Supplementary materials:
Clinical Pharmacokinetics
Title: Physiologically-based pharmacokinetic modeling of macitentan: prediction of drugdrug interactions
Authors: Ruben de Kanter, Patricia N. Sidharta, Stéphane Delahaye, Carmela Gnerre, Jerome
Segrestaa, Stephan Buchmann, Christopher Kohl and Alexander Treiber
Corresponding Author:
Ruben de Kanter
Preclinical Pharmacokinetics and Metabolism
Actelion Pharmaceuticals Ltd.
Gewerbestrasse 16
CH-4123 Allschwil
Switzerland
phone: +41 61 565 5618
e-mail: ruben.de-kanter@actelion.com
Details on experimental in vitro procedures
Physical-chemical properties:
The distribution-coefficient logD was determined using a slightly modified shake flask
method which is based on a OECD guideline [1]. Briefly, the test compound was dissolved
at 4 mM in the organic phase (n-octanol), which was further mixed with the aqueous buffered
phase (67 mM phosphate buffer pH 7.4). After shaking overnight, the two phases were
separated by centrifugation and the concentration of the test compound in each phase was
measured by HPLC-UV. D is given by the direct quotient of the compound concentration in
the organic and aqueous phases.
The ionization constant pKa was determined with a multiwavelength spectrophotometric
method, using the GLpKa / D-PAS (macitentan) or Sirius T3 (ACT-132577) instrument from
Sirius Analytical (Forest Row, UK) [2].
Plasma protein binding:
The fraction unbound in human plasma was determined using a Spectrum equilibrium
dialyzer (Spectrum Chemicals & Laboratory Products) [3]. Pooled plasma was spiked with
100 - 300,000 ng/mL 14C-labeled macitentan or ACT-132577 and dialyzed against 0.02 M
phosphate-buffered saline (pH 7.4) at 37 °C for 6 hours using a filter membrane with a
molecular weight cut-off of 12-14 kDa. Aliquots taken from both donor and buffer
compartments were analyzed by liquid scintillation counting and the fraction unbound was
calculated as described [3].
P450 inhibition:
P450 inhibition was studied using selective marker reactions as specified below. Substrates
were used at a single concentration around their Km (Michaelis-Menten constant) and both
macitentan and ACT-132577 were incubated at eight different concentrations, up to 50 µM.
Solvent (negative) controls and well-known inhibitors (positive) controls were included.
Microsomal incubations were initiated by the addition of NADPH-regenerating system and
terminated by adding an excess of ice-cold organic solvent. A summary of experimental
conditions is given in the table below.
P450
isoform
1A2
2A6
2B6
2C8
2C9
2C19
2D6
2E1
3A4
3A4
marker reaction
enzyme concentration, system
and incubation period
phenacetin-Odeethylation
coumarin 7hydroxylation
(S)-mephenytoin
N-desmethylation
paclitaxel
6-hydroxylation
diclofenac 4’hydroxylation
(S)-mephenytoin
4’-hydroxylation
dextrometorphanO-demethylation
chlorzoxazone 6hydroxylation
midazolam 1’hydroxylation
testosterone 6hydroxylation
1 mg/mL HLM, 30 min
marker
substrate
concentration
(~Km)
50 µM
analytical method
0.025 mg/mL HLM, 6 min
1 µM
HPLC-UV
200 pmol/mL recombinant
CYP2B6, 40 min
0.25 mg/mL HLM, 20 min
50 µM
8 µM
HPLC-radioactivity
detection
LC-MS/MS
0.1 mg/mL HLM, 6 min
5 µM
LC-MS/MS
50 pmol/mL recombinant
CYP2C19, 40 min
0.2 mg/mL HLM, 30 min
50 µM
8 µM
HPLC-radioactivity
detection
LC-MS/MS
0.35 mg/mL HLM, 20 min
50 µM
HPLC-UV
0.25 mg/mL HLM, 5 min
5 µM
LC-MS/MS
0.3 mg/mL HLM, 7 min
40 µM
HPLC-radioactivity
detection or LCMS/MS
LC-MS/MS
HLM, human liver microsomes (pooled from 51 donors; Becton Dickinson, Basel,
Switzerland); recombinant enzymes, SupersomesTM (BD Biosciences, Allschwil,
Switzerland). HPLC-UV High-Pressure Liquid Chromatography with UV Detector; LCMS/MS Liquid chromatography-tandem mass spectrometry.
The Ki values for CYP2C9 inhibition of both macitentan and ACT-132577, as well as Ki
value for CYP3A4 inhibition of ACT-132577, were determined using four different substrate
concentrations, between 2-fold below to 10-fold above the Km. Inhibitor concentrations were
the same as used in the IC50 experiments.
IC50 and Ki values were calculated by fitting the rate of metabolite production against
inhibitor concentration using the transformed Michaelis-Menten equation.
Time dependent inhibition was studied with or without a 30 min pre-incubation period in the
presence of NADPH but without the probe substrate. At the end of the pre-incubation period,
probe substrates were added at a single concentration of about 10 times the respective Km
values, as indicated the table above. No shift in IC50 values was observed for any of the P450
enzymes studies (CYP2C9, CYP2D6 and CYP3A4).
2
Identification of P450 enzymes involved in macitentan and ACT-132577 metabolism:
14
C-labeled macitentan and ACT-132577 were incubated at 10 µM with human liver
microsomes in the absence or presence of P450 enzyme-selective chemical inhibitors at
concentrations 10 times their respective Ki's, as specified in the table below, or with
recombinant enzymes in SupersomesTM (BD Biosciences, Allschwil, Switzerland) or
BactosomesTM (Cypex, Dundee, UK). Pooled human liver microsomes (Becton Dickinson,
Basel, Switzerland) at a protein concentration of 1 mg/mL or recombinant P450 enzymes at
100 pmol/mL were used in 0.1 M phosphate buffer (pH 7.4). Incubations were initiated by
the addition of a NADPH-regenerating system and continued for 40 min at 37 °C, in a
thermostatic shaker. Incubations were terminated by the addition of ice-cold methanol and
samples were analyzed after protein precipitation by HPLC with radiodetection.
P450 enzyme
inhibitor
concentration
CYP2C8
CYP2C9
CYP2C19
CYP3A4
montelukast
sulfaphenazole
S-(+)-N-3-benzylnirvanol
ketoconazole
5 µM
5 µM
5 µM
1 µM
Details on dosing regimens:
Dosing regimens in the simulations were matched to the referenced studies and were
performed in the fed state, except the interaction study with rifampicin, which was done
under fasted conditions [4]. Concomitantly dosed drugs were given at the same time, e.g.
daily macitentan was dosed together with the first daily dose of twice daily rifampicin.
Modeling was done assuming 250 mL fluid intake with dosing.
Once a day ketoconazole interaction: 400 mg ketoconazole was dosed once daily from day 1
until day 24. Concomitantly, 10 mg macitentan was dosed once on day 5 [5].
Twice a day ketoconazole interaction: 200 mg ketoconazole was dosed twice daily from day
1 until day 24. Concomitantly, 10 mg macitentan was dosed once on day 5.
Rifampicin interaction: subjects received a loading dose of 30 mg macitentan on the first day,
followed by 10 mg macitentan once daily for 12 days. Concomitantly, 600 mg rifampicin was
dosed daily from day 6 to day 12 [4].
Cyclosporine interaction: subjects received a loading dose of 30 mg macitentan on the first
day followed by 10 mg macitentan once daily for 16 days. Concomitantly, 100 mg rifampicin
was dosed twice daily from day 6 to day 12 [4].
Sildenafil interaction: subjects received a loading dose of 30 mg macitentan on the first day
followed by 10 mg macitentan once daily for 3 days. Sildenafil was concomitantly dosed at
20 mg three times daily for 3 days and once on day 4 [6].
Warfarin interaction: subjects received a loading dose of 30 mg macitentan on the first day
followed by 10 mg macitentan once daily for 8 days. On day 4, warfarin was given
concomitantly as a single dose of 25 mg [7].
The drug interaction studies with erythromycin, itraconazole, ritonavir, diltiazem, verapamil,
clarithromycin, saquinavir, phenytoin and carbamazepine were simulated using the dosing
3
scheme of the perpetrator drug presented in Table 9, for 24 days and a single concomitant
dose of 10 mg macitentan on day 5.
Details on the model population:
Mean demographic data and scaling factors of the simulations (n=100 healthy male
volunteers)
geometric mean
C.V.
27
25 %
weight (kg)
80
17 %
cardiac output (L/h)
365
10 %
liver weight (g)
1711
17 %
age (years)
hepatocelularity (million cells / g liver)
110
22 %
CYP3A4 liver content (pmol)
8573017
55 %
CYP3A4 gut content (pmol)
60372
54 %
C.V. coefficient of variation
Geometric mean predicted tissue-partition coefficients for healthy subjects (n=100):
macitentan
ACT132577
Adipose
Bone
Brain
Gut
Heart
Kidney
Liver
Lung
Muscle
Skin
Spleen
0.45
0.32
0.25
0.31
0.20
0.20
0.18
0.23
0.34
0.37
0.17
0.16
0.11
0.06
0.17
0.16
0.14
0.09
0.21
0.28
0.28
0.10
4
Summary of the used interacting drug physiologically-based pharmacokinetic models - physical chemistry, absorption, distribution and
interaction
ketoco
nazole
rifampi
cin
cyclosp
orine
sildena
fil
Swarfari
n
erythro
mycin
itracon
azole
hydrox
yitracon
azole
ritonav
ir
200 q.d.
formed
from
itracona
zole
diltiaze
m
desmet
hyldilti
azem
verapa
mil
clarith
romyci
n
saquin
avir
phenyt
oin
carba
mazepi
ne
500
b.i.d.
60 t.i.d.
formed
from
diltiaze
m
120
t.i.d.
250
b.i.d.
1200
t.i.d.
300 q.d.
400
b.i.d.
carba
mazepi
ne10,11epoxid
e
formed
from
carbam
azepine
400 q.d.
or 200
b.i.d.
600 q.d.
100 q.d.
20 t.i.d.
25 q.d.
500
t.i.d.
531.4
823
1202
474.6
308.3
733.9
705.6
721.7
720.95
414.5
400
454.6
748
670.86
252.28
236.27
252.27
3.28
4.3
2.97
2.7
2.5
5.66
4.78
4.3
2.8
2.01
3.81
1.7
3.9
2.47
2.22
1.44
7.9
-
6.5
-
8.8
3.7
2.5, 4.9
2.0
8.1
8.06
8.9
8.99
6.67
-
-
-
acidic pKa
4.04
2.94,
6.51
-
1.7
-
-
5.1
-
-
-
-
-
-
-
-
10.94
8.15
-
-
fu,p
0.029
0.15
0.037
0.036
0.009
0.31
0.016
0.021
0.02
0.25
0.323
0.10
0.18
0.025
0.1
0.25
0.48
blood to plasma ratio
0.62
0.90
1.36
0.63
0.59
0.85
0.58
1
0.59
0.96
1
0.76
1
0.6
0.61
1.1
1.53
1
NA
1
1
1
0.9
0.84
NA
oral dose (mg) and
dosing frequency
molecular weight
(g/mol)
lipophilicity (logP)
basic pKa
fa
1
1
1
1
1
1
1
NA
1
(h-1)
0.78
0.51
1.49
2.58
1.85
0.52
0.62
NA
0.24
6
NA
1.22
2.37
2.55
0.53
0.5
NA
Qgut (L/h)
13.2
10
8.16
10
11.6
1.67
15.7
NA
11.2
13.5
NA
14.4
9.34
4.91
13.2
12.6
NA
fu,gut
0.06
0.15
1
0.036
1
1
0.016
1
0.02
0.25
1
1
1
0.025
1
1
1
0.345
0.015 /
0.97
0.33
5.6
3.46
0.114
0.75
10.7
69.5
3.09
3.9
3.4
1.75
0.78
0.78
1.5
-
-
82 / 0.9
0.0013
0.0144
36.1
2.43
-
2.43
3.63
3.5 /
0.58
0.57
10.5
0.41
0.025 /
0.662
-
-
-
-
-
-
-
-
23.2
-
-
-
4.75
1.74
2.21
12
-
-
-
-
-
-
-
-
-
2.25
-
-
-
0.70
1.09
2
2.13
-
-
-
-
-
0.32
-
-
-
-
-
-
-
-
-
-
-
-
7.7
-
8
-
-
-
-
-
-
-
-
-
-
-
-
6.2
slope
0.16 / h
slope
0.14 / h
[8, 9]
[10]
[11]
[12, 13]
[18]
[14]
[14]
[19]
ka
Vss (L/kg)
Ki CYP3A4 (µM) /
fu,inc
KI CYP3A4 (µM) /
fu,inc
kinact CYP3A4 (h-1)
IndC50 CYP3A4
(µM)
Indmax CYP3A4
(fold)
reference*
[14]
[15]
[14, 16]
[17]
[10]
* Describing the details such as data source and/or performance of the PBPK models; data are from unchanged Simcyp v12 compound library PBPK model files, with the exception of the fu,gut of sildenafil, which
was set equal to its plasma protein binding and the CYP3A4 Ki for cyclosporine [20]. Absorption was modeled using the 1st order model. Distribution was modeled using a minimal PBPK model. Free fraction of
the in vitro input data was 1, unless indicated otherwise.
fu,p, fraction of unbound drug in plasma; pKa, acid dissociation constant; fa, fraction absorbed; Qgut hybrid parameter of enterocytic blood flow and drug permeability; f u,gut, free fraction in the enterocyte; Vss
apparent volume of distribution at steady state; K i inhibition constant; fu,inc, free fraction in the incubation; KI, concentration at half-maximal kinact; kinact maximum rate constant for inactivation; IndC50, inducer
concentration at half-maximal Indmax; Indmax, maximal induction; NA, not applicable.
6
Summary of the used interacting drug PBPK models - elimination
drug
ketoconazole
rifampicin
cyclosporine
sildenafil
S-warfarin
erythromycin
itraconazole
hydroxyitraconazole
ritonavir
diltiazem
desmethyldiltiazem
verapamil
clarithromycin
saquinavir
phenytoin
carbamazepine
carbamazepine10,11-epoxide
elimination mechanism
active uptake into hepatocytes was 2.07 fold and oral clearance of 7.4 L/h
and a renal clearance of 0.147 L/h.
i.v. clearance of 7 L/h and a renal clearance of 1.2 L/h.
CYP3A4 with a Km of 1.17 μM and a Vmax of 200 pmol/min/pmol enzyme
and a renal clearance of 0.018 L/h.
CYP2C9 and CYP3A4 using a CLint 0.486 and 2.2 μL/min/pmol enzyme,
respectively and a renal clearance of 0.72 L/h.
renal and polymorphic specific CYP2C9 clearance as described
previously.
i.v. CL of 27.8 L/h and a renal clearance of 3.13 L/h.
CYP3A4 with a Km of 0.039 μM and a Vmax of 0.065 pmol/min/pmol
enzyme towards its metabolite hydroxyitraconazole.
CYP3A4 with a Km of 0.027 μM and a Vmax of 0.13 pmol/min/pmol
enzyme.
oral clearance of 9.1 L/h and a renal clearance of 0.32 L/h.
CYP3A4 with a Km of 50 μM and a Vmax of 1880 pmol/min/pmol enzyme
and by CYP1A2 using a CLint 2.4 μL/min/pmol enzyme towards
desmethyldiltiazem and using a human liver microsomal CLint of 109
μL/min/mg protein.
CYP3A4 using a CLint 43.2 μL/min/pmol, using a human liver microsomal
CLint of 1.65 μL/min/mg protein and a renal clearance of 2.88 L/h.
i.v. CL of 52.2 L/h and a renal clearance of 2.37 L/h.
CYP3A4 using two pathways, with a K m of 20.6 and 63.9 μM and a V max
of 37.9 and 63.9 pmol/min/pmol enzyme, respectively and a renal
clearance of 8.05 L/h.
CYP3A4 with a Km of 0.51 μM and a Vmax of 2860 pmol/min/pmol
enzyme and a renal clearance of 0.95 L/h.
CYP2C9 and CYP2C19 with a Km of 4.1 and 1.53 μM and a Vmax of 0.24
and 1.53 pmol/min/pmol enzyme, respectively, using a human liver
microsomal CLint of 0.97 μL/min/mg protein and a renal clearance of
0.015 L/h.
CYP3A4, CYP3A5 and CYP2C8 with a K m of 180, 332 and 741 μM and a
Vmax of 0.72, 1.44 and 0.03 pmol/min/pmol enzyme, respectively, towards
its metabolite carbamazepine-10,11-epoxide and additional clearance by
CYP3A4 using a CLint of 0.0106 µL/min/pmol enzyme, a human liver
microsomal CLint of 0.255 μL/min/mg protein and a renal clearance of
0.0084 L/h.
reference*
[21]
[10]
[11]
[12, 13]
[22]
[14]
[15]
[16]
[17]
[18]
[17]
[16]
[19]
[23]
oral clearance of 6.07 L/h and a renal clearance of 0.14 L/h.
* Describing the details of the compound PBPK model elimination processes; data are from unchanged Simcyp v12
compound library PBPK model files
i.v. intravenous, Km Michaelis-Menten constant, Vmax maximum rate CLint intrinsic clearance of drug from
plasma
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