Macitentan does not interfere with hepatic bile salt...

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TITLE PAGE

Macitentan does not interfere with hepatic bile salt transport Alexander Treiber, Päivi Aeänismaa, Ruben de Kanter, Stephane Delahaye, Marianne Treher,

Patrick Hess, Patricia Sidharta

Departments of Preclinical Drug Metabolism and Pharmacokinetics (A.T., P.A., R.K. S.D.),

Toxicology (M.T.), Pharmacology (P.H.) and Clinical Pharmacology (P.S.), Actelion

Pharmaceuticals Ltd, Allschwil, Switzerland

JPET Fast Forward. Published on April 25, 2014 as DOI:10.1124/jpet.114.214106

Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 25, 2014 as DOI: 10.1124/jpet.114.214106

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RUNNING TITLE PAGE

Running title: macitentan and hepatic bile salt transport

Corresponding author: Alexander Treiber Actelion Pharmaceuticals Ltd

Gewerbestrasse 16 4123 Allschwil Switzerland +41 61 565 65 92 (phone) +41 61 565 89 03 (fax) [email protected] Text pages: 50

Number of tables: 8

Number of figures: 5

Number of references: 51

Abstract: 187 words

Introduction: 595 words

Discussion: 1959 words

Section assignment: Toxicology

ABBREVIATIONS

ALT: alanine aminotransferase, ATP: adenosine triphosphate, AUC: area under the plasma

concentration vs. time curve, bid: bis in die (twice a day), BLQ: below limit of quantification,

BSEP: bile salt export pump, CHO: Chinese hamster ovary, CLint : intrinsic clearance, CLpo:

oral clearance, Cmax: (observed) peak plasma concentration, DMEM: Dulbecco's Modified

Eagle's Medium, DMSO: dimethyl sulfoxide, EDTA: ethylenediaminetetraacetic acid, ET-1:

endothelin-1, FCS: fetal calf serum, HBSS: Hank's balanced salt solution, HEPES: N-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid, HIV: human immunodeficiency virus, IC50:

concentration of an inhibitor that reduces the measured response by 50%, ID: internal

diameter, Ki: inhibition constant, NAD: nicotinamide adenine dinucleotide, NADH:

nicotinamide adenine dinucleotide, reduced form, NTCP: sodium taurocholate co-

transporting polypeptide, OATP: organic anion-transporting polypeptide, PBPK: physiology-

based pharmacokinetic (modeling), PK: pharmacokinetic(s), SD: standard deviation, Sf9:

Spodoptera frugiperda cell line, Tmax: time to reach maximum observed plasma

concentration, Tris: tris(hydroxymethyl)aminomethane.


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ABSTRACT

Treatment of pulmonary arterial hypertension with the endothelin receptor antagonist

bosentan has been associated with transient increases in liver transaminases. Mechanistically,

bosentan inhibits the bile salt pump BSEP leading to an intrahepatic accumulation of

cytotoxic bile salts eventually resulting in hepatocellular damage. BSEP inhibition by

bosentan is amplified by its accumulation in liver as bosentan is a substrate of OATP

transport proteins. The novel endothelin receptor antagonist macitentan shows a superior liver

safety profile. Introduction of the less acidic sulfamide moiety and increased lipophilicity

yield a hepatic disposition profile different from other endothelin receptor antagonists.

Passive diffusion rather than OATP-mediated uptake is the driving force for macitentan

uptake into the liver. Interaction with the NTCP and BSEP transport proteins involved in

hepatic bile salt homeostasis is therefore limited due to the low intrahepatic drug

concentrations. Evidence for this conclusion is provided by in vitro experiments in drug

transporter-expressing cells lines, acute and long-term studies in rat and dogs, absence of

plasma bile salt changes in healthy human volunteers after multiple dosing, and finally the

liver safety profile of macitentan in the completed phase III morbidity/mortality SERAPHIN

trial.


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INTRODUCTION

About a decade ago, endothelin receptor antagonists were introduced as a therapeutic concept

for the treatment of pulmonary arterial hypertension, a debilitating and finally fatal disease

for which no oral treatment option was available before. The dual endothelin receptor

antagonist bosentan (Tracleer®) was approved in 2001 as the first member of this new class,

followed by sitaxentan (Thelin®) in 2006 and ambrisentan (Letairis®/Volibris®) in 2007.

Macitentan (Opsumit®, N-[5-(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-

pyrimidinyl-N'-propylsulfamide) has been developed as a new generation endothelin receptor

antagonist with sustained receptor binding and improved receptor potency, pharmacokinetic

and liver safety profile (Iglarz et al., 2008; Raja, 2010). Most of these improvements result

from a modified tissue distribution as macitentan can freely diffuse into tissues rather than

being dependent on active transport.

Bosentan was approved at doses of 62.5 mg and 125 mg bid, but was initially studied at

higher doses for the treatment of hypertension (Krum et al., 1998) and chronic heart failure

(Sütsch et al., 1998). Chronic heart failure patients treated with bosentan 500 mg bid had an

18% incidence of elevated ALT levels vs. 4% on placebo. In a subset of patients

concomitantly treated with the antidiabetic glyburide, 29% had elevated ALT vs. 4% and 0%

on either placebo alone or placebo and glyburide. Changes in liver transaminases were

accompanied by dose-dependent increases in plasma bile salts and alkaline phosphatase.

Inhibition of the bile salt export pump BSEP by bosentan and its metabolites has been

identified as the likely mechanism underlying the observed changes in plasma transaminases

(Fattinger et al., 2001). BSEP is an ATP-dependent transport protein located at the

hepatocanalicular membrane and mediates the rate-limiting step in bile salt secretion from


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blood into bile (Gerloff et al., 1998; Stieger et al., 2000). Bosentan and its metabolites

inhibited taurocholate transport in vitro in canalicular rat liver membrane vesicles and in Sf9

cell vesicles overexpressing rat bsep. In rats, plasma bile salts increased in a dose-dependent

manner after intravenous dosing of bosentan (Stieger et al., 2000; Kis et al., 2009). These

initial findings in rats were later confirmed with human BSEP (Mano et al., 2007) and led to

the hypothesis that bosentan treatment initially triggers a disruption of bile salt homeostasis

through dose-dependent blockade of BSEP-mediated bile salt excretion into bile, eventually

resulting in their accumulation in liver cells. As bile salts are cytotoxic at high concentrations,

the observed liver transaminase elevations in man are believed to result from the secondary

bile salt toxicity in hepatocytes.

The hepatic disposition of bosentan is mediated by OATP transport (Treiber et al., 2007)

followed by extensive metabolism through CYP3A4 and CYP2C9 and finally excretion of

the metabolites into bile (Weber et al., 1999). As a consequence, bosentan pharmacokinetics

are sensitive to concomitant CYP3A4 and/or OATP inhibitors. While the potent CYP3A4

inhibitor ketoconazole increased bosentan in plasma by only about 2-fold (van Giersbergen et

al., 2002), more pronounced elevations were observed with the OATP inhibitor rifampicin

(van Giersbergen et al., 2007), the HIV protease inhibitor ritonavir/lopinavir (Kaletra®)

(Dingemanse et al., 2010) and cyclosporin A (Binet et al., 2000), the latter two being

combined CYP3A4/OATP inhibitors.

Conceptually, there are several options to design drugs with an improved side effect profile.

On one hand, improving receptor affinity and pharmacokinetic properties might yield drugs

that are effective at lower doses. The alternative approach is to avoid interactions with targets

critically involved in toxicity. Both approaches were combined in the discovery of


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macitentan. The present report summarizes the experimental evidence demonstrating that

macitentan does not interact with hepatic transport proteins critically involved in bile salt

trafficking and drug accumulation in the liver.


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MATERIALS AND METHODS

Chemicals and Reagents

Macitentan (N-[5-(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]-ethoxy]-4-pyrimidin-

yl]-N′-propylsulfamide) was obtained from Lonza AG (Visp, Switzerland) with a purity of

99.8%. 14C-Radiolabled macitentan with a specific activity of 55 mCi/mmol was purchased

from GE Healthcare (Little Chalfont, United Kingdom). Metabolite ACT-132577 was either

obtained from the chemistry department of Actelion Pharmaceuticals Ltd or from

SynphaBase (Pratteln, Switzerland), with purity in excess of 97%. 14C-ACT-132577 with a

specific activity of 56 mCi/mmol was obtained from Quotient Bioresearch (Rushden,

Northamptonshire, United Kingdom). Both radiolabeled compounds were supplied as

acetonitrile solutions with radiochemical purities in excess of 97%. Bosentan was obtained

from the chemistry department of Actelion Pharmaceuticals Ltd. Sodium taurocholate was

from Sigma (Buchs, Switzerland) whereas 3H-taurocholic acid with specific activity of

4.6-5.0 Ci/mmol was purchased from Perkin Elmer (Boston, Massachusetts, USA) as a

solution in methanol:ethanol (1:3) at a concentration of 1 mCi/ml. Estrone-3-sulfate and

atorvastatin calcium trihydrate were from Sigma-Aldrich (Buchs, Switzerland). 3H-estrone-3-

sulfate and 3H-atorvastatin calcium with specific activities of 50 Ci/mmol and 10 Ci/mmol,

respectively, were purchased from American Radiolabeled Chemicals (St. Louis, Missouri,

USA) as solutions in ethanol or ethanol:water (1:1). Cyclosporin A was purchased from

Fluka and rifampicin from Sigma-Aldrich. Liquid scintillation cocktails Filter-Count and

IRGA Safe Plus were purchased from Perkin Elmer (Zürich, Switzerland). Baculovirus-

infected Sf9 cell membrane vesicles overexpressing human BSEP were obtained from

SOLVO Biotechnology (Budapest, Hungary). All media and supplements for CHO and CHO

Flp InTM cells were obtained from Invitrogen AG (Basel, Switzerland).


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Transport experiments

Preparation of stock solutions

For BSEP and NTCP inhibition experiments, macitentan and ACT-132577 stock solutions

were initially prepared in DMSO in concentration ranges of 1 μM-100 mM (BSEP) and

1 μM-100 mM (NTCP), and then diluted with the buffer used in the transport experiments

(see below). For cellular transport experiments, macitentan and ACT-132577 DMSO stock

solutions were prepared in a range from 0.1 µM to 100 mM and again diluted with transport

buffer. Stock solutions of cyclosporin A and rifampicin were prepared in DMSO in a

concentration range from 1-50 mM. DMSO was also used to prepare the 10 mM and 100 mM

stock solutions of atorvastatin, taurocholic acid and estrone-3-sulfate.

Cell culture

CHO Flp InTM cells overexpressing human NTCP were cultured at passage numbers 5 to 19

on tissue culture dishes of 55 cm2 growth area (Sarstedt, USA) at 37 °C in a humidified

atmosphere containing 5% carbon dioxide. Cells were maintained in Ham's F-12 medium

supplemented with 10% fetal calf serum, penicillin/streptomycin (100 IU/ml), L-glutamine

(1 mM) and hygromycin B (500 μg/ml). For transport experiments, cells from a maximally

90% confluent 58 cm2 tissue culture dish were detached with trypsin-EDTA, uniformly

resuspended in Ham's F-12 medium and seeded on tissue culture dishes (Corning, USA). The

cells were used for transport experiments 72 h to 96 h later, when they were 80-90%

confluent. 24 h before starting the transport experiments, cells were additionally induced by

adding 5 mM sodium butyrate (Sigma-Aldrich, Buchs, Switzerland) to the medium.

CHO cells overexpressing human OATP1B1, OATP1B3 and OATP2B1 and wild-type

CHO cells were cultured at passage numbers 9 to 60 on tissue culture dishes (Corning, USA)

at 37 °C in a humidified atmosphere containing 5% carbon dioxide. All cell lines were


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maintained in DMEM containing 1 g/L glucose and supplemented with 10% fetal calf serum,

penicillin/streptomycin (100 IU/ml) and L-proline (0.05 mg/ml). The culture medium for the

OATP-expressing CHO cells additionally contained geneticin (500 μg/ml). For transport

experiments, cells from a confluent 55 cm2 tissue culture dish were detached with trypsin-

EDTA, uniformly resuspended in DMEM, and seeded on tissue culture dishes of 8 cm2

growth area. Cells were used for transport experiments 72 h to 96 h later, when they were 90-

100% confluent. 24 h before starting the transport experiment, cells were additionally

induced by adding 5 mM sodium butyrate to the medium.

Cryopreserved human hepatocytes (Bioreclamation IVT, Brussels, Belgium, lot SSR) were

seeded on collagen-coated 24-well plates (Nunc, Thermo Scientific, Wohlen, Switzerland) at

a density of 0.2 x 106 viable cells per well. Cells were allowed to attach for about 4 hours in

William's medium E (Life Technologies Europe B.V., Zug, Switzerland) supplemented with

10% FCS, 10 mg/mL insulin and 10 mg/mL penicillin/streptomycin prior to use in uptake

experiments.

Transport experiments with overexpressing CHO cells

Transport experiments with CHO Flp InTM cells expressing NTCP were run using three 8 cm2

tissue culture dishes for each concentration investigated. After washing the cells three times

with 2 ml of pre-warmed (37 °C) sodium or choline buffer, the uptake experiment was

initiated by adding 1 ml of buffer containing either 14C-labeled macitentan at various

concentrations or 5 μM 3H-labeled taurocholic acid (appropriately diluted with non-labeled

material). The sodium-containing buffer was composed of 20 mM HEPES (pH 7.4), 116.4

mM NaCl, 1 mM NaH2PO4, 5.3 mM KCl, 0.8 mM MgSO4 and 5.5 mM D-glucose. The

choline-containing buffer had the overall same composition but sodium chloride and

NaH2PO4 were replaced with 116.4 mM choline chloride and 1 mM KH2PO4, respectively.

After incubation at 37 °C for 40 s, cellular uptake was stopped by addition of two times 2 ml


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of ice-cold choline buffer containing 0.5% bovine serum albumin (Sigma-Aldrich, Buchs,

Switzerland). Bovine serum albumin was included in the washing buffer to minimize

unspecific binding. Cells were washed four times with approximately 2 ml of ice-cold choline

buffer and then solubilized by addition of 1 ml of 1% (w/v) Triton X-100. After incubation

for at least 20 min, 0.5 ml of the cell lysate was mixed with 3.5 ml of scintillation cocktail

IRGA Safe Plus and total radioactivity determined using a Tri-Carb 2300 TR liquid

scintillation analyzer (Packard Bioscience, Zürich, Switzerland). 25-μl aliquots of the cell

lysates were used to determine the protein content of each sample (see below). Inhibition

experiments were performed by simultaneous addition of 5 μM 3H-taurocholic acid and pre-

defined concentrations of macitentan or ACT-132577. Incubations and sample work-up were

done as outlined above. NTCP-mediated transport rates were calculated as the difference

between sodium and choline buffer.

Transport experiments with OATP-expressing and wild-type CHO cells were run using

three 8 cm2 tissue culture dishes for each concentration. After washing the cells three times

with 2 ml of pre-warmed (37 °C) transport buffer, the uptake experiment was initiated by

adding 1 ml of buffer containing macitentan or ACT-132577 at concentrations of 0.01-100

μM and 0.01-300 μM, respectively. Cellular uptake was determined at 37°C and stopped

after 40 s by addition of two times 2 ml of ice-cold transport buffer containing 0.5% bovine

serum albumin. The latter was included in the washing buffer to minimize non-specific

binding of radioactive compounds. Cells were then rapidly washed four times with each 2 ml

of ice-cold transport buffer and solubilized by addition of 1 ml of 1% (w/v) Triton X-100.

After incubation for at least 20 min, 0.5 ml of the cell lysate was mixed with 5 ml of

scintillation cocktail IRGA Safe Plus and total radioactivity determined using a Tri-Carb

2300 TR liquid scintillation analyzer. 25-μl aliquots of the cell lysates were used to determine


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total protein content. Prior to each transport experiment, the time dependence of cellular

uptake was individually determined in order to optimize experimental conditions.

The effect of the OATP inhibitors cyclosporin A and rifampicin on the uptake of 1 μM

macitentan was investigated for all three OATP transporters. The inhibition experiment was

started by addition of 1 ml pre-warmed transport buffer containing radiolabeled macitentan

and the inhibitor in a concentration range from 0.05-100 μM. After incubation at 37 °C for

20 s, cellular uptake was terminated by addition of two times 2 ml of ice-cold transport buffer

containing 0.5% bovine serum albumin. The sample work-up in these inhibition experiments

was done as outlined above. The final content of organic solvent in the transport experiments

never exceeded 1%. 3H-Estrone-3-sulfate was used as a positive control.

Transport experiments with membrane vesicles

For transport experiments, membrane vesicles expressing human BSEP (50 μg total protein)

were incubated in the presence and absence of 5 mM ATP. Incubations were carried out at

37 °C for 1 min or 3 min in transport buffer containing 10 mM HEPES (pH 7.4), 50 mM

sucrose, 100 mM KNO3, 10 mM Mg(NO3)2, and 5 μM 3H-taurocholic acid. Taurocholate

uptake was stopped by addition of ice-cold washing buffer containing 10 mM Tris-HCl (pH

7.4), 50 mM sucrose and 100 mM KCl, followed by collection of membrane vesicles on a

cellulose nitrate membrane filter (pore size 0.45 µm) using a rapid filtration system

(Millipore, Zug, Switzerland). Prior to the experiments, filters were saturated with 1 mM

non-labeled taurocholic acid to minimize non-specific binding of radiolabeled compound.

Retained membranes were then washed twice with ice-cold buffer, and transferred into

scintillation vials. After addition of 3.5 ml scintillation cocktail, total radioactivity was

determined on a Tri-Carb 2300 TR liquid scintillation analyzer. Inhibition experiments were

performed by incubating membrane vesicles simultaneously with 5 μM 3H-taurocholic acid


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and various concentrations of macitentan or ACT-132577. BSEP-mediated transport rates

were calculated as the difference of results obtained in the presence and absence of ATP.

Macitentan partitioning in human hepatocytes

Cellular uptake of macitentan was determined in triplicate with plated, cryopreserved human

hepatocytes. After removing the William's medium E, cells were washed twice with 0.5 mL

pre-warmed Hank's balanced salt solution (HBSS). Medium and washing solutions were

pooled and the number of unattached cells counted using a Vi-CELL counter (Beckman

Coulter, Nyon, Switzerland) to estimate the number of plated hepatocytes in the well. The

hepatic uptake experiment was started by addition of 200 µL pre-warmed (37 °C) incubation

solution containing macitentan in HBSS with 1% DMSO at a final concentration of ca. 100

nM. After 10 min incubation at 37 °C on an orbital shaker at 300 rpm, uptake was terminated

by removal of the supernatant followed by washing of the cells with twice 0.5 mL ice-cold

phosphate-buffered saline (pH 7.4). Supernatants were fortified with one volume equivalent

of acetonitrile containing tetra-deuterated macitentan as analytical standard. Hepatocytes

were lysed by addition of 200 µL of a 2:3 mixture of HBSS and acetonitrile containing

tetradeuterated macitentan, and incubation at room temperature for 15 min. Calibration

samples were prepared and worked up in parallel in a concentration range from 2 - 1000 nM

by diluting the macitentan stock solution in DMSO with a 1:1 (v/v) mixture of either

acetonitrile and HBSS, or hepatocyte lysate. All samples were placed in 96-well plates

pending analysis by LC-MS/MS.

Macitentan binding in human hepatocytes

Macitentan binding to human hepatocyte homogenate was determined using rapid

equilibrium dialysis and a membrane with a molecular weight cut-off of 8 kDa (Thermo

Fisher Scientific, Reinach, Switzerland). Prior to equilibrium dialysis, human hepatocytes (1

x 106 cells/mL) were metabolically inactivated by initial incubation at 37 °C and 800 rpm on


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a thermomixer for 48 h, followed by three freeze-thaw cycles at room temperature and

-20 oC, and finally sonication for 10 s (Vibracell 75043, Bioblock Scientific, Illkirch, France).

Macitentan at a final concentration of 0.5 μM was added to the hepatocyte homogenate as a

0.5 mM stock solution in DMSO. 200 µL-aliquots of this mixture were transferred into the

donor compartment of the rapid equilibrium device and dialyzed against 350 µL HBSS at 37

°C for 4 h on an orbital shaker in an atmosphere containing 5% CO2. At the end of dialysis,

50 μL aliquots of the donor compartment were diluted with HBSS, while 50 µL of the

receiver compartment were diluted with 50 µL of blank hepatocyte homogenate in order to

generate samples with the same analytical matrix. Three independent experiments were

performed with three replicates each. Sample work-up for LC-MS/MS analysis consisted of

protein precipitation with three volume equivalents of methanol containing tetradeuterated

macitentan as analytical standard. After centrifugation at 3220 g and 4 °C for 20 min, 5 µL-

aliquots were transferred in a 96-well plate pending analysis. Calibration samples were

prepared and worked up in parallel in a concentration range from 0.5 -1000 nM by diluting

the macitentan stock solution in DMSO with a 1:1 (v/v) mixture of hepatocyte homogenate

and HBSS.

Quantification of macitentan by LC-MS/MS

The analytical equipment consisted of a Shimadzu HPLC System (Shimadzu, Reinach,

Switzerland) connected to an API5000 (AB SCIEX, Concord, Ontario, Canada). Data

acquisition was done using the Analyst software package (version 1.5.1). The

chromatographic analysis was achieved on a Phenomenex Luna C8 column (5 µm, 2.0 x

20 mm ID) at room temperature with a flow rate of 0.6 mL/min. Mobile phases consisted of

0.1 % aqueous formic acid and acetonitrile. The mass transitions used for macitentan and its


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tetradeuterated internal standard were 589 to 201 and 593 to 205, respectively, both with a

scan time of 50 ms.

Determination of total protein content

Total protein content was determined using the Pierce bicinchoninic acid assay (Pierce

Science, Lausanne, Switzerland) with quantification at a wavelength of 590 nm on a

SpectraCount spectrophotometer (Packard Bioscience) according to the supplier’s protocol.

Bovine serum albumin was used as a standard. Raw data were analyzed using the

PlateReader software I-Smart (version 3.0 for Windows, Packard Bioscience).

Data evaluation

Data from the inhibition experiments were evaluated by plotting the inhibitor concentration

(logarithmic scale) against the BSEP- or NTCP-mediated transport of taurocholic acid. IC50

values were then determined from the plot by non-linear regression using equation 1 with a

constraint Bottom > 0 (Giacomini et al., 2010):

equation 1 � ��

��

��

in which y is the transport expressed as % inhibition relative to control, x is the inhibitor

concentration (μM), s is the slope at the point of inversion, and Top and Bottom are the

maximum and minimum transport rates. For all graphical data evaluations, the GraphPad

Prism software package (version 5.0, GraphPad Software Inc., La Jolla, USA) was used. The

fitted parameters are presented as best-fit parameter and standard error.

Cellular uptakes were normalized to total protein content and are either expressed as

(pmol/mg protein) or are further normalized for incubation time and expressed as

(pmol/mg·min). OATP-mediated net uptake rates are calculated as the difference of OATP-


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expressing and wild-type CHO cells for each individual concentration and are presented as

mean and standard deviation (SD). Uptake ratios were calculated from the OATP-expressing

and wild-type cells.

The partitioning ratio of macitentan between human hepatocytes and in the incubation

medium (Kp) was calculated using equation 2:

equation 2 solutionincubation

hepatocytehepatocytep c

VAK

/=

where Ahepatocyte is the amount of macitentan in hepatocyte lysate, Vhepatocyte is the hepatocyte

volume and cincubation solution is the macitentan concentration in the incubation medium at the

end of the experiment. The hepatocyte volume was estimated from cell diameters measured

before plating (23 µm). This value is in good agreement with previously published data, i.e.,

16.2 µm for human hepatocytes (Mateus et al., 2013), and 24 µm for rat hepatocytes (Treijtel

et al., 2005).

The unbound fraction in the hepatocyte homogenate (fu,homogenate) was calculated using

equation 3:

equation 3 ogenate

bufferogenateu c

cf

homhom, =

where chomogenate and cbuffer are the macitentan concentrations in the donor and receiver

compartments at the end of dialysis. The unbound fraction in human hepatocytes (fu, hepatocyte)

was derived from equation 4:

equation 4 1)1/1(

1

hom,, +−

=ogenateu

hepatocyteu fDf


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where D is the homogenate dilution factor. The volume of human hepatocytes was again

determined from cell diameters (20 µm) prior to homogenization. The ratio of unbound

macitentan concentrations between cells and medium (Kp,uu) as a measure for drug

accumulation was calculated using equation 5:

equation 5 psolutionincubationu

hepatocyteuuup K

f

fK ×=

,

,,

where fu, incubation solution is the unbound macitentan concentration of the incubation medium. Fu,

incubation solution is assumed to be 1 as the medium does not contain proteins.

Bile salt measurements in animals and man

Quantification of bile salts in plasma and serum

Bile salt concentrations in plasma and serum were determined using an enzymatic assay

based on the reduction of NAD to NADH, which is subsequently used to reduce

nitrotetrazolium blue to formazan, followed by colorimetric quantification of the latter at a

wavelength of 530 nm. For samples from the intravenous rat model, bile salts in plasma were

quantified using a kit from Sigma Diagnostics (St. Louis, USA) and a set of calibration

samples ranging from 0-100 μM that was run on the same 96-well plate as the unknown

samples. The commercial kit is designed to quantify bile salts in serum but can equally be

used for plasma (validation data not shown). Bile salts in sera from the externally performed

rat and dog toxicity studies and the multiple-ascending dose study with macitentan in human

healthy subjects (Sidharta et al., 2013) were analyzed in the respective preclinical and clinical

research labs.


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Animals

For the acute cholestasis model, male Wistar rats, 8-12 weeks of age, were delivered from

RCC Ltd, Biotechnology and Animal Breeding Division, Füllinsdorf, Switzerland, and used

after an acclimatization period of at least 7 days. Body weights were between 221-345 g at

the day of the experiment. All animals were housed under climate-controlled conditions with

a 12-hour light/dark cycle in accordance with the guidelines of the Basel Cantonal Veterinary

Office (license no. 169). All animals were maintained under identical conditions and had free

access to drinking water and food (batch 3418, Provimi Kliba, Kaiseraugst, Switzerland).

The multiple-dose toxicity studies in Wistar or Sprague Dawley rats and Beagle dogs were

conducted in certified contract research organizations in compliance with principles of Good

Laboratory Practice. All animal experiments adhered to the "Principles of Laboratory Animal

Care" (NIH publication #85-32, revised in 1985).

Bosentan, macitentan and ACT-132577 in the acute rat model

Macitentan, its metabolite ACT-132577, and bosentan were formulated as microsuspensions

in 7.5% gelatin and intravenously administered via the tail vein at a dose of 25 mg/kg (n=6)

and a dosing volume of 1 ml/kg. All formulations were prepared freshly on the day of

experiment and stirred well prior to administration. About 0.5 ml of blood was collected into

EDTA-containing vials from the sublingual vein before dosing and at 10, 45, and 120 min

after dosing. The effect of the gelatin vehicle was investigated in a control experiment.

Plasma was prepared by centrifugation at ca. 4000 rpm and stored frozen at -20 oC pending

analysis.

Bile salt measurements in the rat and dog toxicity studies

In the multiple-dose oral toxicity studies, male and female animals were treated once daily

with macitentan either by gavage (rats) or with capsules (dogs). A control group receiving the

methylcellulose vehicle or empty capsules was an integral part of all study designs. Doses


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were selected based on previous dose-range finding studies and preceding studies of shorter

duration. Treatment duration was 4, 13 and 26 weeks in the rat, and 4, 13, and 39 weeks in

the dog. Bile salts in plasma were determined at the end of the toxicity study as part of the

regular clinical chemistry program.

Bile salt measurements in the multiple ascending dose study in man

The multiple ascending dose study with macitentan in healthy human subjects was designed

as a double-blind, placebo-controlled, randomized study to investigate the tolerability, safety,

pharmacokinetics and pharmacodynamics of macitentan (Sidharta et al., 2013). The study

followed the principles of the Declaration of Helsinki and Good Clinical Practice. The

protocol and informed consent form was approved by an independent Ethics Committee

(Ethics Committee of the Landesärztekammer Baden-Württemberg, Stuttgart, Germany). A

total of 32 male subjects received doses of 1, 3, 10 and 30 mg macitentan or placebo for 10

days once daily in fasted state. Each dose was administered sequentially to a group of eight

subjects (six on macitentan, two on placebo). Safety evaluation comprised the collection of

adverse event data including assessments of seriousness, severity, relationship to study drug

and outcome. The safety assessment comprised laboratory variables, vital signs, 12-lead

ECG, physical condition, and body weight. Pharmacokinetic parameters of macitentan and its

metabolite ACT-132577 were assessed for all doses. Bile salts in serum were determined

pre-dose on day 1 and day 10 of treatment.

Physiologically-based pharmacokinetic (PBPK) modeling

The Simcyp Population-Based ADME Simulator (version 12, Simcyp Limited, Sheffield,

UK), a PBPK computer model combined with genetic, physiological, and demographic

variables using Monte Carlo methods and equations derived from population databases

obtained from literature sources, was used.


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The physicochemical properties and blood binding of macitentan, i.e., molecular weight

588 g/mol, logD 2.9, pKa 6.2, plasma protein binding 99.6% and blood/plasma ratio 0.55

were entered into Simcyp. The corresponding values for ACT-132577 were molecular weight

546 g/mol, logD 1.5, pKa 6.1, plasma protein binding 99.5% and blood/plasma ratio 0.55.

Published data were used as source for the pharmacokinetic, metabolism and excretion data

used for the development of the PBPK model (Bruderer et al., 2012b; Atsmon et al., 2013).

Only 4% of the oral dose was excreted in feces as unchanged macitentan after oral dosing of

14C-labeled macitentan to healthy volunteers, suggesting almost complete absorption of the

dose from the gut. Unchanged macitentan was not detected in urine. Oral absorption was

modeled using a simple first-order model, with the fraction of the dose absorbed from the gut

(fa) set to 1 without variation. The rate constant of absorption (ka), absorption lag time and

Qgut, a hybrid term including both villous blood flow and permeability through the enterocyte

membrane (Yang et al., 2007; Pang and Chow, 2012), were optimized to fit the observed

plasma concentration profile of macitentan. Optimized values were: ka 0.3 h-1, lag time 1 h,

and Qgut 9.5 L/h. The volume of distribution was calculated based on a full PBPK model

using the tissue partitioning equations of Rodgers et al. (Rodgers et al., 2005; Rodgers and

Rowland, 2006; Rodgers and Rowland, 2007). The predicted volume of distribution at steady

state using tissue volumes for a healthy volunteer population was 0.36 L/kg for macitentan

and 0.22 L/kg for ACT-132577. Distribution was assumed to be perfusion-limited for all

organs. The liver to plasma partitioning coefficients for macitentan and ACT-132577 were

predicted as 0.18 and 0.09, based on physiological liver volume, intra- and extracellular water

content, neutral and acidic (phospo)lipid content, binding to albumin and (predicted) binding

to lipoproteins.

For the purpose of modeling, it was assumed that macitentan excretion into feces was the

result of biliary excretion rather than incomplete absorption. Consequently, 4% of the


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clearance was set to occur via unchanged excretion into the bile and the remaining 96% was

set to be cleared by hepatic metabolism. Renal clearance was set to zero as no unchanged

macitentan was detected in urine. Macitentan blood clearance in man is unknown. However,

oral clearance can be calculated using equation 6:

equation 6 ��

Mean plasma AUC of macitentan was 5759 ng·h/ml at a dose of 10 mg (Atsmon et al., 2013).

The oral clearance, CLpo, was calculated as 1.8 l/h. The metabolic oral clearance (96% of the

total oral clearance) of macitentan was scaled to ‘μl/min/mg protein’ and ‘µl/min/million

hepatocytes’ with the assumption, based on in vitro and clinical interaction data, that 62% of

macitentan is metabolized to ACT-132577 (Actelion, data on file) using the scaling factors in

Simcyp. The intrinsic clearance CLint was derived from CLpo using the well-stirred liver

model and equation 7:

equation 7 ��

,��

��

This resulted in a liver microsomal clearance CLint of 0.48 μl/min/pmol CYP3A4 for the

formation of ACT-132577. The intrinsic clearance of 1.5 μl/min/million hepatocytes was

used to calculate biliary clearance in the PBPK model. The remainder of the metabolic

clearance of macitentan was not assigned to a specific enzyme.

ACT-132577 has not been dosed intravenously to humans; therefore the human clearance is

unknown. However, the clearance of a metabolite can be calculated using equation 8

(Rowland and Tozer, 1989):

equation 8

��−��

��

= ��

��

��−��


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Application of equation 8 using the observed AUC values for macitentan and ACT-132577

(corrected for the difference in molecular weight) after a single 10 mg macitentan dose

(Atsmon et al., 2013) and the 62% fraction of macitentan metabolized to ACT-132577

resulted in a metabolite clearance of 0.32 L/kg.

A coefficient of variation of 30% was assumed for the input parameters single compartment

absorption rate constant, lag time, Qgut, and intrinsic clearance. For all other parameters,

variation during the simulations was based on (physiological) variation of the population

database within Simcyp. The population selected for the trial design was a healthy volunteer

population, male subjects, age 18-45 years, in fed state. Ten virtual trials of ten subjects each

were run (total size: 100) for a single dose of 10 mg and for steady state simulations with a 30

mg macitentan loading dose followed by 11 daily doses of 10 mg.


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RESULTS

Inhibition of taurocholate uptake by macitentan and metabolite ACT-132577 was

investigated using membrane vesicles expressing human BSEP at concentrations up to

100 μM and 300 μM, respectively (Fig. 1). IC50 values of macitentan and ACT-132577

derived from an analysis of pooled data were 18 ± 5 μM (n=6) and 60 ± 14 μM (n=4),

respectively. Taurocholate was also the substrate in the inhibition experiments with human

NTCP overexpressed in CHO cells (Fig. 2). Macitentan inhibited taurocholate uptake with a

mean IC50 value of 18 ± 2 μM (n = 3), whereas ACT-132577 showed a mean IC50 of 14 ± 2

μM (n = 2).

We have previously reported on the cellular uptake of macitentan in OATP1B1 and

OATP1B3-overexpressing cells in the context of the clinical drug-drug interaction studies

with cyclosporin A and rifampicin (Bruderer et al., 2012a). Table 1 summarizes these results

of the uptake experiments with macitentan and ACT-132577 together with new data on

OATP2B1. Estrone-3-sulfate was used as a positive control. Uptake ratios between OATP-

overexpressing and wild-type cells were calculated. Based on these data, neither macitentan

nor ACT-132577 are considered substrates for OATP1B1 or OATP2B1. Cellular uptake rates

of both compounds into CHO wild-type cells exceeded that of the OATP substrate estrone-3-

sulfate by at least 150-fold (at 5 μM), indicating that their cellular uptake is mostly driven by

high passive diffusion. Figure 3 displays the net uptake rates for macitentan in OATP1B3-

overexpressing CHO cells. Net uptake rates consistently differed from those in wild-type

CHO cells and the linear increase up to the highest concentration of 100 μM suggests that

macitentan is likely a substrate of OATP1B3. No saturation in macitentan uptake was

observed in this concentration range indicating that the affinity of macitentan for OATP1B3

transport is rather low. Passive permeation was again the major contributor to overall cellular


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uptake as uptake ratios never exceeded 1.2. The role of OATP in the overall cellular uptake is

therefore considered of little clinical relevance. Based on the results in Table 1, metabolite

ACT-132577 is not a substrate for OATP1B3.

To verify the above conclusion, macitentan uptake into OATP-overexpressing cells was

additionally determined in the presence of the known OATP inhibitors cyclosporin A (Shitara

et al., 2003) and rifampicin (Vavricka et al., 2002; Hirano et al., 2006). The results are

summarized in Table 2. No consistent effect of cyclosporin A was observed over the

concentration range up to 100 μM, i.e., at concentrations largely exceeding its Ki value of 0.2

μM (Shitara et al., 2003). Cyclosporin A inhibition was not evident in OATP1B3 cells, nor

was there any consistent effect of rifampicin in either cell line. Control experiments with both

inhibitors using 3H-atorvastatin as an OATP substrate yielded concentration-dependent

decreases in net uptake rates for both compounds (data not shown). Overall, these OATP

inhibition experiments support the conclusion that macitentan cellular uptake is mostly

dependent on passive diffusion with only a small component of OATP1B3-mediated uptake.

Macitentan uptake into CHO Flp InTM cells overexpressing the sodium-dependent

taurocholate co-transporting polypeptide NTCP was investigated in the same concentration

range as used for OATP transporters. Uptake ratios were determined from experiments in the

presence and absence of sodium using taurocholic acid at 5 μM as a positive control. The

results are summarized in Table 3. Uptake ratios were around unity over the entire

concentration range indicating that macitentan is not a NTCP substrate.

The potential for intracellular accumulation of macitentan has been determined in human

hepatocytes using a previously published method with small modifications (Mateus et al.,


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2013). Two parallel experiments were performed, in which first the partitioning ratio (Kp) of

macitentan between hepatocytes and culture medium was determined. Mean Kp was 724 ± 96

indicating a significant partitioning of macitentan into human hepatocytes. Macitentan

binding in hepatocytes (fu,hepatocytes) was derived from binding in hepatocyte homogenates

(fu,homogenate) as determined by rapid equilibrium dialysis. The corresponding values for

fu,homogenate and fu,hepatocytes were 0.189 ± 0.009 and 0.00099 ± 0.00004, respectively. This data

suggests that macitentan is highly bound in human hepatocytes and that the free fraction

therein is only around 0.1%. The ratio of unbound macitentan concentrations in hepatocytes

and medium (Kp,uu) as a measure for hepatocellular accumulation was calculated as 0.7 ± 0.1.

Macitentan does therefore not accumulate in human liver cells as the unbound concentrations

in liver cells and incubation medium are similar. The significant partitioning of macitentan

into liver cells is likely the consequence of its elevated lipophilicity and compensated by the

high binding to cellular components.

The acute effects of macitentan and ACT-132577 on bile salt homeostasis were tested upon

intravenous dosing to the rat. This model was developed by Fattinger et al. (Fattinger et al.,

2001) to mechanistically rationalize the increased plasma bile salts in clinical trials with

bosentan. Macitentan and ACT-132577 were individually tested in this model at an

intravenous dose of 25 mg/kg, with plasma samples taken before and at 10, 45, and 120 min

after dosing. As individual plasma bile salt concentrations varied significantly between

animals prior to drug administration, results are also expressed as individual differences from

pre-dose values. Bosentan was included as positive control. Results for all three endothelin

receptor antagonists are summarized in Table 4. Bosentan increased plasma bile salts by 18 ±

13 μM at 10 min post-dose which then returned to pre-dose values within 45 min after

dosing. Neither macitentan nor ACT-132577 elicited such an increase in plasma bile salts.


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After 10 min, mean increases were 3.7 ± 4.4 μM for macitentan and 2.5 ± 6.7 μM for ACT-

132577, and thus not different from vehicle (0.4 ± 3.4 μM).

Bile salts in serum were systematically determined in the oral toxicology program of

macitentan in the rat and dog as part of the clinical chemistry program. Table 5 summarizes

the data collected for all dose groups at the end of the respective study period. In the 4-week

rat study, there was no increase in mean bile salts in the male animals up to the highest dose

of 1500 mg/kg, and in female rats up to 450 mg/kg. Similar to the observations in the

intravenous rat model, significant inter-individual variability was evident in these rat studies,

most likely resulting from differences in food consumption as animals had free access to food

over the entire study. No difference between dose groups was noted in the bile salt

concentrations in the 26-week toxicity study, in which rats received macitentan doses up to

250 mg/kg. In the 4-week dog study, macitentan doses up to 250 mg/kg were given. Inter-

animal variability was significantly lower compared to the rat. There was no difference in

mean serum bile salts between dose groups in male or female animals. A similar picture was

obtained for doses up to 100 mg/kg in the 13-week study, during which bile salt data were

collected after 4 weeks of treatment and at study end. No change in bile salts was observed

across dose groups for the entire study duration. Doses in the 39-week dog study were 5, 30,

and 100 mg/kg at the start of the study. After 20 weeks, the high dose had to be reduced to 75

mg/kg. Serum bile salt data were collected at the end of the study and confirmed the

observations from the studies of shorter duration.

Changes in serum bile salts were also monitored in the multiple-ascending dose study with

macitentan in which healthy volunteers received macitentan doses of 1, 3, 10 and 30 mg for a

period of 10 days (Sidharta et al., 2013). Each dose group consisted of six individuals on


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active treatment and two on placebo. The placebo data were pooled from the four active dose

groups. Bile salts were collected on the first day prior to macitentan dosing and on day 10 at

the end of study. Results are shown in Table 6. Bile salt concentrations in serum were in a

narrow range from 7.7-19 μM and there was no discernible trend toward increased serum

levels at any dose. Inspection of the individual data revealed a maximum difference of 5 μM

between measurements on day 1 and day 10, which was observed in a subject receiving

placebo. These data confirm the above animal data and show that macitentan treatment in

man is not associated with changes in serum bile salts.

A physiology-based pharmacokinetic (PBPK) model of macitentan was developed in order to

allow comparison of the in vitro transport data to concentrations of macitentan and

ACT-132577 in the portal vein, systemic circulation, and liver. Table 7 summarizes the

observed and predicted human pharmacokinetic parameters of macitentan and ACT-132577

after single and repeat dosing as derived from the PBPK model. The derived plasma

concentration vs. time profiles for both compounds after a single oral dose are depicted in

Fig. 4 (panels A and B). Projected plasma exposure (AUC) and peak plasma concentration

(Cmax) data were close to the mean observed data. The model predicts a mean Tmax of 7 h, i.e.,

slightly earlier than the observed mean of 9 h but still well within the range of individual

values. Similarly, the Tmax for ACT-132577 was predicted at 26 h vs. observed 48 h. Overall,

the PBPK model adequately describes the observed plasma concentration profiles of

macitentan and ACT-132577. It was therefore considered suitable to predict portal vein and

liver concentrations as well.

Panel C in Fig. 4 shows the time course of mean macitentan and ACT-132577 concentrations

in the portal vein, plasma, and liver. Peak concentrations of macitentan in the portal vein and


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liver after a single 10 mg dose to healthy male subjects were estimated as 354 nM (208

ng/ml) and 62 nM (36 ng/ml), respectively. The corresponding values for ACT-132577 were

420 nM (223 ng/ml) and 39 nM (21 ng/ml). As ACT-132577 is generated by hepatic

metabolism, plasma and portal vein concentrations are identical. The concentration vs. time

profiles for both compounds at steady-state are illustrated in panel D. Predicted total peak

concentrations for macitentan at steady-state were 400 nM (235 ng/ml) in plasma, 419 nM

(247 ng/ml) in portal vein and 72 nM (43 ng/ml) in liver. The corresponding values for

ACT-132577 were 1722 nM (915 ng/ml) for plasma/portal vein and 159 nM (85 ng/ml) for

the liver.

A parameter sensitivity analysis of the PBPK model was performed to investigate the effect

of various degrees of active hepatic uptake on the pharmacokinetic parameters of macitentan,

with the liver as a permeability-limited instead of a perfusion-limited organ. Passive uptake

clearance CLint, passive into human hepatocytes was estimated from the macitentan logD of 2.9

as 5.3 μL/min/106 cells (Menochet et al., 2012). Four different values for active uptake

clearance CLint, active, i.e. 0, 10, 25, and 50 μL/min/106 cells, were then arbitrarily assigned to

OATP1B1 as a prototypical hepatic uptake transporter. Ten virtual trials of ten subjects each

were again run (total size: 100) for a single dose of 10 mg without any other changes to the

PBPK model. A comparison between predicted and observed pharmacokinetic parameters is

shown in Table 8. In the permeability-limited PBPK liver model, the best fit between

modeled and observed macitentan AUC0-24h is achieved with a CLint, active of 25 μL/min/106

cells, for which a ratio of unbound liver and plasma concentrations of 1.05 is predicted. The

outcome is then closest to the perfusion-limited PBPK model, in which the ratio of unbound

concentrations is per definition set to unity.


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DISCUSSION

‘Dosis sola facit venenum’ is the scientific legacy of the Swiss physician, alchemist,

astrologer, and philosopher Paracelsus (1493-1541). About 500 years later, its longer version

‘all things are poison, and there is nothing without poisonous qualities…it is only the dose

which make a thing poison’ is still a major paradigm in modern pharmacology in its attempt

to find drugs with adequate risk-benefit profiles. Nowadays, the interplay of target affinity

and local drug concentrations has replaced the historical dose term as a measure for drug

quantity. Whether a drug reaches its target in sufficient concentrations is mostly dependent on

its physicochemical properties, as drug distribution to the majority of organs and tissues is

driven by passive diffusion. While total drug concentrations can vary significantly between

tissues as a consequence of differing binding properties, free drug concentrations strive to

equilibrate. This general concept needs to be expanded for organs expressing transport

proteins that are capable of maintaining non-equilibrium conditions and consequently,

depending on their transport direction, result in lower or higher free drug concentrations on

one side of a membrane. Liver, kidney, brain, and placenta are typical examples of such

organs, and the discovery and characterization of transporters like the ATP-dependent efflux

pump P-gp or the organic anion-transporting polypeptide family OATP has fundamentally

changed our understanding of drug disposition.

Endothelin receptor antagonists have been established as a therapeutic concept for the

treatment of pulmonary arterial hypertension. From a chemical perspective, they require a

negative charge (Wu, 2000; Boss et al., 2002) for their interaction with Arg326 of the

endothelin receptors (Breu et al., 1995). In the case of sitaxentan and ambrisentan, this

negative charge is provided by a carboxylic acid whereas bosentan contains an aromatic

sulfonamide. As a consequence, the majority of drug molecules are negatively charged at


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physiological pH which – together with high binding to plasma proteins - limits distribution

into tissues. On the other hand, the negative charge makes them candidate substrates for

anion transporters, and interactions with OATP transport proteins have indeed been described

(Katz et al., 2006; Treiber et al., 2007; Spence et al., 2010). Hepatic side effects – mostly

manifest as transient reversible elevations of liver transaminases in plasma – have also been

reported for some marketed endothelin receptor antagonists (Humbert et al., 2007; Galie et

al., 2011). Active drug uptake by OATP transporters has the potential to increase hepatic

drug concentrations and thus to contribute to liver injury. The combination of BSEP

inhibition and OATP-mediated accumulation in liver cells is the likely mechanism for the

cholestatic effect of bosentan (Fattinger et al., 2001; Treiber et al., 2007). In line with this

hypothesis is the observation in pulmonary arterial hypertension patients concomitantly

receiving the HIV protease inhibitor lopinavir/ritonavir, a potent inhibitor of both CYP3A4

metabolism and OATP transport (Hull et al., 2009; Annaert et al., 2010): bosentan trough

plasma concentrations were increased by 48-fold in this patient population but were not

associated with a higher frequency of liver injury (Dingemanse et al., 2010), most likely

because hepatic drug burden was reduced as a consequence of blocked OATP uptake.

The above points illustrate the necessity to discover novel endothelin receptor antagonists

with an improved safety profile. The medicinal chemistry program leading to the discovery of

macitentan and its pharmacological profile in animal models have been published previously

(Iglarz et al., 2008; Bolli et al., 2012). Key structural changes in macitentan versus bosentan

constitute the replacement of the sulfonamide by a sulfamide moiety and the increase in

overall compound lipophilicity. The sulfamide function in macitentan is less acidic than the

sulfonamide in bosentan as evidenced by the difference in pKa values of 6.2 and 5.1 (Iglarz et

al., 2008), respectively, thus increasing the proportion of molecules present in non-ionized


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state at physiological pH. The octanol/water partition coefficient logD7.4, a measure of

lipophilicity, is 2.9 for macitentan and 1.3 for bosentan. Both factors facilitate membrane

permeability and penetration into tissues. These changes were achieved without

compromising target affinity. Macitentan antagonizes the specific binding of ET-1 to

recombinant ETA and ETB receptors with IC50 values of 0.5 nM and 391 nM, respectively

(Iglarz et al., 2008). In ex vivo models using rat aorta and trachea preparations, macitentan

behaved as a more dual endothelin receptor with pA2 values of 7.6 and 5.9 for ETA and ETB

receptors, respectively (Iglarz et al., 2008). Metabolic stability was optimized in vitro and

favorably translated in animals (Bolli et al., 2012) and man (Sidharta et al., 2011; Sidharta et

al., 2013). As a consequence, macitentan was used at daily doses of 3 mg and 10 mg in the

completed phase III SERAPHIN trial in patients with pulmonary arterial hypertension

(Pulido et al., 2013). Macitentan has an active metabolite, ACT-132577, that is formed in a

cytochrome P450-catalyzed reaction, circulates in human plasma and likely contributes to the

overall efficacy.

Beyond improved receptor binding affinities and pharmacokinetic properties over bosentan,

the structural changes in macitentan have altered the hepatic disposition and interaction

profile with hepatic transporters. As illustrated in Figs. 1 and 2, macitentan and ACT-132577

are inhibitors of the two major transport proteins responsible for hepatic bile salt trafficking,

i.e., NTCP and BSEP. NTCP (SLC10A1) is the major transporter responsible for moving bile

salts from blood into liver cells (Doring et al., 2012) whereas BSEP mediates the rate-limiting

step in bile salt secretion from blood into bile (Gerloff et al., 1998; Stieger et al., 2000) (Fig.

5). NTCP was equally inhibited by macitentan and ACT-132577 with IC50 values in the range

of 14–18 μM, whereas BSEP was inhibited with IC50 ranging from 18–60 μM. These in vitro

data are thus not different from those of bosentan with reported IC50 values on BSEP and


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NTCP of 25–77 μM (Mano et al., 2007; Dawson et al., 2012; Warner et al., 2012) and 24-30

μM (Leslie et al., 2007), respectively. However, the mechanism of hepatic uptake differs

between bosentan and macitentan. Bosentan uptake into liver cells is largely dependent on

OATP transport in animals and man (Treiber et al., 2004; Treiber et al., 2007). In contrast, its

increased lipophilicity allows macitentan to enter cells by passive diffusion as shown in the

uptake experiments with OATP-expressing and wild-type cells (Table 1). The same

conclusion was drawn in a recent study on macitentan interactions with other hepatic

transport proteins (Weiss et al., 2013). Macitentan is a weak substrate for OATP1B3 (Fig. 3),

but not for OATP1B1, OATP2B1 or NTCP. Active transport contributes less than 20% to

overall cellular uptake. Consequently, macitentan uptake is not vulnerable to OATP

inhibition by cyclosporin A or rifampicin (Table 3). These conclusions were confirmed in a

clinical drug-drug interaction study with cyclosporin A, an established potent inhibitor of

OATP- (Shitara et al., 2003) and NTCP-mediated transport (Mita et al., 2006). Concomitant

cyclosporin A increased macitentan exposure in man by only 1.1-fold (Bruderer et al.,

2012a), compared to a 30-fold increase observed with the proven OATP substrate bosentan

(Treiber et al., 2007) in the presence of cyclosporin A (Binet et al., 2000).

Bosentan elicited a dose-dependent increase in plasma bile salts in the rat after single

intravenous administration (Fattinger et al., 2001). Macitentan and ACT-132577 were both

tested in this model alongside with bosentan, all at a dose of 25 mg/kg. Results with bosentan

reproduced literature data, while no effect was observed with macitentan or ACT-132577

(Table 4). These data provide evidence in a more physiological setting that neither compound

interferes with bile salt homeostasis under conditions that led to an acute cholestatic effect

with bosentan. Supportive data upon chronic dosing was provided by the bile salt data from

the rat and dog toxicity studies. After 26 or 39 weeks of treatment with macitentan at doses


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up to 250 mg/kg (rat) and 100/75 mg/kg (dog), there was no pattern of elevated plasma bile

salts in any toxicity study nor were there any histological findings pointing to a cholestatic

potential of macitentan (Actelion, data on file). In contrast, bosentan-treated animals showed

elevated bile salt and alanine aminotransferase levels, which were considered to be

consequences of a functional cholestasis (Actelion, data on file).

A PBPK model was developed for macitentan and ACT-132577 with the aim to estimate

blood and liver concentrations. Total mean macitentan concentrations projected from PBPK

modeling at steady-state in plasma, portal vein and liver are 400 nM, 419 nM and 72 nM. The

corresponding values for ACT-132577 were 1722 nM for plasma/portal vein and 159 nM for

the liver. Correction for plasma protein binding leads to predicted unbound peak macitentan

concentrations of 1.60 nM in plasma, 1.68 nM in the portal vein, and 0.29 nM in the liver.

Free concentrations for ACT-132577 in plasma/portal vein and liver were 8.61 nM and 0.80

nM, respectively. These concentrations are significantly below those observed in the bile salt

transport experiments with NTCP- and BSEP-expressing cells. The key assumption of free

partitioning of macitentan between blood and liver and equal unbound drug concentrations in

both compartments is supported by the experimentally determined Kp,uu of 0.7 in human

hepatocytes, and the sensitivity analysis of the PBPK model probing for the impact of various

degrees of active hepatic uptake on macitentan pharmacokinetics. In this permeability-limited

PBPK model, the best fit between observed and predicted macitentan pharmacokinetics in

achieved when the ratio of unbound plasma and liver concentrations approaches unity

(Table 8).

Serum bile salts were measured in the multiple ascending dose study with macitentan in

which daily doses up to 30 mg were given to healthy volunteers for 10 days (Sidharta et al.,


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2013). As shown in Table 6, there was no difference in bile salts between dose groups and

compared to placebo. All individual values were in a narrow range from 7.7–19 μM. Liver

transaminases but not bile salts were measured in the long-term phase III SERAPHIN trial

with macitentan (Pulido et al., 2013). The incidence of liver enzyme elevations more than

three times the upper limit of normal was not different between placebo and the 3 mg and 10

mg dose groups. The incidences of alanine or aspartate aminotransferase elevations more

than three times above the upper limit of normal were 4% and 3% in the 3 mg and 10 mg

dose group, respectively, compared to 4% on placebo (Pulido et al., 2013). Similarly, the

incidence of plasma bilirubin elevations was equally distributed over all dose groups and

placebo.

In conclusion, there is cumulative evidence demonstrating the superior liver safety profile of

the next generation endothelin receptor antagonist macitentan. Unlike bosentan, macitentan is

devoid of a pattern of functional cholestasis in long-term clinical trials. On a molecular basis,

this difference is the result of discrete changes in chemical structure. While macitentan

maintains equal inhibitory potency to bosentan on NTCP and BSEP transport in vitro,

replacement of the sulfonamide by a less acidic sulfamide moiety and an increased

lipophilicity result in a complete change in the hepatic disposition profile. Bosentan uptake

into the liver is an active process, mediated by OATP transport proteins, likely leading to

accumulation in liver cells. In contrast, macitentan partitions into the liver mostly by passive

diffusion. Local drug concentrations are thus limited by the extensive binding of macitentan

to plasma proteins to levels that are unlikely to exert an inhibitory potential on hepatic bile

salts. These conclusions from biochemical and drug disposition data were confirmed in an

acute rat model and long-term toxicity studies in the rat and dog. In clinical trials, no change

in plasma bile salts was observed in healthy volunteers upon multiple-dose treatment up to

30 mg per day, nor was the incidence of liver transaminase elevations different in macitentan-


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vs. placebo-treated patients in the long-term morbidity/mortality SERAPHIN trial.

Macitentan at a daily dose of 10 mg is therefore not expected to interfere with hepatic bile

salt transport in clinical practice.

ACKNOWLEDGMENTS

The authors would like to thank Aude Weigel, Eric Soubieux, Julia Friedrich and Stephanie

Bernhard for their dedication and experimental contributions. We are also grateful to Susan

Flores and Charlotte Gonzales for their help in preparing the manuscript.

AUTHORSHIP CONTRIBUTIONS

Participated in research design: Treiber, Aeänismaa, Delahaye, Treher, Hess, Sidharta

Conducted experiments: Treiber, Aeänismaa, Delahaye, Treher, Hess, Sidharta

Performed data analysis: Treiber, Aeänismaa, Treher, de Kanter, Delahaye, Hess, Sidharta

Wrote or contributed to the writing of the manuscript: Treiber, Aeänismaa, Treher, de Kanter,

Hess, Sidharta


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FOOTNOTES

Contact for reprint requests: Alexander Treiber, Actelion Pharmaceuticals Ltd,

Gewerbestrasse 16, 4123 Allschwil, Switzerland, e-mail: [email protected]


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LEGENDS TO FIGURES

Fig. 1 Inhibition of vesicular taurocholate uptake into membrane vesicles expressing human

BSEP by (A) macitentan and (B) ACT 132577. Data are mean ± SD of 3 replicates.

Data of one representative experiment are shown.

Fig. 2 Inhibition of taurocholate uptake into CHO cells expressing human NTCP by (A)

macitentan and (B) ACT-132577. Data are mean ± SD of 3 replicates. Data of one

representative experiment are shown.

Fig. 3 Net cellular uptake of macitentan in OATP1B3-expressing CHO cells. Data are mean

± SD of 3 replicates.

Fig. 4 Liver, portal vein and systemic plasma concentration-time profiles of macitentan and

ACT-132577 after (A, B, C) single and (D) repeat 10 mg oral dosing. Observed data

(panels A and B) from (Atsmon et al., 2013).

Fig. 5 Hepatic transport proteins involved in bile salt transport. NTCP as well as OATP1B1,

OATP1B3 and OATP2B1 are located at the basolateral membrane of hepatocytes and

facilitate the uptake of bile salts in liver cells. BSEP is located at the canalicular

membrane and promotes the exports of bile salts into bile canaliculi.


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TABLES

Table 1 Concentration dependence of macitentan and ACT-132577 cellular uptake into OATP-expressing and wild-type CHO cells a

Concentration (μM)

Wild-type CHO (pmol/min·mg)

OATP1B1 (pmol/min·mg)

Ratio OATP1B3 (pmol/min·mg)

Ratio Wild-type CHO (pmol/min·mg)

OATP2B1 (pmol/min·mg)

Ratio

macitentan

0.01 3.3 ± 0.5 3.1 ± 0.3 0.9 3.9 ± 0.7 1.2 3.0 ± 0.3 2.9 ± 0.1 1.0

0.05 13 ± 1 14 ± 0.3 1.0 14 ± 0.3 1.0 12 ± 1 14 ± 0.4 1.2

0.1 23 ± 1 24 ± 1 1.1 27 ± 1 1.2 24 ± 1 26 ± 2 1.1

0.5 120 ± 7 126 ± 3 1.1 137 ± 6 1.1 119 ± 1 117± 5 1.0

1 228 ± 5 236 ± 1 1.0 280 ± 9 1.2 214 ± 2 222 ± 6 1.1

5 1290 ± 43 1290 ± 67 1.0 1540 ± 37 1.2 1050 ± 40 1060 ± 18 1.0

15 3200 ± 108 3300 ± 183 1.0 3700 ± 144 1.2 3600 ± 128 3400 ± 136 1.0

50 10600 ± 306 10700 ± 291 1.0 12100 ± 449 1.1 9600 ± 189 8900 ± 336 0.9

100 14800 ± 600 14900 ± 426 1.0 17900 ± 479 1.2 20000 ± 755 19400 ± 629 1.0

5 b 3.8 ± 0.2 26 ± 0.3 6.9 32 ± 0.9 8.3 3.8 ± 0.2 77 ± 1 20.4

ACT-132577

0.01 4.0 ± 1.0 3.9 ± 0.3 1.1 4.5 ± 0.9 1.2 2.8 ± 0.3 2.6 ± 0.2 0.9

0.05 7.9 ± 0.8 8.3 ± 0.6 1.1 9.2 ± 0.8 1.2 7.1 ± 0.5 7.6 ± 0.1 1.1

0.1 12 ± 1 13 ± 1 1.1 13 ± 2 1.1 12 ± 1 14 ± 1 1.1

0.5 57 ± 3 57 ± 4 1.0 64 ± 1 1.1 57 ± 1 66 ± 1 1.2

1 107 ± 5 121 ± 8 1.1 145 ± 8 1.4 35 ± 2 44 ± 1 1.3

5 590 ± 22 650 ± 26 1.1 720 ± 10 1.2 530 ± 27 560 ± 3 1.1

15 1930 ± 93 1920 ± 58 1.0 2200 ± 80 1.1 1650 ± 20 1790 ± 16 1.1

50 6200 ± 152 6240 ± 33 1.0 6700 ± 393 1.1 6800 ± 238 6400 ± 138 0.9

100 12000 ± 514 12000 ± 377 1.0 13100 ± 542 1.1 12200 ± 564 12800 ± 551 1.1

150 19500 ± 383 17900 ± 108 0.9 20000 ± 805 1.0 17800 ± 972 17200 ± 359 1.0

300 36700 ± 623 34900 ± 805 0.9 37000 ± 2410 1.0 34000 ± 1084 31000 ± 1648 0.9

5 b 3.8 ± 0.6 15 ± 0.8 4.0 21 ± 0.4 5.6 nd c nd nd

a uptake rates are expressed as mean ± SD of n = 3; b positive control estrone-3-sulfate; c nd: not determined.

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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Table 2 Effect of cyclosporin A and rifampicin on the net uptake rate of 1 μM macitentan

in OATP-overexpressing CHO cells a

Concentration

(μM) Net uptake rate OATP1B1

(pmol/min·mg) Net uptake rate OATP1B3

(pmol/min·mg)

Net uptake rate OATP2B1

(pmol/min·mg)

cyclosporin A rifampicin cyclosporin A rifampicin cyclosporin A rifampicin

0 80 ± 25 170 ± 43 70 ± 21 100 ± 33 20 ± 22 -4 ± 26

0.05 41 ± 9 40 ± 54 48 ± 5 50 ± 82 20 ± 20 -20 ± 51

0.1 50 ± 22 70 ± 39 40 ± 12 40 ± 35 17 ± 9 -20 ± 39

0.5 80 ± 24 60 ± 8 110 ± 17 110 ± 22 50 ± 15 10 ± 23

1 60 ± 27 90 ± 61 60 ± 19 80 ± 57 40 ± 24 2 ± 47

5 70 ± 19 120 ± 25 50 ± 24 100 ± 19 10 ± 14 90 ± 50

10 70 ± 18 90 ± 21 70 ± 21 100 ± 23 20 ± 17 20 ± 14

30 30 ± 37 60 ± 32 20 ± 33 70 ± 23 10 ± 65 30 ± 24

60 50 ± 18 230 ± 17 40 ± 25 70 ± 17 20 ± 16 10 ± 19

100 24 ± 1 90 ± 19 48 ± 8 90 ± 22 10 ± 23 10 ± 24

a net uptake rates are expressed as mean ± SD of n = 3.


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Table 3 Concentration dependence of macitentan cellular uptake into NTCP-expressing

CHO Flp InTM cells a

Concentration

(μM)

NTCP without Na+

(pmol/min·mg)

NTCP with Na+

(pmol/min·mg)

Ratio

0.01 3.7 ± 0.3 3.4 ± 0.2 0.9

0.05 17 ± 1 18 ± 1 1.0

0.1 35 ± 1 33 ± 2 0.9

0.5 162 ± 4 168 ± 2 1.0

1 310 ± 12 324 ± 1 1.0

5 1520 ± 98 1500 ± 59 1.0

15 4270 ± 43 4500 ± 240 1.1

50 13800 ± 124 14000 ± 207 1.0

100 22500 ± 905 23500 ± 314 1.0

5 b 14 ± 4 310 ± 10 21

a uptake rates are expressed as mean ± SD of n = 3. b positive control taurocholic acid.


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Table 4 The effect of bosentan, macitentan and its metabolite ACT-132577 on plasma bile

salts after intravenous administration at a dose of 25 mg/kg

Compound Plasma bile salts a

pre-dose (μM) 10 min (μM) 45 min (μM) 120 min (μM)

Vehicle b 16 ± 5.4 (8.2 – 19)

16 ± 1.6 (7.7 – 17)

12 ± 5.2 (7.7 – 16)

13 ± 3.1 (7.0 – 21)

Bosentan 10 ± 0.8 (4.1 – 29)

29 ± 1.2 (5.2 – 57)

15 ± 3.5 (6.3 – 23)

12 ± 6.3 (5.7 – 20)

Macitentan 8.7 ± 2.1 (7.2 – 20)

17 ± 4.9 (12 – 21)

13 ± 3.5 (11 – 25)

18 ± 1.1 (15 – 19)

ACT-132577 20 ± 1.4 (7.4 – 41)

21 ± 9.4 (13 – 45)

20 ± 1.9 (8.7 – 26)

23 ± 17 (11 – 35)

Δ Plasma bile salts c

Vehicle --

0.4 ± 3.4 (-4.3 – 5.5)

-2.0 ± 5.7 (-11 – 4.0)

-0.5 ± 5.9 (-9.0 – 7.5)

Bosentan --

18 ± 13 (-3.9 – 36)

1.1 ± 5.1 (-6.2 – 7.2)

-1.6 ± 3.1 (-16 – 6.1)

Macitentan --

3.7 ± 4.4 (-1.5 – 10)

1.8 ± 4.1 (-5.5 – 5.5)

3.5 ± 4.8 (-2.5 – 9.5)

ACT-132577 --

2.5 ± 6.7 (-6.0 – 11)

-4.3 ± 11 (-21 – 7.4)

-2.0 ± 13 (-20 – 14)

a Results are means ± SD (range) of n = 6 b Vehicle: 0.5% methylcellulose in water c Results are calculated as individual differences from pre-dose values and presented as mean ± SD (range).


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Table 5 Serum bile salts (in μM) in rat and dog toxicity studies with macitentan

Species Duration Sex Dose (mg/kg)

Rat 4 weeks a 0 50 150 450 1500

M 25 ± 11 (11 – 46)

20 ± 6.6 (6.6 – 45)

21 ± 8.9 (9.4 – 34)

25 ± 9.1 (12 – 40)

24 ± 12 (10 – 40)

F 20 ± 8.1 (12 – 40)

25 ± 8.8 (8.8 – 45)

27 ± 9.9 (8.9 – 34)

28 ± 9.3 (16 – 39)

37 ± 33 (13-128)

26 weeks b 0 10 50 250

M 32 ± 15 (11 – 57)

17 ± 6.6 (9.4 – 27)

23 ± 10 (10 – 52)

19 ± 9.3 (6.5 – 28.9)

F 49 ± 27 (19 – 124)

32 ± 16 (13 – 81)

37 ± 31 (11 – 125)

30 ± 16 (11 – 58)

Dog 4 weeks c 0 10 50 250

M 8.3 ± 1.3 (7.2 – 10)

7.3 ± 0.4 (7.0 – 7.7)

6.6 ± 0.8 (6.0 – 7.5)

7.7 ± 3.3 d

(6.3 – 12)

F 15 ± 10 (8.3 – 33)

15 ± 8.2 (8.1 – 24)

7.8 ± 1.6 (6.6 – 9.6)

9.0 ± 3.9 (6.2 – 16)

13 weeks 0 2 5 30 100

M e 2.8 ± 2.3 (BLQ – 7.7) f

1.0 ± 0.6 (BLQ – 2.3)

1.6 ± 0.9 (BLQ – 2.7)

1.2 ± 0.9 (BLQ – 3.1) BLQ h

F 1.5 ± 0.5 (BLQ – 2.0)

3.5 ± 3.7 (BLQ – 2.3)

1.9 ± 0.9 (BLQ – 2.3)

1.9 ± 0.8 (BLQ – 3.4)

1.8 ± 1.5 (BLQ – 3.9)

M g BLQ h BLQ h 1.3 ± 1.1 (BLQ – 3.5)

1.9 ± 1.4 (BLQ – 4.0)

1.8 ± 2.4 (BLQ – 7.2)

F 3.2 ± 3.6

(BLQ – 9.7) 1.7 ± 1.2

(BLQ – 3.1) BLQ h BLQ h 4.9 ± 8.7

(BLQ – 6.2) i

39 weeks j 0 5 30 100/75 k

M 1.4 ± 1.9 (0.2 – 5.9)

2.0 ± 3.7 (0.3 – 11.1)

0.5 ± 0.3 (0.1 – 1.0)

0.7 ± 0.8 (BLQ – 1.9) l, m

a n = 10, data are from day 28 of the 4-week study, all data are mean ± SD (range); b n = 15, data are

from the last day of macitentan treatment; c n = 5 in the control and 250 mg/kg dose groups and n = 3

in the 10 and 50 mg/kg dose groups, data are from day 23 of the 4-week study; d one sample missing,

mean and SD of n = 4; e n = 7, data are from week 4 of the 13-week study; f BLQ: below limit of

quantification (1.5 μM); g n = 7, data are from the last treatment week of the 13-week study; h all

animals BLQ; i one apparent outlier with plasma bile salts of 24 μM at study end, included in the

calculation of mean and SD but not in range, liver specimens were unremarkable in the subsequent

histological examination; j n = 8, data are from the last treatment week of the 39-week study; k the

high dose was reduced from 100 mg/kg to 75 mg/kg in week 20; l n = 6-7; m BLQ : 0.5 μM.


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Table 6 Serum bile salts in the multiple ascending dose study with macitentan in healthy

human subjects

Dose (mg) Bile salts (μM) a

day 1 Bile salts (μM) a

day 10

placebo 10.7 ± 2.4 (7.7 – 14) 12.2 ± 4.0 (8.2 – 19)

1 9.8 ± 1.7 (8.2 – 13) 10.8 ± 1.2 (9.7 – 13)

3 9.8 ± 1.4 (7.7 – 11) 11.1 ± 1.6 (8.8 – 13)

10 11.8 ± 1.2 (11 – 14) 14.8 ± 2.7 (12 – 18)

30 11.8 ± 2.7 (9.5 – 15) 13.3 ± 1.4 (11 – 15)

a Bile salts in serum were determined before dosing on both dosing days, BLQ: below limit of

quantification (1 μM). Results are expressed as mean ± SD (range) of n = 6–8.


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Table 7 Simulated and observed pharmacokinetic parameters of macitentan and

ACT-132577 after single and multiple oral doses of 10 mg

Compound PK parameter Observed a Predicted b

macitentan SD c Cmax (ng/ml) 227 (205-252) 199 (190 - 209)

Tmax (h) 9.0 (5.0-12) 7 (7 - 7)

AUC0-336h (ng·h/ml) 5'759 (4'914-6'749) 4'966 (4'634 - 5'321)

ACT-132577 SD Cmax (ng/ml) 178 (155-204) 226 (217 - 237)

Tmax (h) 48 (24-48) 26 (24 - 27)

AUC0-336h (ng·h/ml) 19'749 (17'969-21'705) 18'903 (17'899 - 19'963)

macitentan SS d Cmax (ng/ml) 371 (290-475) 227 (213 - 242)

Tmax (h) 6.0 (5.0-12) 6 (6 - 6)

AUC0-24h (ng·h/ml) 5'400 (4107-7099) 3'956 (3676 - 4257)

ACT-132577 SS Cmax (ng/ml) 802 (585-1100) 884 (836 - 935)

Tmax (h) 9.0 (8.0-12) 9 (9 - 9)

AUC0-24h (ng·h/ml) 15'541 (11931-20244) 20'067 (18'908 - 21298)

a observed single dose data are from (Atsmon et al., 2013) and multiple-dose data are from

(Sidharta et al., 2013); b results are geometric mean (95% confidence interval); c SD: single

dose, d SS: steady state.


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JPET #214106

50

Table 8 Parameter sensitivity analysis of active hepatic uptake on macitentan human

pharmacokinetic parameters for a single oral dose of 10 mg a

PBPK model CLint, active

(μL/min*106 cells)

cu, liver/ cu, plasma

b AUC0-24h

(ng*h/mL) Cmax

(ng/mL)

perfusion-limited liver

-- 1 4'966 (4'634 - 5'321)

199 (190 - 209)

permeability-limited liver

0 0.20 10'322 (9'828 - 10'840)

236 (226 - 247)

10 0.55 7'557

(7'151 - 7'985) 218

(208 - 229)

25 1.05 5'395

(5'025 - 5'793) 199

(189 - 209)

50 1.85 3'661

(3'357 - 3'991) 175

(165 - 185)

observed -- -- 5'759

(4'914-6'749) 227

(205-252)

a results are geometric mean (95% confidence interval); b unbound macitentan concentrations

in liver and plasma at time of peak concentrations in the liver.


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Have a nice day and see you on Monday.

Have a nice day and see you on Monday.

l

basolateral membrane

bile canaliculus

canalicular membrane


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Macitentan does not interfere with hepatic bile salt...

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