6-chloro-
pyrimidin-4-amine (730 mg, 2.57 mmol, see M19) was added.
Stirring was continued at 80 ꢀC for 18 h before the mixture
was cooled to room temperature, diluted with water and
extracted with ethyl acetate. The organic extract was dried
over MgSO4, filtered and concentrated. The crude product
was purified by prep. HPLC (Waters AtlantisTM T3, 10 mm,
30 ꢁ 75 mm ID, gradient of acetonitrile in water containing
0.5% of formic acid, flow: 75 ml/min) to give M18
(580 mg, 73%) as a white solid. 1H NMR (400 MHz,

256 A. Treiber et al.
Xenobiotica, 2016; 46(3): 253–267
7-(Aminosulfonyl-imino)-8-(4-bromophenyl)-2,3-dihydro-7H-
oxazolo[3,2-c]pyrimidine (M27)
disturbed and the isolated cells passed through bolting cloth.
The cell suspension was centrifuged for 4 min at 50 g, the
supernatant was removed and the pellet was washed 2–3 times
with pre-incubation medium (see below). Dog hepatocytes
were obtained from an end-of-lobe perfusion. The connective
tissue was removed to separate the liver into individual lobes.
Each lobe was cannulated and connected to a peristaltic pump
and perfused according to the procedures outlined above.
Liver cells were re-suspended in pre-incubation medium and
viability determined by trypan blue exclusion. Only batches
with viabilities in excess of 80% were used for further
experiments.
O
H N
2
S
Br
10
N
N
O
4
N
9
7
5
8
2
6
O
12
11
To
a
solution of N-[5-(4-bromophenyl)-6-[2-hydro-
xyethoxy]-4-pyrimidinyl]-sulfamide (76 mg, 195 mmol, M21)
in dichloromethane (5 ml), N,N-diisopropylethylamine
(DIPEA) (25 mg, 33 ml, 195 mmol) followed by methanesulfo-
nyl chloride (22 mg, 15 ml, 195 mmol) was added. The mixture
was stirred at room temperature for 15 min before another
portion of DIPEA (13 mg, 17 ml, 100 mmol) and methanesul-
fonyl chloride (11 mg, 8 ml, 100 mmol) was added. Stirring was
continued at room temperature for 45 min before the mixture
was diluted with dichloromethane (20 ml) and washed with
water (10 ml). The organic layer was separated, dried over
MgSO4, filtered and concentrated. The crude product was
purified on a preparative TLC plate (silica gel, 0.5 mm) with
dichloromethane containing 15% of methanol to give M27
(5 mg, 7%) as a white solid. LC–HRMS: tR ¼ 0.69 min,
[M+H]+ ¼ 370.9820/372.9800 (79/81Br isotopes), calc. mass
(C12H12N4O3SBr): 370.9813; 1H NMR (500 MHz, D6-
DMSO): 8.54 (s, 1 H, H-C2), 7.58–7.62 (m, 2 H, H-C9),
7.44–7.47 (m, 2 H, H-C8), 6.01 (s, 2 H, NH2, exchangeable
with D2O), 4.79 (t, J ¼ 8.2 Hz, 2 H, H2-C11), 4.52 (t,
J ¼ 8.2 Hz, 2 H, H2-C12); 13C NMR (125 MHz, D6-DMSO) ꢀ:
162.2 (C4), 157.7 (C6), 146.1 (C2), 132.8 (C8), 131.1 (C9),
130.3 (C7), 121.0 (C10), 97.8 (C5), 71.3 (C11), 46.8 (C12).
1H–13C heteronuclear multiple-bond correlation (HMBC): H-
C2 ! C2 (1J), C4, C5, C6, C12; H-C9 ! C7, C8, C9 (1J),
C10; H-C8 ! C5, C8 (1J), C10; H2-C11 ! C6, C12; H2-
Macitentan incubations with liver microsomes and
hepatocytes
In a reaction volume of 200 ml, 14C-labeled macitentan was
incubated at a concentration of 10 mM in 100 mM phosphate
buffer (pH 7.4) with 1 mg/ml of microsomal protein. After a
pre-incubation period of 5 min at 37 ꢀC, the reaction was
started by the addition of 1 mM NADPH and terminated
after 0, 10, 20, 30 or 40 min by the addition of 0.1 ml ice-
cold methanol. The protein precipitate was removed by
centrifugation and total radioactivity was assessed by liquid
scintillation counting. Control experiments in the absence of
NADPH or microsomal protein were run in parallel under
otherwise identical conditions. All incubations were con-
ducted in duplicate and the supernatants were stored frozen
at ꢂ20 ꢀC prior to analysis. Hepatocytes were suspended in
pre-incubation medium, i.e. William’s medium E without
phenol red containing 0.0001% (w/v) insulin and 10% (v/v)
newborn calf serum, at a density of 1.2 ꢁ 106 cells/ml. A
0.6 ml aliquot was added to each well of BIOCOAT 12-well
plates and incubated at 37 ꢀC in a humidified atmosphere
containing 5% CO2 for a period of 2–4 h. At the end of this
attachment period, the medium was replaced with 0.6 ml of
pre-warmed 14C-macitentan stock solution diluted in incu-
bation medium, i.e. William’s medium E without phenol red
supplemented with 100 IU/ml penicillin, 100 mg/ml strepto-
mycin and 100 mg/ml neomycin. Macitentan was incubated
at a single concentration of 10 mM and duplicate samples
were taken at 0, 2, 6 and 24 h. Control experiments in the
absence of liver cells were run in parallel under otherwise
identical conditions. At the end of incubation, 1 ml of
methanol was added to the wells, the cell layer was scraped
from the well, the entire content was transferred into cryo-
vials, vortex-mixed, centrifuged and then frozen at ꢂ20 ꢀC
pending analysis.
1
C12 ! C2, C6, C11. H rotating frame nuclear Overhauser
effect (ROESY): H-C2 ! H-C11, H-C12, NH2; H-C8 !
H-C9; H-C9 ! H-C8; H2-C11 ! H2-C12; H2-C12 ! H-C2,
H2-C11.
Isolation of rat and dog hepatocytes
Male Wistar rats (6–8 weeks of age, 200–250 g body weight)
were anaesthetized by injection of an appropriate dose of a
1:1:6 solution of HypnormÕ:HypnovelÕ:sterile water (v/v/v)
containing 1000 IU/ml sodium heparin. The hepatic portal
vein was then cannulated and the inferior vena cava clamped
superior to the kidney. The diaphragm of the animal was cut,
the ribcage removed and the superior vena cava cannulated.
The liver was perfused with buffer 1 containing 120 mM
NaCl, 5.3 mM KCl, 7.4 mM NaHCO3, 0.3 mM Na2HPO4,
5.6 mM glucose, 10 mM HEPES, 0.5 mM EGTA in purified
water at (pH 7.4) at a flow rate of 30 ml/min for 10 min.
Buffer 2, i.e. buffer 1 without EGTA but supplemented with
collagenase in 5 mM aqueous calcium chloride at a final
concentration of 0.25 mg/ml, was perfused through the liver at
a flow rate of 20–30 ml/min. Prior to the perfusion, buffers
were saturated with oxycarbon for about 5 min. The perfusion
was ceased once the reticular pattern of the liver was
Animal experiments
Male and female Wistar rats with body weights ranging from
210 to 340 g were supplied by RCC Ltd., Biotechnology and
Animal Breeding Division (Fu¨llinsdorf, Switzerland). The
animals were acclimatized to the experimental unit for at least
7 days before use in the study. During this period, animals
were carefully monitored to ensure good health and suitability
for inclusion in the study. All animals, including those used
for the preparation of hepatocytes, were housed under the
same conditions in accordance with the National Institutes of
Health guidelines and as approved by the cantonal veterinary

DOI: 10.3109/00498254.2015.1070302
Macitentan metabolism in rat and dog 257
office (license nos. 169 and 222). During pretrial holding
periods, rats were housed in groups of up to five animals per
cage in Makrolon type-3 cages with wire mesh tops and
standardized softwood bedding. For metabolic profiling, rats
were housed individually post-surgery in the same cage type
as before. After dose administration, rats were kept individu-
ally in metabolism cages (Techniplast Gazzada, Buguggiate,
Italy) specially designed for the separate, quantitative collec-
tion of urine, bile and faeces. During the entire course of
the study, standard laboratory diet of known composition
(Provimi Kliba, Kaiseraugst, Switzerland) and domestic
mains tap water were available ad libitum.
least 5 days after cannulation of the bile duct, and 2 days after
implantation of jugular vein cannulae.
Two male Beagle dogs with a body weight range of 14.7–
15.8 kg were surgically prepared with indwelling bile duct
cannulae under general isoflurane anaesthesia. The common
bile duct was cannulated with a T-piece catheter between the
last hepatic junction and the entrance to the duodenum. Bile
was allowed to flow continuously into the duodenum until the
animals were required for dosing. A balloon at the end of the
central cannula was inflated approximately 1 h prior to dose
administration to divert bile into the proximal cannula for
collection via the access port. During experiments, artificial
bile was infused via the distal cannula at an hourly rate of
3 ml/kg for 8 h per day.
Two male Beagle dogs, ca. 12 months of age with body
weights of 14.7 and 15.8 kg, which had been previously
prepared with indwelling bile-duct cannulae, were used in the
study. These animals were supplied by Harlan UK Limited
and were individually identified by tattoo. Animals were
observed daily during the acclimatization period and clinical
signs were recorded. Following surgery, the animals were
observed 3–4 times daily for up to 10 days after surgery.
Haematology and clinical chemistry parameters were mea-
sured prior to dosing and were shown to be within normal
limits. During the study period, the animals were housed
individually in stainless steel metabolism cages specially
designed for the separate, quantitative collection of urine and
faeces. With the exception of a period of fasting the night
before and until 4 h after dose administration, animals were
given a daily allowance of 400 g of a standard laboratory diet
of known formulation (Harlan Teklad Global Diet Code
2021). Domestic mains quality water was available ad
libitum.
Artificial bile was prepared by dissolving 25 mmol of
taurocholic acid, 12.5 mmol of lecithin and 0.3 mmol of
cholesterol in a mixture of 140 ml of chloroform and 70 ml of
methanol. After evaporation of the organic solvents, the dry
substance was re-dissolved in 200 ml of physiological saline,
aliquoted and stored at ꢂ20 ꢀC. A buffer solution containing
40 mmol of sodium hydrogen carbonate and 90 mmol of
sodium chloride was prepared in 2 l of bidistilled water and
stored frozen at ꢂ20 ꢀC. Both stock solutions were mixed
freshly in a ratio of 1:9 (v/v) prior to the start of the infusion.
Macitentan formulations and dosing
For intravenous dosing to rats, the 14C-macitentan stock
solution in acetonitrile containing 0.2 mCi/ml of radioactivity
was initially transferred into a glass vial and the solvent was
allowed to evaporate overnight. The remaining solid was
dissolved in 450 ml of PEG400 followed by the addition
of 550 ml of an aqueous 50 mM Tris-HCl buffer (pH 8.3)
to achieve final drug and radioactivity concentrations of
0.6 mg/ml and 50 mCi/ml, respectively. The targeted dosing
volume was 1.0 ml/kg. For oral dosing, the specific activity
was adjusted by the addition of non-labeled macitentan. For
the preparation of 5 ml of oral dosing solution, 2.4 mg of
non-labeled macitentan was added to 255 mL of the
14C-macitentan stock solution in acetonitrile. After complete
solubilisation of the solid, the acetonitrile was allowed to
evaporate overnight. The solid was dissolved in 2.25 ml of
PEG400 followed by the addition of 2.75 ml of an aqueous
50 mM Tris-HCl buffer (pH 8.3) to achieve target drug and
radioactivity concentrations of 0.6 mg/ml and 10 mCi/ml,
respectively. This solution was administered orally by gastric
gavage at a dosing volume of 5.0 ml/kg. Oral and intravenous
target doses for the metabolic profiling in bile duct-
cannulated rats were 3 and 0.6 mg/kg, respectively, while
the radioactive dose was 60 mCi/kg for both dosing routes. All
formulations were prepared freshly on the day of the
experiment.
Animal surgery
For the simultaneous collection of urine, bile and faeces, a
permanent, double bile fistula was implanted into rats. About
30 min prior to general anaesthesia, rats received a subcuta-
neous dose of TemgesicÕ (buprenorphine, 0.01 mg/kg). Rats
were then anesthetized by an intraperitoneal injection of a
mixture of ketamine (100 mg/kg) and xylazine 2% (10 mg/
kg). All microsurgical procedures were performed under
aseptic conditions. Following a laparotomy from the linea
alba, the common bile duct was prepared and then
bidirectionally cannulated for the simultaneous collection of
natural bile and re-infusion of artificial bile. Two pieces of 4.0
silk suture were used to secure both cannulated ends of the
bile duct. Both catheters were tunneled subcutaneously and
the ends of the catheters were plugged together with a plastic
seal, which was then fixed at the neck musculature. During
the recovery period, this set-up allowed for a normal bile
circulation. For bile collection, both cannulae were inter-
rupted and natural bile was collected into appropriate
containers while artificial bile was infused at a rate of 1 ml/h
in order to maintain normal bile homeostasis. To allow for
serial blood sampling, a satellite group of Wistar rats had
jugular vein cannulae implanted 2 days prior to the experi-
ment. After surgery and during recovery from general
anaesthesia, all animals were additionally offered Solid
Drink Energy (Triple A Trading, Tiel, The Netherlands).
Rats were included into the study after a recovery period of at
14C-macitentan was administered to Beagle dogs at total
oral and intravenous doses of 3 and 0.6 mg/kg, respectively,
both containing a radioactive dose of 200 mCi. For oral
dosing, the specific activity of 14C-macitentan was adjusted
by the addition of non-labeled drug and then formulated as a
solution in PEG400. A 50 mM aqueous Tris buffer (pH 8.3)
containing each 25% of PEG400 and N-methylpyrrolidone
was used for intravenous dosing.

258 A. Treiber et al.
Xenobiotica, 2016; 46(3): 253–267
suspending aliquots of the faeces in three equivalents (w/v)
of a 0.1 M phosphate buffer (pH 7.4) and homogenization
using an Ultra Turrax T25 (IKA, Staufen, Germany). To a
200 mg aliquot of this suspension, three equivalents (v/w) of a
1:1 (v/v) mixture of acetonitrile and water were added and the
mixture was homogenized with a Vortex mixer for about 60 s.
The resulting suspension was centrifuged at 20 800 g and
room temperature for 6 min. The supernatant was removed
and the pellet was extracted twice, each time with 600 ml of
the acetonitrile/water mixture. For the determination of total
radioactivity and HPLC analysis, the supernatants of all
three extraction steps were combined, evaporated to dryness,
reconstituted in 550 ml of a 1:1 (v/v) methanol/water mixture,
filtered (PTFE, pore size 0.45 mm, Infochroma, Zug,
Switzerland) and submitted to HPLC analysis.
Sample collection and work-up
Serial blood samples of 0.25 ml volume were taken from
jugular vein-cannulated rats (n ¼ 2 per route) over a period of
32 h after intravenous and oral dosing. Blood was collected on
ice into vials containing EDTA as anticoagulant. Plasma was
separated by centrifugation at 3220 g and 4 ꢀC for 20 min and
samples were stored at ꢂ20 ꢀC pending analysis. For the
determination of total radioactivity, an aliquot of 50 ml rat
plasma was mixed with 4 ml of liquid scintillation cocktail
and analyzed using a Tricarb 2300 TR liquid scintillation
analyzer (Perkin Elmer, Zu¨rich, Switzerland) with lumines-
cence correction and on-line quenching correction by means
of an internal standard. For the recording of metabolic
profiles, 100 ml of rat plasma was mixed with 200 ml methanol
for protein precipitation, homogenized on a Vortex mixer
(Merck, Zu¨rich, Switzerland) for about 60 s and centrifuged
at 2000 g for 5 min at room temperature. 200 ml of the
supernatant was submitted to HPLC analysis.
Analytical method
Metabolic profiles were recorded using HPLC coupled to
radioactivity detection. The analytical device consisted of two
Shimadzu HPLC pumps model LC-10AD-VP equipped with a
membrane degasser (DGU-14A), a Shimadzu system con-
troller model SCL-10A VP, a Shimadzu UV detector SPD-
10AV VP, a Berthold Radioflow Detector LB509 and a
Shimadzu autosampler model SIL-HTc. Radioactivity was
quantified with an 150 ml yttrium/glass solid detection cell
Dog blood was collected from the jugular vein at pre-
defined time points over a period of 96 h after oral and
intravenous dosing into EDTA-containing tubes. One aliquot
was retained for analysis of total radioactivity using liquid
scintillation counting. Plasma was separated from the remain-
ing sample by centrifugation. For the recording of metabolic
profiles, 500 ml of dog plasma was mixed with 1 ml methanol,
vortex-mixed for about 30 s and then centrifuged at 20 800 g
for 5 min at room temperature. The supernatant was
evaporated to about 50% of its initial volume and submitted
to HPLC analysis.
YG-150-S4D
(Berthold
Technologies,
Regensdorf,
Switzerland). Data acquisition was done using the RadioStar
software (version 3.0; Berthold Technologies). The chroma-
tographic separation of macitentan and its metabolites was
achieved on a Phenomenex Luna C18 (5 mm, 250 ꢁ 4.6 mm
ID) at 30 ꢀC with a flow rate of 1.0 ml/min. Mobile phases
consisted of 0.1% (v/v) aqueous trifluoroacetic acid (phase A)
and acetonitrile (phase B). Two different linear gradient
methods with run times of 50 and 60 min, respectively, were
used for the analysis of in vitro and in vivo samples. Using
these chromatographic conditions, macitentan exhibited
retention times of 43 and 53 min. The effect of the different
biological matrices on the shift in retention times did not
exceed 0.4 min.
Rat urine was collected in metabolic cages over a period of
48 h. For the determination of total radioactivity, 10 ml
aliquots of rat urine were mixed with 4 ml of liquid
scintillation cocktail and analyzed on a Tricarb 2300 TR
liquid scintillation analyzer. For HPLC analysis, the urine
samples were centrifuged at 20 800 g for 5 min at room
temperature and an aliquot of the supernatant was submitted
to HPLC analysis without further treatment. Dog urine was
collected into containers over a period of 96 h. During the first
48 h, containers were cooled by dry ice. For the recording of
metabolic profiles, urine aliquots were centrifuged at 20 800 g
for 5 min at room temperature and then directly submitted to
HPLC analysis.
Metabolite structure identification
Metabolite structures were investigated using liquid chroma-
tography combined with high resolution mass spectrometry
and radiodetection (split ratio 1:5). The LC–MS system
consisted of two LC-20ADXR pumps, a SIL-20AC/HT
Bile sampling in the rat and dog was performed continu-
ously after intravenous or oral dosing and collected in
fractions over periods of 48 and 96 h, respectively. Dog bile
was collected into containers cooled by dry ice during the first
48 h after dosing. For the determination of total radioactivity,
10 ml bile was supplemented with 990 ml of a 1:1 (v/v) mixture
of methanol and water and homogenized with a Vortex mixer
for about 30 s. A 10 ml of this mixture was added to 4 ml of
scintillation cocktail and radioactivity quantified by liquid
scintillation counting. For the recording of metabolic profiles,
rat bile was mixed with one volume equivalent of a 1:1 (v/v)
mixture of methanol and water, homogenized with a Vortex
mixer for about 30 s and centrifuged at 20 800 g at room
temperature for 5 min.
autosampler,
(Shimadzu, Reinach, Switzerland) and a Hot Dog 200/300
column oven (ThermoFisher, San Jose, CA).
Chromatographic separation was achieved using
a
five channel on-line degasser unit
a
Phenomenex Luna C-18 (250 ꢁ 4.6 mm ID, 5 mm) kept at
30 ꢀC with a flow rate of 1 ml/min and a gradient method over
65 min. Mobile phase A consisted of 0.1% (v/v) aqueous
formic acid and mobile phase B of acetonitrile. The HPLC
was coupled with an LTQ Orbitrap Velos Pro mass spec-
trometer equipped with a heated electrospray source H-ESI II
(ThermoFisher, San Jose, CA). The capillary potential was set
to 3 kV and the S-Lens was set to 67%. Sheath and auxiliary
gas were supplied using 40 and 5 arbitrary units, respectively,
and desolvation was supported by a capillary operated
Faeces were collected in parallel to urine and bile over
periods of 48 or 96 h in the rat and dog, respectively.
Radioactivity was extracted from these samples by

DOI: 10.3109/00498254.2015.1070302
Macitentan metabolism in rat and dog 259
at 200 ꢀC. Full scan high resolution LC–MS chromatograms
were recorded in positive ion mode at a resolving power
of 100 000 and further evaluated using Xcalibur 2.2 and
Metworks 1.3 as software packages to identify potential
metabolites (Thermo Electron, San Jose, CA). Selected ion
chromatograms of standard metabolic transformations were
generated and checked for the presence of signals in the
respective chromatograms. The presence of metabolites was
confirmed by accurate mass measurements. Putative metab-
olites were further investigated by MS/MS experiments in
positive ion mode using collision-induced dissociation (CID)
in the ion trap with 40% normalized collision energy, and high
energy collision dissociation (HCD) in the collision cell at
40% normalized collision energy as fragmentation tool. On-
line H/D exchange experiments were performed to investigate
the number of acidic protons using D2O with 0.1% (v/v) of
formic acid as eluent. Radiochemical detection was performed
using a Radioflow detector LB513 with a 500 ml liquid cell
Z-500 and a LB5035 pump for supplying liquid scintillation
cocktail at 3 ml/min (Berthold AG, Regensdorf, Switzerland).
the slope of a ln-linear regression of the unweighted data
considering the last concentrations versus time points ꢃ lower
limit of quantification). AUC0–inf was accepted if the
AUC%extp, i.e. the percentage of area extrapolated to infinity,
did not exceed 20%, otherwise AUC0–last was reported. Values
below the lower limit of quantification were set to ‘‘zero’’
before the first measurable concentration, and to ‘‘missing’’
after the last measurable concentration.
Results
The metabolism and excretion of macitentan have been
studied in bile-duct cannulated rats and dogs after oral and
intravenous dosing of 14C-labeled drug and parallel sampling
of bile, urine and faeces over periods of 48 and 96 h,
respectively. Blood was sampled at selected time points from
bile-duct cannulated dogs while for rats blood was sampled
from satellite animals. Table 1 summarizes the total radio-
activity recovered from bile, urine and faeces per sampling
interval and over the entire collection period.
Biliary excretion was the major elimination route of
macitentan in the rat as 71.0% and 48.5% were recovered
from bile after intravenous and oral dosing. The vast majority
of radioactivity was excreted within the first 24 h after
intravenous dosing while biliary excretion was somewhat
delayed after oral dosing. Renal excretion accounted for 15.8–
17.8% of the dose, with no relevant differences in the time
course between dosing routes. 9.5% and 23.1% of the dose
were recovered from faeces after intravenous and oral dosing,
respectively. Total excretion reached 89.4–96.3% over the
48-h collection period.
Pharmacokinetic analysis
Pharmacokinetic parameters of macitentan and M6 were
estimated using the WinNonlin software package
(Professional Version 3.0, Pharsight Corporation, Mountain
View, CA). The following parameters were calculated: Cmax
(peak plasma concentration), Tmax (time to reach peak plasma
concentration), T1/2 (apparent terminal half-life, calculated as
T1/2 ¼ ln(2)/lz); AUC0–last (area under the plasma concentra-
tion versus time curve up to the last measurable concentration,
calculated by the log-linear trapezoidal rule); AUC0–inf (area
under the plasma concentration versus time curve extrapo-
lated to infinity, calculated as AUC0–inf ¼ AUC0–last + Clast/lz
where lz (terminal rate constant, lambda z) is the negative of
Biliary excretion was also the predominant elimination
pathway in the dog, accounting for 33.8–39.2% of the dose,
while renal excretion was 21.9–27.2% over the 96-h collection
interval. 18.3% of the dose was recovered from faeces, with
Table 1. Excretion of total radioactivity in bile, urine and faeces of rats and dogs after intravenous and oral dosing of
14C-macitentan.
Matrix
Bile
Sampling period (h)
Rat iv (%)
Rat oral (%)
Dog iv (%)
Dog oral (%)
0–8a
8–24b
24–48
48–72
72–96
Total
0–8
31.7
35.7
3.6
9.7
24.5
14.3
–
8.5
19.6
8.6
1.9
0.6
39.2
3.6
12.1
8.5
2.1
0.9
27.2
50.1
6.9d
8.6
1.7
1.1
18.3
84.7
5.7
16.7
9.2
2.1
0.1
33.8
3.1
7.3
8.3
c
–
–
71.0
3.6
7.7
4.5
–
–
48.5
4.0
8.6
5.2
–
Urine
8–24
24–48
48–72
72–96
Total
0–8
2.5
0.7
–
–
15.8
1.0
3.3
5.2
–
–
9.5
96.3
17.8
0.1
15.1
7.9
–
–
23.1
89.4
21.9
50.1
5.8d
11.0
0.6
0.9
18.3
74.0
Faeces
8–24
24–48
48–72
72–96
Total
All excreta
Data are presented as means (n ¼ 2) and expressed as percent of dose. iv, intravenous.
a0–6 h collection period for the dog.
b6–24 h collection period for the dog.
cNo sample collection.
dCollection period: 0–24 h.

260 A. Treiber et al.
Xenobiotica, 2016; 46(3): 253–267
no difference between dosing routes. Total excretion was
84.7% and 74.0% over 96 h after intravenous and oral dosing,
respectively. No difference in the time course of excretion was
observed between dosing routes. The incomplete recovery of
total radioactivity during the 96 h collection period is likely
the result of the long half-life of metabolite M6 (Table 4 and
Figure 6, panel B).
spectroscopic analyses, such as MS and NMR. Structural
1
assignment of M2 was based on 1D and 2D H and13C NMR
spectroscopy, comparison with literature data of close analogs
(Griffin & Stevens, 1994; Jovanovic, 1984) and 13C chemical
shift predictions calculated using ChemBioDraw Ultra, ver-
sion 14.0.0.117. N-oxidation in 1-position of the pyrimidine is
the structural proposal with best fit of spectroscopic data. The
structure of the 7-imino-2,3-dihydro-7H-oxazolo[3,2-c]pyri-
midin-7-imine core of metabolite M27 was supported by the
Metabolic patterns were recorded from all matrices using
HPLC coupled to radiodetection. Radiochromatograms of
representative rat and dog plasma, bile, urine and faeces
samples are depicted in Figures 2 and 3. The relative abundance
of each metabolite in these radiochromatograms together with
total radioactivity data was used to estimate the amount of each
metabolite as a fraction of macitentan dose. The results of this
analysis are summarized in Table 2 for both species.
1
chemical shift of the observed H and 13C NMR signals, the
two exchangeable protons, 1H–13C heteronuclear multiple-
bond correlations (HMBC) and 1H rotating frame nuclear
Overhauser effect (ROESY) NMR spectra, in particular by the
HMBC correlations of H-C2 with C12 and H-C12 with C2
and C6 (3J); and the ROESY correlation of H-C2 with H2-C12
and H2-C11. The metabolic pathways of macitentan in the
rat and dog are depicted in Figure 5. Most macitentan
metabolites are products of combined dealkylation and
hydrolysis reactions. Figure 5 therefore aims to outline the
chemical relationship between the various biotransformation
products rather than claiming knowledge about the exact
sequence of metabolic steps.
The chemical nature of macitentan metabolites was
identified with the help of high resolution mass spectrometry.
Table 3 gives an overview of the experimentally determined
exact masses and compares them to their theoretical value.
The fragmentation pattern of parent macitentan is depicted in
Figure 4. Key MS fragments were m/z 201 and m/z 413
resulting from cleavage of the proximal and terminal C–O
bond of the ethylene glycol linker. Out of a total of 23
metabolites in rat and dog, structural proposals were made for
19 biotransformation products. For most of the phase I
metabolites, i.e. M2, M3, M4, M5, M6, M18, M19, M21 and
M27, the proposed structures were confirmed with the help of
synthetic references on the basis of identical retention times
and mass spectrometry fragmentation patterns. For M2, M3,
M4 (Bolli et al., 2012), M5, M6 (Bolli et al., 2009, 2012),
M18, M19 and M21, the structure of the chemically
synthesized reference compounds was evident from their
synthetic access and was confirmed by the data obtained from
Beyond parent macitentan, the depropylated metabolite M6
was the only entity circulating in rat and dog plasma (Figures 2
and 3, panel A). The individual concentrations of macitentan
and M6 were estimated from the relative abundance of both
entities in the respective radiochromatograms and the total
radioactivity in each plasma sample, and used to calculate
basic pharmacokinetic parameters and construct plasma
concentration versus time plots (Table 4 and Figure 6). As
macitentan was not detected in rat plasma taken later than 8 h
post-dose, pharmacokinetic parameters derived for macitentan
are only an approximation of their true values.
Figure 2. Metabolic profiles of macitentan in rat plasma (panel A), bile (panel B), urine (panel C) and faeces (panel D) after oral dosing of
14C-macitentan.

DOI: 10.3109/00498254.2015.1070302
Macitentan metabolism in rat and dog 261
Figure 3. Metabolic profiles of macitentan in dog plasma (panel A), bile (panel B), urine (panel C) and faeces (panel D) after oral dosing of 14C-
macitentan.
Table 2. Prevalence of macitentan metabolites in bile, urine and faeces after oral dosing of 14C-macitentan to rats and
dogs.
Rat
Doga
Compound
Bile
7.2
Urine
Faeces
0.9
Bile
Urine
Faeces
1.9
Macitentan
M1
M2
0.1
2.7
0.2
0.2
M3
M6
M8
M9
9.7
7.2
2.4
1.3
8.7
24
0.6
2.4
2.9
0.3
0.1
0.2
0.2
2.8
0.8
M14
M15
M16
M17
M18
M19
M20
M21
M24
M25
M26
M27
M29
0.7
0.1
7.4
0.2
0.6
4.4
4.0
2.3
1.0
3.3
5.7
0.6
1.4
4.0
2.4
0.2
2.6
1.4
2.2
2.8
1.3
0.1
1.1
Data are presented as percentage of dose.
aDue to the low total amount of radioactivity, samples from the 48–96 h collection period in dog urine, 72–96 h in dog bile
and 24–96 h in dog faeces were not included.
Macitentan mean plasma clearance (CL) was 7.4 and
5.2 ml/(min kg) in rat and dog, respectively. Considering the
blood/plasma ratios of 0.63 and 0.54 for rat and dog (de
Kanter et al., 2015), blood clearance of macitentan was about
17–22% of the respective liver blood flows and qualifies
macitentan as a low clearance drug in both species. Volume of
distribution at steady-state (Vss) was 1.0 l/kg in the rat and
1.6 l/kg in the dog, indicating good overall penetration into
tissues. Apparent terminal plasma half-lives after intravenous
dosing were 1.8 and 5.7 h in the rat and dog, respectively.
Macitentan was slowly absorbed after oral dosing and peak
plasma concentrations were only reached after 6.0 and 2.8 h in

262 A. Treiber et al.
Xenobiotica, 2016; 46(3): 253–267
the rat and dog. Oral bioavailability was in the range of 11–
but again no parent drug were found in rat urine. M19,
Table 3. Mass spectrometry characteristics of macitentan metabolites in rat and dog.
Compound
Experimental exact massa
Theoretical exact mass
Elemental composition
C19H20Br2N6O4S
Key fragments
H/D exchange
Macitentan
M1
M2
M3
M4
M5
M6
M7
M8
586.9704
706.9765
481.9456
465.9508
431.0384
445.0176
544.9237
602.9656
720.9558
749.0234
736.9509
323.9980
310.0188
265.9924
560.9186
388.9917
641.9829
778.9981
778.9980
370.9800
586.9706
706.9765
481.9458
465.9509
431.0383
445.0176
544.9237
602.9655
720.9560
749.0234
736.9507
323.9978
310.0186
265.9923
560.9185
388.9914
641.9829
778.9976
778.9976
370.9808
413, 201
545, 201
308, 201
n/ab
n/a
6
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
22H24Br2N6O9S
16H13Br2N5O3
16H19Br2N5O2
15H19BrN4O4S
15H17BrN4O5S
16H14Br2N6O4S
19H20Br2N6O5S
n/a
n/a
n/a
n/a
1
n/a
6
n/a
8
n/a
n/a
n/a
3
n/a
5
387
386, 324
371, 292, 201
466, 429, 292,201
547,466, 292, 201
587, 201
371, 292, 217
266, 199
266
187
308, 201
–
468, 292, 201
292, 201
292, 201
292, 211
22H22Br2N6O10
S
M9
25H30Br2N6O9S
M16
M17
M18
M19
M20
M21
M24
M25
M26
M27
22H22Br2N6O11
12H10BrN3O3
12H12BrN3O2
10H8BrN3O
S
16H14Br2N6O5S
12H13BrN4O4S
22H21Br2N5O8
25H28Br2N6O11
25H28Br2N6O11
12H11BrN4O3S
S
S
6
6
2
aAll exact masses refer to the protonated non-labeled entities.
bn/a: not determined.
resulting from combined hydrolysis of the sulfamide moiety
and oxidative cleavage of the ethylene glycol linker, M21,
dealkylated at both the sulfamide moiety and the ethylene
glycol linker, M14 and M20 were the major products in urine,
each accounting for 2.3–4.4% of the dose. None of the other
urinary metabolites individually exceeded 1%. No structural
proposals are available for metabolites M14, M15 and M29.
Parent macitentan, M6 and the common hydrolysis product
M3 were present in rat faeces. Ex vivo experiments with
freshly collected faeces confirmed that macitentan and M6
were both vulnerable to hydrolysis in the presence of
intestinal content (data not shown).
H+
Br
Br
N
H
H
N
N
O
H3C
S
O
N
O
O
N
N
m/z 587
m/z 413
m/z 201
Thirteen metabolites were found in dog bile together with
trace amounts (ca. 0.1%) of unchanged macitentan. M16, the
glucuronide of M20, was the major entity, accounting for
7.4% of the dose while most of the other products were
present in a range from 1% to 3%. All biliary metabolites
observed in the rat were also found in the dog. Dog urine
contained nine metabolites. The aminopyrimidine M18, M19
and M21 were the most prevalent products, representing 2.4–
5.7% of the dose, while none of the other urinary metabolites
exceeded 1.1%. Unchanged macitentan was not observed in
dog urine. Similar to the rat, macitentan, M3 and M6 were
found in dog faeces, all in the range of 1.3–2.4% of dose.
The metabolism studies with macitentan in the rat and dog
were complemented with in vitro experiments using liver
microsomes and hepatocytes of both species (Figure 7). M6
was the major product in experiments with liver microsomes
(panels A and B) followed by metabolites M3 and M7. To a
lesser extent, M3 was also found in control experiments
without liver microsomes (data not shown), which further
supports the hypothesis of a mixed chemical as well as
enzyme-catalyzed hydrolysis. M7 is the product of hydrox-
ylation in the b-position of the propyl side chain. In rat and
dog hepatocytes (panels C and D), the alcohol M4, resulting
Figure 4. MS fragmentation pattern of macitentan.
14% for both species but is likely underestimated for the
abovementioned reasons.
Plasma exposure to metabolite M6 exceeded that of parent
macitentan by 3.6- to 7.1-fold in both species, irrespective of
the dosing route. Its apparent terminal half-life in the rat was
7.9 and 10 h after intravenous and oral dosing. In the dog, M6
half-life was 14 h irrespective of the dosing route and
exceeded that of parent macitentan by about 2.5-fold. Peak
plasma concentrations of M6 were reached about 7 h after
dosing in both species.
Five metabolites but no parent macitentan were observed
in rat bile (Table 2). With exception of the minor product M9,
a glucose conjugate of parent macitentan, all other biliary
metabolites were secondary products of the depropylated M6.
The glucuronide conjugate M8 was by far the major entity,
accounting for 24% of the dose, accompanied by the glucose
conjugate M1, M6 itself, and the oxidized derivative M20,
each accounting for 4.0–8.7% of the dose. Eleven metabolites

DOI: 10.3109/00498254.2015.1070302
Macitentan metabolism in rat and dog 263

264 A. Treiber et al.
Table 4. Pharmacokinetic parameters of macitentan and M6 in the rat and dog after intravenous and oral dosing of 14C-macitentan.
Xenobiotica, 2016; 46(3): 253–267
Rata
Dogb
Route
iv
Parameter
Macitentan
M6
Macitentan
M6
AUC0–inf (nmol h/ml)
CL (ml/min kg)
Vss (L/kg)
3.10 (2.98–3.26)
7.4 (7.3–7.5)
1.0 (0.8–1.1)
1.8 (1.4–2.2)
1.73 (1.53–1.95)
0.34 (0.34–0.35)
6.0 (6.0–6.0)
NC
14.1 (13.6–14.5)
ND
ND
7.9 (7.8–8.1)
12.3 (11.7–13.0)
0.69 (0.66–0.73)
7.0 (6.0–8.0)
10 (9.9–10)
ND
3.10 (2.97–3.23)
5.2 (5.0–5.4)
1.6 (1.5–1.7)
5.7 (5.3–6.2)
2.11 (1.81–2.46)
0.30 (0.30–0.31)
2.8 (1.5–4.0)
5.5 (4.4–6.9)
14 (12–15)
11.3 (9.76–13.2)
ND
ND
14 (13–15)
9.31 (7.70–11.3)
0.41 (0.39–0.44)
7.0 (6.0–8.0)
14 (12–16)
ND
T1/2 (h)
oral
AUC0–last (nmol h/ml)
Cmax (nmol/ml)
Tmax (h)
T1/2 (h)
F (%)c
11 (10–12)
All data are given as mean (n ¼ 2) and range. iv, intravenous; ND, not determined; NC, not calculable.
aIntravenous and oral doses were 0.6 and 3 mg/kg containing 60 mCi/kg 14C-macitentan formulated in a 55:45 mixture of 50 mM TRIS buffer (pH 8.3)
and PEG400 for intravenous and oral dosing and administered in fed state.
bIntravenous and oral doses were 0.6 and 3 mg/kg nominally containing 200 mCi 14C-macitentan, formulated in PEG400 for oral use and in a 2:1:1
mixture of 50 mM TRIS buffer (pH 8.3), PEG400 and N-methylpyrrolidone for intravenous dosing, and administered in fasted state.
cOral bioavailability was estimated from the mean AUC after intravenous dosing.
Figure 6. Plasma concentration versus time
profile of macitentan and M6 after oral
dosing of 14C-macitentan to rats and dogs.
from oxidative cleavage of the ethylene glycol linker, was
observed in addition.
depicted in Figure 5. Macitentan undergoes five primary
metabolic pathways. Conjugation with glucose, most likely at
one of the nitrogen atoms of the sulfamide moiety, yields M9.
Oxidative depropylation, catalyzed by microsomal enzymes
via initial hydroxylation at the a carbon atom of the propyl
chain, gives M6, while hydroxylation of the b carbon atom
generates M7. Hydroxylation at the terminal carbon atom was
Discussion
The metabolic pathways of macitentan in the rat and dog have
been identified in bile-duct cannulated animals and are

DOI: 10.3109/00498254.2015.1070302
Macitentan metabolism in rat and dog 265
Figure 7. Metabolic profiles of macitentan with liver microsomes (panels A and B) and hepatocytes (panels C and D) of rat and dog.
ruled out by co-chromatography with an authentic reference
of the corresponding alcohol (Boss et al., 2006) based on a
retention time difference of about 2.5 min (35.2 min for M7
versus 32.8 min for the terminal alcohol, data not shown). As
the b carbon atom in macitentan is prochiral, non-selective
hydroxylation generates both enantiomeric alcohols. No direct
evidence for formation of both alcohols was provided by the
radiochromatograms of the hepatocyte incubations (Figure 7)
as analysis was done by non-chiral chromatography. However,
the glucuronides of M7, i.e. M25 and M26, have been
identified in dog bile. Conjugation with glucuronic acid
converts the enantiomeric alcohols into diastereoisomeric
glucuronides which were well separated by the analytical
method (Figure 3, panel B). The isomers M25/M26 therefore
provide indirect evidence that the hydroxylation to M7 is not
stereoselective but preferentially yields the aglycon of M26.
Another primary pathway leads to the alcohol M4 via loss
of the 5-bromopyrimidine moiety. The molecular mechanism
of this biotransformation is unknown. Aliphatic hydroxylation
of the terminal carbon atom of the ethylene glycol linker
would yield the alkoxyacetaldehyde but not the alcohol M4.
Alternatively, oxidation at the 2-position of the pyrimidine
ring would release M4 in an ipso-substitution type reaction
which has been previously described for other alkoxypyr-
imidines (Liu et al., 2005). M4 but not the acetaldehyde was
observed with rat and dog hepatocytes but was absent in
microsomal incubations. Neither M4 nor the corresponding
acid M5 was directly detected in rat or dog excreta. M17, M18
and M21 are secondary metabolites resulting from oxidative
depropylation and/or hydrolysis of M4 and M5, but none of
them allows for a conclusion on the nature of the primary
intermediate.
Finally, macitentan and M6 undergo hydrolysis of the
sulfamide moiety to yield M3. M3 has been observed in rat
and dog faeces and is formed in vitro with liver microsomes
and hepatocytes. Control experiments in the absence of
microsomal protein or liver cells indicate that M3 formation is
at least partly a chemical process but might also result from
enzyme-catalyzed hydrolysis.
M3 and M6 are both subject to secondary metabolism.
M3 undergoes N-oxidation at the 1-position of the central pyr-
imidine ring to yield M2, as evidenced by identical retention
time and fragmentation pattern of an authentic reference,
and is glucuronidated to M24. M6 is conjugated with either
glucose or glucuronic acid to M1 and M8, respectively.
Oxidation of M6 leads to M20 which is subsequently
glucuronidated to M16. The exact site of oxidation in M20
is unknown.
The structure of the bicyclic metabolite M27 has been
assigned based on exact mass spectra, 1H and 13C NMR
1
1
chemical shifts, H–13C HBMC correlations and H ROESY
NMR spectra. The ring-closed structure was further sub-
stantiated by the presence of only two exchangeable protons.
Formation of M27 requires nucleophilic attack of the
pyrimidine nitrogen at the terminal oxygen atom of the
ethylene glycol linker and the presence of a good leaving
group. Chemically, this is achieved by tosylation of the
terminal alcohol (cf. Materials and Methods section). From a
metabolic perspective, the alcohol could possibly be activated
by sulfation or glucuronidation. While there is no experimen-
tal evidence for such conjugation products, the mechanism
underlying M27 remains unclear.
Macitentan excretion in rat and dog was strongly depend-
ent on metabolism on the basis of low amounts of unchanged

266 A. Treiber et al.
Xenobiotica, 2016; 46(3): 253–267
parent drug detected in urine and bile. The overall metabolic
pattern of macitentan was similar in both species. However,
the relative importance of the individual metabolic pathways
showed differences between species. M6 and its secondary
products, i.e. M1, M8, M16 and M20, represented about 75%
of the rat metabolite pool, calculated from the relative
abundance of each metabolite and normalized to the total
amount of radioactivity for which structural information was
available. The M3 and M4 pathways were equally important
in the rat, representing about 13% and 10% of the overall
metabolite pool, respectively. Glucosidation of macitentan to
M9 was a minor pathway accounting for only about 1%, and
hydroxylation to M7 was not observed at all. In the dog, the
M6 pathway was also the most important metabolic route but
only represented about 40% of the dog metabolite pool. M4-
derived products, i.e. M17, M18, M19 and M21, were more
prominent in the dog than in the rat, accounting for ca. 27% of
all structurally identified products. The glucuronides M25 and
M26, secondary products of M7, represented 8% of the dog
pool while they were absent in the rat. The importance of the
M3 and M9 pathways was similar between species.
metabolite (Figures 2 and 3, panel A). After single oral
dosing, its exposure exceeds that of parent drug by 7.1- and
4.4-fold in the rat and dog, respectively. Metabolite M5 could
not be detected in the present study with low doses of
radiolabeled macitentan. Analysis of rat and dog plasma
samples from toxicity studies at higher doses using more
sensitive MS technology confirmed the presence of M5 in
both main animal species used in the non-clinical safety
assessment of macitentan (data not shown).
Metabolism and disposition of macitentan in man have
been investigated in healthy subjects after a single oral 10 mg
dose of 14C-labeled macitentan (Bruderer et al., 2012).
Following extensive biotransformation, macitentan metabol-
ites are equally excreted into urine and feces. Macitentan is
metabolized along four distinct primary pathways, i.e. (1)
oxidative depropylation of the sulfamide group to M6, mainly
catalyzed by CYP3A4 and a minor contribution of the
CYP2C family, (2) oxidative cleavage of the ethylene glycol
to M4 catalyzed by CYP2C9. M4 then undergoes oxidation to
the acid M5 followed by hydrolysis to M17, (3) hydroxylation
of the propyl chain to form M7, catalyzed by CYP2C enzymes
and (4) hydrolysis of macitentan and ACT-132577 to form
M3. M17 was the most prominent metabolite detected in
human urine, followed by M1, the glucose conjugate of M6,
the hydrolysis product M3 and the acid M5. Metabolites M3,
M5 and M17 were also observed in human faeces,
accompanied by smaller amount of M6 and M7. Though
quantitative comparisons are hampered by methodological
differences, as no biliary pattern is available from the
human ADME study, all primary metabolic pathways of
macitentan are covered in the rat and dog and therefore
qualify both species for the preclinical safety assessment of
macitentan.
The pharmacokinetics and metabolism of macitentan in
healthy subjects and PAH patients have been summarized in a
recent review (Sidharta et al., 2015). At the marketed dose of
10 mg, macitentan is slowly absorbed with a median Tmax of
8 h. Drug levels decline thereafter with an apparent terminal
elimination half-life of 16 h. As a consequence of its long-half
life, macitentan accumulates by about 1.4-fold after multiple
dosing. Pharmacokinetic data after intravenous administration
are not available but estimates on plasma clearance, volume
of distribution and oral bioavailability have been made using
physiology-based pharmacokinetic modeling (de Kanter et al.,
2015). On this basis, macitentan was predicted with a low
plasma clearance of 2.26 l/h, a Vss of 0.3 l/kg and an oral
bioavailability of 74%. The pharmacologically active metab-
olite M6 is slowly formed in man and reaches peak plasma
concentrations only about 30 h after macitentan administra-
tion. Plasma half-life of M6 is 40–66 h leading to an
accumulation of 7.1-fold after multiple dosing. M6 exposure
at steady-state exceeds that of parent macitentan by 2.7-fold.
The pharmacologically inactive metabolite M5 was observed
as a minor entity in human plasma.
Acknowledgements
The authors would like to thank Aude Weigel, Sybille
´
¨
¨
Flaschel, Stephanie Combes, Julien Grimont, and Jurgen
Seifert for their dedication and experimental contributions,
and Susan Flores and Makda Fisseha for their assistance in
the preparation of this manuscript.
Pharmacokinetic properties of macitentan in both animal
species (Table 4) resemble those in man with respect to slow
oral absorption and low plasma clearance. The slightly higher
Vss in animals likely reflects the species differences in plasma
protein binding (data not shown). Oral bioavailability in
animals was only 11–14% and thus significantly below
predicted values in man. Methodologically, these values
likely underestimate true oral bioavailability as radioactivity
measurements were used to derive macitentan plasma
concentrations. As this method is less sensitive compared to
MS-based analytics, full plasma concentration versus time
profiles could not be generated over the entire collection
interval. On the other hand, macitentan is vulnerable to
hydrolysis in the gut yielding the aminopyrimidine M3. This
pre-systemic degradation reduces the drug amount available
for oral absorption and might contribute to the lower
bioavailability observed in animals. Similar to man, M6 was
also present in plasma of rat and dog as the only circulating
Declaration of interest
This work was funded by Actelion Pharmaceuticals Ltd,
Allschwil, Switzerland. All authors are employees of Actelion
Pharmaceuticals Ltd.
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