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Drug interaction potential of osilodrostat (LCI699) based on its effect
on the pharmacokinetics of probe drugs of cytochrome P450 enzymes
in healthy adults
Sara Armani MD, Lillian Ting PhD, Nicholas Sauter MD, Christelle Darstein PhD,
Anadya Prakash Tripathi PhD, Lai Wang MSc, Bing Zhu MSc, Helen Gu MSc,
Dung Yu Chun BSc, Heidi J Einolf PhD, Swarupa Kulkarni PhD
Supplemental Information
Materials and methods
Materials
Osilodrostat and [14C]osilodrostat was provided by Novartis (Basel, Switzerland). The
specific activity of [14C]osilodrostat was 56.3 mCi/mmol with 99% radiochemical purity.
Pooled human liver microsomes (HLM) were purchased from XenoTech, LLC (Lenexa, KS,
USA) or Corning Life Sciences – Discovery Labware (Woburn, MA, USA). Recombinant
human cytochrome P450 (CYP), uridine 5′-diphospho-glucuronosyltransferase (UGT), and
flavin-containing monooxygenase (FMO) enzymes were purchased from Corning Life
Sciences – Discovery Labware. Cryopreserved human hepatocytes were obtained from
BioreclamationIVT (Westbury, NY, USA) or Corning Life Sciences – Discovery Labware.
The following chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA):
acetonitrile, alamethicin, ammonium formate, amodiaquine hydrochloride dehydrate,
bupropion hydrochloride, chlorzoxazone, coumarin, diclofenac, dimethyl sulfoxide (DMSO),
furafylline,
magnesium
diphenyltetrazolium
bromide
chloride,
(MTT),
midazolam,
nicotinamide
3-(4,5-dimethylthiazol-2-yl)-2,5adenine
dinucleotide
phosphate
(NADPH), uridine 5′-diphospho-glucuronic acid (UDPGA), omeprazole (OME), paroxetine,
phenacetin, potassium phosphate (mono- and dibasic), rifampin (RIF), S-mephenytoin,
testosterone, ticlopidine, troleandomycin, and warfarin. Bufuralol hydrochloride, paclitaxel,
and S-mephenytoin were obtained from Ultrafine Chemicals (Manchester, UK). Gemfibrozil
glucuronide was obtained from LC Scientific (Concord, ON, USA). Tienilic acid was
1
obtained from Novartis. IN FLOW 2:1 was purchased from LabLogic Systems Inc.
(Brandon, FL, USA).
The internal standards used for the CYP inhibition assay, [2H4]acetaminophen, [2H6]
hydroxybupropion,
[2H4]-α-hydroxymidazolam, and
[2H3]6β-hydroxytestosterone,
were
obtained from Cerilliant (Round Rock, TX, USA). [2H3]Desethylamodiaquine and [2H5]7hydroxycoumarin
were
obtained
from
Corning
(Corning,
NY,
USA).
[2H3]4′-
hydroxymephenytoin, [13C22H3]-4′-hydroxy-S-mephenytoin, and [13C6]-4′-hydroxydiclofenac
were prepared by the isotope laboratory of Novartis (East Hanover, NJ, USA).
The metabolite standards for the CYP inhibition assay, acetaminophen, 7-hydroxycoumarin,
N-desethylamodiaquine dihydrochloride, and 6β-hydroxytestosterone, were obtained from
Sigma-Aldrich. Hydroxybupropion, 6-hydroxypaclitaxel, and 4′-hydroxydiclofenac were
purchased from Corning. 1′-Hydroxybufuralol maleate, 6-hydroxychlorzoxazone, 4′-hydroxyS-mephenytoin, and 1′-hydroxymidazolam were obtained from Ultrafine Chemicals.
In vitro cytochrome CYP inhibition
The potential for reversible and/or time-dependent inhibition (TDI) of CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A by osilodrostat
was investigated in vitro using pooled HLM. CYP activity was assessed using the probe
reactions phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6),
bupropion hydroxylation (CYP2B6), paclitaxel 6α-hydroxylation (CYP2C8), diclofenac
4′-hydroxylation
(CYP2C9),
S-mephenytoin
4′-hydroxylation
(CYP2C19),
bufuralol
1′-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam-1′hydroxylation or testosterone 6β-hydroxylation (CYP3A). For reversible inhibition,
incubations (37°C, 10–20 min) were composed of (final concentrations) potassium
phosphate buffer (100 mM, pH 7.4), NADPH (1 mM), magnesium chloride (5 mM), HLM
protein (0.05–0.5 mg protein/mL), probe substrate (1 µM midazolam or coumarin, 5 µM
phenacetin, diclofenac, or bufuralol, 10 µM paclitaxel or chlorzoxazone, 15 µM
S-mephenytoin, or 25 µM bupropion or testosterone), varying concentrations of osilodrostat
(0–100 µM), and organic solvent (≤1%). After pre-incubation for 3 min, the reactions were
initiated by addition of NADPH and terminated by addition of acetonitrile (2 volumes).
2
Reactions were previously shown to be linear with respect to time and protein concentration
(results not shown). Formation of probe substrate metabolites from the above samples,
acetaminophen,
7-hydroxycoumarin,
4′­hydroxydiclofenac,
6-hydroxychlorzoxazone,
hydroxybupropion,
4′-hydroxy-S-mephenytoin,
1′-hydroxymidazolam,
and
6α-hydroxypaclitaxel,
1′-hydroxybufuralol,
6β-hydroxytestosterone,
was
determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) after
concentration and reconstitution of the samples in acetonitrile/water containing an internal
standard. Values for 50% inhibitory concentration (IC50) for the inhibition of CYP enzyme
were determined by visual inspection of the data (percentage of control CYP activity vs
osilodrostat concentration).
For assessments of TDI of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,
and CYP3A activity, osilodrostat (0–50 or 0–100 µM) was pre-incubated (37°C) with HLM
(0.5–10 mg microsomal protein/mL) in the same buffer components as described above.
The pre-incubations were initiated by addition of NADPH. After various pre-incubation
times, aliquots were removed and transferred to an enzyme activity assay mixture (20- to
100-fold dilution of the pre-incubation reaction) containing the same buffer components as
the pre-incubation and CYP probe substrates to determine the activity remaining. The
concentrations of probe substrates in the enzyme activity assay were 100 µM phenacetin
(CYP1A2), 1.5 mM bupropion (CYP2B6), 20 µM amodiaquine (CYP2C8), 25 µM diclofenac
(CYP2C9), 300 µM S-mephenytoin (CYP2C19), 50 µM bufuralol (CYP2D6), and 10 µM
midazolam (CYP3A4). The enzyme activity assay reactions were incubated at 37°C for
6–10 min and terminated as above. Positive-control time-dependent inhibitors included
furafylline (CYP1A2), ticlopidine (CYP2B6 and CYP2C19), gemfibrozil glucuronide
(CYP2C8), tienilic acid (CYP2C9), paroxetine (CYP2D6), and troleandomycin (CYP3A4).
Preparation of samples for analysis of probe substrate metabolite formation by LC-MS/MS
was as described above. The probe metabolites analyzed were as described above, with
the exception that amodiaquine N-desethylation was the probe reaction used for CYP2C8.
The inactivation parameters kinact (maximum inactivation rate) and KI (concentration at
½ kinact) were determined by plotting the natural log of the percentage of control activity
remaining following incubations with increasing inhibitor concentration against time of preincubation. The absolute value of the observed rate of inactivation (kobs) was then plotted
3
against inhibitor concentration ([I]) and the data analyzed by non-linear regression using the
equation:
kobs = kinact[I]/KI+[I]
In vitro cytochrome CYP induction
The potential for induction of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 mRNA and
enzyme activities by osilodrostat was initially assessed in vitro using cryopreserved
hepatocytes from three donors (Corning Life Sciences – Discovery Labware). The cells
were treated with osilodrostat (0.25, 2.5, 10, or 25 µM), the positive controls RIF (10 µM) or
OME (50 µM), and the vehicle control (0.1% DMSO) for 48 h. The medium was changed
with fresh addition of the compounds or vehicle control 24 h after the first treatment dose.
Induction of mRNA was determined by real-time polymerase chain reaction (PCR) using the
comparative CT method, enzyme activity was measured in situ using CYP-selective probe
substrates, and cell viability was assessed using the MTT assay after the treatment period.
The method was essentially as described in Flarakos et al, 2016 [1]. Based on this initial
assessment, values for half-maximal effective concentration (EC50) and maximum effect
(Emax) for CYP1A2, CYP2B6, and CYP3A4 mRNA induction were determined in one
cryopreserved hepatocyte donor. Hepatocytes were treated with 0.05, 0.5, 2.5, 10, 25, 100,
200, or 400 µM of osilodrostat for 48 h, the mRNA isolated, and CYP induction assessed by
real-time PCR as described above. The induction parameters EC50 and Emax were
determined by non-linear regression of the mRNA induction data using a sigmoidal dose–
response model:
Effect = Emin+(Emax–Emin)/[1+(EC50/X)N]
where X is the inducer concentration in vitro, Emin and Emax are the minimal and maximal
induction levels in vitro (fold increase over vehicle control), respectively, EC50 is the
concentration of inducer at half-maximal induction level in vitro, and N is the Hill slope. If the
results of the fit to this model were ambiguous (as was the case only for CYP1A2 induction),
the EC50 and Emax values were estimated by visual inspection of the data, assuming that the
4
Emax value was the maximal induction effect observed at the highest osilodrostat
concentration tested.
In vitro enzyme reaction phenotyping
The metabolism of [14C]osilodrostat was examined in pooled HLM in the presence of
NADPH and/or UDPGA. HLM (1 mg microsomal protein/mL) in 100 mM potassium
phosphate buffer (pH 7.4) were pre-incubated with alamethicin (60 µg alamethicin/mg
protein, final concentration) for 15 min on ice. MgCl2 (5 mM, final concentration) and
[14C]osilodrostat (50 µM, 0.5% DMSO, final concentrations) were then added and the
samples thermally equilibrated at 37ºC for 3 min. The reactions were initiated with 4 mM
UDPGA and/or 1 mM NADPH (final concentrations), and the samples (in singlets) were
incubated for 30 min at 37°C. The reactions were terminated by the addition of an equal
volume of cold acetonitrile, and the precipitated protein was removed by centrifugation at
39,000 x g for 10 min at ~4°C in an Avante 30 high-speed microcentrifuge (Beckman
Coulter, Fullerton, CA, USA). Aliquots of the supernatants were analyzed by reversedphase high-performance liquid chromatography (HPLC; see below).
To identify the oxidative metabolic enzyme(s) involved in the metabolism of osilodrostat in
humans, [14C]osilodrostat (50 µM, final concentration) was incubated with: the recombinant
human CYP enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4A11,
CYP4F2, CYP4F3A, CYP4F3B, or CYP4F12 (100 pmol CYP/mL); the recombinant human
FMO enzymes FMO1, FMO3, or FMO5 (1 mg microsomal protein/mL); or control
microsomes in 100 mM potassium phosphate buffer (pH 7.4) containing 5 mM MgCl2 (final
concentrations). The reactions were thermally equilibrated and initiated by the addition of
NADPH for the CYP reactions or [14C]osilodrostat for the FMO reactions. The samples were
incubated for 30 min at 37°C and the reactions quenched by the addition of an equal
volume of cold acetonitrile. The precipitated protein was removed by centrifugation as
described above, and an aliquot of each sample was analyzed by HPLC with both in-line
and off-line radioactivity detector as described below. Kinetic parameters of [14C]osilodrostat
metabolism were determined for CYP3A4, CYP2D6, and CYP2B6. Recombinant human
CYP3A4, CYP2D6, and CYP2B6 (100 pmol CYP/mL) were thermally equilibrated with
5
varying concentrations of [14C]osilodrostat in the buffer components described above. The
reactions were initiated with NADPH and incubated for 30 min. Control samples at each
concentration of [14C]osilodrostat did not contain NADPH. The reactions were quenched
and prepared for analysis as described above. The samples were analyzed by HPLC and
off-line radioactivity detection. To determine the metabolism activity of [14C]osilodrostat, the
percentage of radioactivity of each peak in the HPLC chromatogram (parent and metabolite)
was quantified (totaling 100%). The amount of osilodrostat metabolite formed (M24.9) in the
reaction was based on the percentage of radioactivity in the product peak with respect to
the total amount of [14C]osilodrostat in the starting reaction. The metabolism activity was
therefore calculated as the amount of product formed per reaction time per total amount of
CYP in the reaction (i.e. nmol/h/nmol CYP). [14C]Osilodrostat metabolism activity was
plotted against substrate concentration, and the kinetic parameters Km and Vmax were
determined by non-linear regression using the hyperbolic function:
v = Vmax x [S]/(Km+[S])
where v is the velocity of the reaction, Vmax is the maximal velocity, [S] is the substrate
concentration, and Km is Michaelis–Menten constant. Total osilodrostat remained below
20% for all reactions.
To identify the UGT enzyme(s) involved in the glucuronidation of osilodrostat in humans, the
following recombinant UGT enzymes were examined for osilodrostat metabolizing activity.
Human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10,
UGT2B4, UGT2B7, UGT2B10, UGT2B15, UGT2B17, or control microsomes (1 mg
protein/mL, final concentration) in 100 mM potassium phosphate buffer (pH 7.4) were
treated with alamethicin (60 μg alamethicin/mg protein, final concentration) for 15 min on
ice. MgCl2 (5 mM, final concentration) and [14C]osilodrostat (50 µM, final concentration)
were then added and the samples thermally equilibrated prior to initiation with UDPGA
(4 mM, final concentration). The samples were incubated for 30 min at 37ºC and the
reactions quenched by the addition of an equal volume of cold acetonitrile. The precipitated
protein was removed by centrifugation as described above, and an aliquot of each sample
was analyzed by HPLC with off-line radioactivity detector as described below.
6
The HPLC chromatographic equipment consisted of a Waters Acquity ultra-performance
liquid chromatography (UPLC) system (Milford, MA, USA). The chromatographic separation
was performed on a Waters Acquity UPLC HSS T3 column (1.8 µm, 2.1 x 150 or 100 mm)
at a temperature of 50ºC. Gradient elution consisted of solvent A (20 mM ammonium
formate) and solvent B (acetonitrile) at a flow rate of 0.5 mL/min. The gradient elution
program (%B) was 0→2 min (2%), 2→7 min (2–6%), 7→17 min (6–15%), 17→27 min
(15–35%), and 27→29 min (35–95%). For the kinetic studies, the gradient elution program
(%B) was 0→0.5 min (5%), 0.5→18 min (5–45%), 18→20 min (45–95%), and 20→22 min
(95%). Radioactivity was measured either in-line or with off-line radioactivity detection.
In-line radioactivity measurement was performed with a β-RAM radioactivity detector
(LabLogic Systems Inc.), with addition of 1 mL liquid scintillant/min (IN FLOW 2:1, LabLogic
Systems, Inc.) to the HPLC eluate prior to flow through a liquid flow cell (250 µL). Off-line
radioactivity measurement was performed with TopCount low-level radioactivity detection.
The HPLC eluate was collected with a fraction collector (Collect PAL, LEAP Technologies,
Carrboro, NC, USA) at 9 s per fraction into Deepwell LumaPlate-96 plates (PerkinElmer Life
and Analytical Sciences). The fractions were dried with a stream of nitrogen, and
radioactivity was counted with a TopCount NXT Microplate Scintillation and Luminescence
Counter (PerkinElmer Life and Analytical Sciences) at a counting time of 10 min per well.
Chromatograms were evaluated using Laura HPLC application software (LabLogic Systems
Ltd, Sheffield, UK).
Osilodrostat PBPK model
Input parameters
The platform used for the physiologically based pharmacokinetic (PBPK) modeling was the
Simcyp® Simulator (Certara Inc., Version 15, Release 1). Physiochemical and clinical
pharmacokinetic parameters, and in vitro CYP perpetrator properties of osilodrostat used for
the PBPK model, are summarized in Supplemental Table 5. The fraction of dose absorbed
from the gastrointestinal tract (f a) was estimated to be 1 as excretion of osilodrostat-related
radioactivity in the feces of humans from the radiolabeled mass-balance study accounted
for <2% of the administered dose, suggesting nearly complete oral absorption (internal
data, clinical study CLCI699C2101). The absorption rate constant (ka) was estimated to be
7
2.8/h based on fitting of the actual clinical data of a 30 or 50 mg single oral osilodrostat
dose. The term fugut (unbound fraction in enterocytes) was defaulted as 1. The effective
terms Peff,man (permeability in man) and Qgut (gut blood flow) were predicted in the software
to be 6.88 x 10–4 cm/s and 14.5 L/h, respectively, based on Caco-2 cell permeability data.
The minimal PBPK model was used with a predicted volume of distribution (Vss) of
1.277 L/kg. Osilodrostat hepatic microsomal intrinsic clearance (CLint) was back calculated
from the clinically observed median plasma clearance after oral dosing (CLpo) of ~16.6 L/h
(30 mg single dose; internal data, clinical study CLCI699A2101) using the enzyme kinetics
retrograde model implemented in Simcyp. The percentage contributions of the individual
CYP-mediated pathways were based on enzyme reaction phenotyping (Supplemental
Table 4) and contribution of CYP-mediated oxidative clearance to total osilodrostat
clearance
determined
from
the
radiolabeled
mass-balance
study
(clinical
study
CLCI699C2101). The percentage of clearance mediated by CYP oxidative metabolism in
humans was estimated to be 26% based on the amount of specific metabolites (normalized
to total dose recovery) characterized in the excreta (internal data, clinical study
CLCI699C2101). The relative contributions of CYP enzymes to this oxidative pathway in
HLM in vitro was determined to be 45% by CYP3A4, 31% by CYP2D6, and 24% by
CYP2B6 (see below; Supplemental Table 4). Therefore, in humans, the relative
contributions of the individual CYP enzymes to total clearance of osilodrostat in vivo was
estimated to be 11.7% for CYP3A4 [(0.26 x 0.45) x 100], 8.07% for CYP2D6 [(0.26 x 0.31) x
100], and 6.25% for CYP2B6 [(0.26 x 0.24) x 100]. The resultant entries in the model for the
individual CYP CLint values and additional HLM clearance calculated in Simcyp are shown
in Supplemental Table 5. Osilodrostat mean renal clearance was entered as 0.86 L/h
(internal data, clinical study CLCI699C2101).
The CYP interaction parameters used in the model are shown in Supplemental Table 5. The
inhibition parameters used were IC50/2 values as they were, for the majority of the CYP
enzymes, the lower value (compared with determined KI values; Supplemental Table 1) and
provided a better prediction of the actual drug–drug interaction (DDI; AUC ratio) of the effect
of osilodrostat on the CYP probe substrates. The induction parameters used for the model
are shown in Supplemental Table 3. The CYP3A4 induction parameters were normalized in
the Simcyp software based on the positive-control RIF induction parameters determined in
8
the in vitro study (RIF Emax of 49-fold and EC50 of 0.61 µM; raw data not shown). Since
normalization functions were not available for CYP1A2 or CYP2B6 induction in the
software, the experimental induction values were used without normalization.
Model development and application
The Simcyp simulator was used for these simulations with the Simcyp ‘healthy volunteer’
population. The proportion of females in the model was set to 0.5. Ten trials of 10 subjects
were simulated for each dosing regimen. Input values for midazolam, OME (enteric coated),
caffeine, and dextromethorphan were provided in the Simcyp simulator. The model was
developed to simulate the pharmacokinetic parameters of single osilodrostat 30 and 50 mg
doses and the osilodrostat (50 mg single dose) DDI effect on the CYP probe substrates
midazolam (2 mg), caffeine (100 mg), OME (20 mg), and dextromethorphan (30 mg),
according to the actual trial design (see main text). The model was then applied to predict
the pharmacokinetics of osilodrostat and the DDIs of the aforementioned CYP substrates
(dosed on day 14) after multiple 30 mg twice-daily doses of osilodrostat (dosed on
days 1–16).
Results
In vitro cytochrome CYP inhibition
The results of the in vitro CYP inhibition assessment are tabulated in Supplemental Table 1.
Osilodrostat was found to be a reversible inhibitor of CYP1A2, CYP2C19, CYP2E1,
CYP2D6, and CYP3A4/5 (IC50 <10 µM) and a weaker inhibitor of CYP2B6 and CYP2C9
(IC50 ≥20 µM). Osilodrostat was not found to inhibit CYP2A6 or CYP2C8 at the
concentrations examined. No TDI of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, or
CYP3A4 by osilodrostat was observed. TDI of CYP2C19 was observed, with the
inactivation parameters KI and kinact estimated to be 52.3 µM and 0.0260/min, respectively.
In vitro cytochrome CYP induction
The results of the preliminary in vitro CYP induction study in three donors of human
hepatocytes are shown in Supplemental Table 2. Dose-dependent increases in CYP1A2,
CYP2B6, CYP2C9, and CYP3A4 mRNA and activity with osilodrostat treatment (up to
9
25 µM) were observed in the three donors. Induction of CYP2C9 mRNA or activity was less
than 2-fold and was therefore deemed negligible. Induction of CYP1A2, CYP2B6, and
CYP3A4 mRNA exceeded 2-fold in the three donors (although CYP3A4/5 activity did not
exceed 2-fold induction); therefore, a follow-up in vitro induction study to determine the
induction parameters Emax and EC50 for mRNA was performed in one hepatocyte donor.
The results of this analysis are shown in Supplemental Table 3.
In vitro enzyme reaction phenotyping
[14C]Osilodrostat was metabolized in HLM in the presence of NADPH and UDPGA to form a
monohydroxylated metabolite, M24.9, and one direct glucuronide metabolite, M16.5 (data
not shown). Of the recombinant CYP enzymes evaluated for osilodrostat metabolism,
M24.9 was observed in incubations with CYP3A4, CYP2D6, and CYP2B6. M16.5 was
formed by the UGT enzymes UGT1A4, UGT2B7, and UGT2B10. Kinetic analysis of M24.9
formation by recombinant human CYP3A4, CYP2D6, and CYP2B6 enzymes and scaling of
the data by the relative abundance of these enzymes in HLM [2] found the relative
contribution of these enzymes to total oxidative clearance in HLM to be 45%, 31%, and
24%, respectively (Supplemental Table 4).
Osilodrostat PBPK model
Based on the physiochemical properties, clinical pharmacokinetic information, and in vitro
CYP inhibition and induction characterization of osilodrostat, a PBPK model was built within
the framework of the Simcyp simulator to: 1) simulate the pharmacokinetics and DDI of
osilodrostat after a single 50 mg dose; 2) simulate the pharmacokinetics of osilodrostat after
a 30 mg single dose; and 3) predict the pharmacokinetics and DDI of osilodrostat after
multiple doses (30 mg twice daily). The simulated and observed concentration–time profiles
of osilodrostat after a 30 and 50 mg oral dose can be found in Supplemental Figure 1a and
Figure 1b, respectively. The observed versus predicted pharmacokinetic parameters
(AUClast and Cmax) are tabulated in Supplemental Table 6. For both doses, the
pharmacokinetic parameters were predicted well within 2-fold of the actual values
(prediction errors of +10% to –13.2%). The concentration–time profiles and pharmacokinetic
parameters were predicted for multiple 30 mg twice-daily doses of osilodrostat. The
10
predicted concentration–time profiles can be found in Supplemental Figure 2, and the
values of the pharmacokinetic parameters can be found in Supplemental Table 6.
The effect of osilodrostat (50 mg single dose) on single doses of the CYP probe substrates
caffeine (CYP1A2), OME (CYP2C19), dextromethorphan (CYP2D6), and midazolam
(CYP3A4/5) was simulated. The predicted versus observed pharmacokinetic parameters
(geometric mean AUClast and Cmax) can be found in Supplemental Table 7. The predicted
geometric mean AUClast and Cmax ratios (and prediction errors) can be found in Table 2 of
the main text. The pharmacokinetic parameters of the probe substrates caffeine, OME, and
midazolam in the presence and absence of osilodrostat were predicted well within 2-fold of
the actual values. The exception was dextromethorphan, whereby the observed
pharmacokinetic variability was very high and the predicted geometric mean AUClast and
Cmax values were much higher than observed. Also included in Supplemental Table 7 are
the arithmetic means observed and the simulated pharmacokinetic parameters for
dextromethorphan. In the case of the arithmetic mean, the pharmacokinetic parameters
were simulated within 2-fold of the actual values (+40 to +63%). Regardless, the predicted
geometric mean AUC and Cmax ratios for dextromethorphan were predicted within 6% of the
observed values (main text, Table 2). The other CYP probe substrate DDIs were also well
predicted, with geometric mean AUC and Cmax ratios within 30% of the observed values.
The predicted geometric mean AUC ratios were within the criteria for prediction accuracy
described by Guest et al [3].
Multiple-dose osilodrostat treatment (30 mg twice daily for 16 days) predicted a similar DDI
effect on the CYP probe substrates (dosed as a single dose on day 14) to that in the actual
single-dose trial. The prediction results can be found in Table 2 of the main text. For the
CYP1A2, CYP2D6, and CYP3A4/5 substrates, there was essentially no change in DDI
magnitude (≤3% change) when co-administered after multiple doses of osilodrostat (30 mg
twice daily) for 14 days. For the CYP2C19 substrate OME, a modest 26% increase in the
AUC ratio was predicted after multiple dosing of osilodrostat. This predicted increase was a
result of the in vitro CYP2C19 TDI parameters included in the PBPK model for osilodrostat.
11
References
1. Flarakos J, Du Y, Bedman T, Al-Share Q, Jordaan P, Chandra P, et al. Disposition
and metabolism of [(14)C] sacubitril/valsartan (formerly LCZ696) an angiotensin
receptor neprilysin inhibitor, in healthy subjects. Xenobiotica. 2016;46:986–1000.
2. Inoue S, Howgate EM, Rowland-Yeo K, Shimada T, Yamazaki H, Tucker GT, et al.
Prediction of in vivo drug clearance from in vitro data. II: potential inter-ethnic
differences. Xenobiotica. 2006;36:499–513.
3. Guest EJ, Aarons L, Houston JB, Rostami-Hodjegan A, Galetin A. Critique of the
two-fold measure of prediction success for ratios: application for the assessment of
drug-drug interactions. Drug Metab Dispos. 2011;39:170–173.
12
Supplemental Table 1. In vitro CYP inhibition parameters for osilodrostat
CYP enzyme
IC50 (IC50/2), µM
KI, µM
TDI KI, µM
TDI kinact, /min
CYP1A2
1 (0.5)
0.776 ± 0.112
No TDI
No TDI
CYP2A6
>100
ND
ND
ND
CYP2B6
20 (10)
ND
No TDI
No TDI
CYP2C8
~100
ND
No TDI
No TDI
CYP2C9
40 (20)
ND
No TDI
No TDI
CYP2C19
4 (2)
4.63 ± 0.353
52.3 ± 29.3
0.0260 ± 0.00695
CYP2D6
4 (2)
1.97 ± 0.397
No TDI
No TDI
CYP2E1
2 (1)
0.482 ± 0.272
ND
ND
6.5 (3.25)
7.00 ± 1.22
No TDI
No TDI
8 (4)
ND
ND
ND
CYP3A4/5 (midazolam)
CYP3A4/5 (testosterone)
CYP, cytochrome P450; IC50, 50% inhibitory concentration; KI, concentration at ½ kinact; kinact, maximum
inactivation rate; ND, not determined; TDI, time-dependent inhibition
13
Supplemental Table 2. Fold change in CYP mRNA and activity in human hepatocytes treated with osilodrostat or
positive control inducers
CYP1A2
Donor
1
2
3
CYP2B6
CYP2C9
CYP3A4
Treatment
mRNA
Activity
mRNA
Activity
mRNA
Activity
mRNA
Activity
Osilodrostat, 0.25 µM
1.13
1.37
0.985
1.08
1.02
1.07
1.20
0.940
Osilodrostat, 2.5µM
2.47
2.78
1.17
1.45
1.04
0.985
1.13
1.08
Osilodrostat, 10 µM
3.33
4.28
1.41
2.04
1.22
1.32
1.32
1.16
Osilodrostat, 25 µM
5.06
6.49
2.20
2.59
1.29
1.48
2.45
1.35
Positive control
207
60.5
11.7
8.17
3.17
3.44
96.2
9.97
Osilodrostat, 0.25 µM
1.81
2.17
1.50
0.932
1.16
1.02
1.42
0.958
Osilodrostat, 2.5µM
5.01
3.62
1.48
1.32
1.08
0.987
1.14
0.940
Osilodrostat, 10 µM
12.7
4.57
1.74
1.71
1.23
1.16
1.48
1.10
Osilodrostat, 25 µM
24.6
5.47
2.26
2.17
1.31
1.31
2.67
1.30
Positive control
1160
11.3
12.2
3.45
3.45
2.05
193
4.12
Osilodrostat, 0.25 µM
1.75
1.66
1.97
1.13
1.55
0.931
1.71
0.922
Osilodrostat, 2.5µM
2.41
3.08
2.31
1.43
1.67
0.976
1.85
0.889
Osilodrostat, 10 µM
3.37
4.64
2.14
2.74
1.60
1.13
1.74
1.11
Osilodrostat, 25 µM
4.55
6.44
3.49
4.41
1.73
1.46
3.03
1.28
Positive control
207
32.3
15.1
6.52
3.79
1.88
112
3.82
The positive-control inducers were omeprazole (50 µM) for CYP1A2 and rifampin (10 µM) for CYP2B6, CYP2C9, and CYP3A4. CYP,
cytochrome P450
14
Supplemental Table 3. Fold change in CYP mRNA and calculated in vitro induction
parameters for osilodrostat
Osilodrostat, µM
Mean fold change in mRNA
CYP1A2
CYP2B6
CYP3A4
0.05
1.35
1.17
0.988
0.50
1.82
1.31
0.909
2.5
2.40
1.47
1.30
10
3.35
1.93
1.77
25
4.30
2.05
3.12
100
8.79
6.46
7.67
200
11.8
10.4
13.5
400
18.7
13.1
19.8
EC50, µM
~100
136 ± 12
375 ± 125
Emax, fold
18.7
15.1 ± 0.80
37.4 ± 6.7
CYP, cytochrome P450; EC50, half-maximal effective concentration; Emax, maximum effect
15
Supplemental Table 4. Kinetic parameters of osilodrostat metabolism by recombinant
human CYP enzymes
Parameter
CYP3A4
CYP2B6
CYP2D6
Km, μM
36.1 ± 6.4
36.1 ± 3.8
30.4 ± 2.4
Vmax, nmol/h/nmol CYP
15.1 ± 1.3
80.6 ± 4.2
122.5 ± 4.5
Vmax/Km, mL/h/nmol CYP
0.42
2.24
4.03
CYP abundance in HLM, nmol CYP/mg protein [2]
0.111
0.011
0.008
0.0466
0.0246
0.0322
45
24
31
,a
HLM scaled CLint mL/h/mg protein
Relative CYP contribution in HLM,b %
aV
max/Km
x CYP abundance; bPercentage individual CYP enzyme contribution to total HLM CLint = HLM
scaled CLint,individual
CYP/ΣHLM
scaled CLint,all
CYP.
CLint, intrinsic clearance; CYP, cytochrome P450; HLM,
human liver microsomes; Km, Michaelis–Menten constant; Vmax, maximal reaction velocity
16
Supplemental Table 5. Simcyp model input parameters for osilodrostat
Parameter
Value
Physical chemistry and blood binding
Molecular weight, g/mol
Parameter
Elimination
227.24
Log P
1.7
pKa
6.9
B/P
0.85
fuplasma
0.636
Model used
CLint CYP3A4,
µL/min/pmol CYP
CLint CYP2B6,
µL/min/pmol CYP
CLint CYP2D6,
µL/min/pmol CYP
Additional HLM clearance,
µL/min/mg protein
CLR, L/h
Model used
First order
fa
ka, /h
Peff,man, x
fugut
Qgut, L/h
Vss, L/kg
0.0229
0.0627
4.60
0.86
cm/s
0.5
1
CYP1A2 EC50, µM
100
2.8
CYP1A2 Emax, fold
18.7
6.88
CYP2B6 KI, µM
10
1
CYP2B6 EC50, µM
136
14.5
CYP2B6 Emax, fold
15.1
CYP2C9 KI, µM
20
CYP2C19 KI, µM
2
Minimal PBPK
CYP2C19 TDI KI, µM
52.3
1.277
CYP2C19 TDI kinact, /h
1.56
CYP2D6 KI, µM
2
CYP3A4 KI, µM
3.25
CYP3A5 KI, µM
3.25
EC50,c µM
12.38
CYP3A4 Emax,c fold
196.7
CYP3A4
aThe
0.00531
CYP1A2 KI, µM
Distribution
Model used
Retrograde modela
Interactionb
Absorption
10–4
Value
CYP CLint and additional HLM clearance was estimated using the Simcyp retrograde model
(Supplemental Materials and Methods); bFor KI values, IC50/2 was used as, for the majority of the
enzymes, this value was more conservative (Supplemental Table 1); cThese values were normalized in
Simcyp with the rifampin positive-control Emax (49-fold) and EC50 (0.61 µM) values. B/P, blood-to-plasma
ratio; CLint, intrinsic clearance; CLR, renal clearance; CYP, cytochrome P450; EC50, half-maximal effective
concentration; Emax, maximum effect; fa, fraction absorbed in gastrointestinal tract; fugut, unbound fraction
in enterocytes; fuplasma, unbound fraction in plasma; HLM, human liver microsomes; k a, absorption rate
constant; KI, concentration at ½ kinact; kinact, maximum inactivation rate; PBPK, physiologically based
17
pharmacokinetic; Peff,man, effective permeability in man; pKa, acid dissociation constant; Qgut, gut blood
flow; TDI, time-dependent inhibition; Vss, volume of distribution
18
Supplemental
Table
6.
Observed
versus
PBPK-model-predicted
osilodrostat
pharmacokinetic parameters
Treatment
30 mg single dose
AUClast, ng·h/mL
Cmax, ng/mL
Observeda
1782 ± 199 (11)
250 ± 42 (17)
Predicted
1965 ± 739 (38)
217 ± 57.9 (27)
+10.3%
–13.2%
Observed
3470 ± 1050 (30) [3330]
400 ± 79 (20) [392]
Predicted
3275 ± 1231 (38) [3046]
361 ± 96.5 [348]
–5.6% [–8.5%]
–9.8% [–11%]
PE%
50 mg single dose
PE%
30 mg twice-daily multiple
dose for 14 days (28 doses)
Predicted
AUC0–12h: 1976 ± 753 (38) [1835]
AUC0–48h: 2629 ± 1282 (49) [2349]
281 ± 83.2 (30) [268]
Data are given as arithmetic mean ± standard deviation (CV%) [geometric mean].
aClinical
study
CLCI699A2101 (unpublished data). AUC, area under the concentration–time curve; AUClast, AUC0–48h for
single dose and AUC0–12h or AUC0–48h after the last dose for multiple dose; Cmax, maximum concentration;
CV%, coefficient of variation; PBPK, physiologically based pharmacokinetic; PE%, calculated prediction
error (%) = [(predicted value – observed value)/observed value] x 100
19
Supplemental Table 7. Observed versus PBPK-model-predicted CYP probe substrate
PK parameters
Probe substrate
Treatment
Caffeine
Substrate alone
(CYP1A2)
AUClast, ng·h/mL
Cmax, ng/mL
Observed
17200 (61.7)
2500 (29.5)
Predicted
16757 (69)
2166 (39)
–2.6%
–13%
Observed
41100 (37.5)
2640 (20.1)
Predicted
27861 (60)
2524 (38)
–32%
–4.4
Observed
460 (97.0)
271 (69.7)
Predicted
495 (109)
242 (69)
+7.6%
–11%
Observed
852 (90.7)
420 (70.8)
Predicted
766 (96)
349 (60)
–10%
–17%
PE%
Substrate + osilodrostat
PE%
Omeprazole
Substrate alone
(CYP2C19)
PE%
Substrate + osilodrostat
PE%
Dextromethorphan
Substrate alone
(CYP2D6)
Substrate + osilodrostat
Midazolam
Substrate alone
(CYP3A4/5)
Observed
Predicted
70.1 (178) [138]
5.19 (106) [7.50]
PE%
+819% [+53%]
+319% [+63%]
Observed
12.6 (1857.4) [123] 1.80 (332.6) [6.13]
Predicted
103 (146) [172]
7.41 (86) [9.87]
PE%
+718% [+40%]
+312% [+61%]
Observed
21.2 (40.2)
8.78 (38.8)
Predicted
23.8 (76)
8.46 (65)
+12%
–3.6%
Observed
31.7 (34.2)
12.8 (26.9)
Predicted
35.3 (74)
11.8 (61)
+11%
–7.8%
PE%
Substrate + osilodrostat
7.63 (1758.1) [90.3] 1.24 (330.9 [4.60]
PE%
Data are given as geometric mean (CV%) [arithmetic mean]. The arithmetic mean was included as an
additional parameter for comparison of the observed with the predicted pharmacokinetics of
dextromethorphan owing to the high pharmacokinetic variability of this probe substrate. AUClast, area
under the concentration–time curve from time zero to last measureable concentration; C max, maximum
concentration; CV%, coefficient of variation; CYP, cytochrome P450; PBPK, physiologically based
pharmacokinetic; PE%, calculated prediction error (%) = [(predicted value – observed value)/observed
value] x 100
20
Supplemental Figure 1. Observed and PBPK-model-simulated concentration–time
profiles of a single (a) 30 mg or (b) 50 mg oral dose of osilodrostat
b)
a)
600
Osilodrostat Concentration (ng/mL)
Osilodrostat Concentration (ng/mL)
350
300
250
200
150
100
50
500
400
300
200
100
0
0
0
4
8
12
16
20
24
28
Time (h)
32
36
40
44
48
0
4
8
12
16
20
24
28
32
36
40
44
48
Time (h)
The black solid line represents the simulated concentration–time profiles of osilodrostat and the grey
dotted lines are the 10% and 90% confidence intervals. The points on the graphs are the observed mean
concentration–time data ± standard deviation (error bars) from clinical study A2101 (a) or the control arm
of the cocktail drug–drug interaction study (b). PBPK, physiologically based pharmacokinetic
21
Supplemental Figure 2. Predicted concentration–time profile of osilodrostat after
multiple 30 mg twice-daily doses for 14 days
Osilodrostat Concentration (ng/mL)
450
400
350
300
250
200
150
100
50
0
0
48
96
144
192
240
288
336
Time (h)
The solid black line represents the simulated concentration–time profiles of osilodrostat and the dotted
lines are the 10% and 90% confidence intervals. The points on the graph are the observed mean
concentration–time data ± standard deviation (error bars) from a single 30 mg dose (clinical study A2101)
22