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BIOPHARMACEUTICS & DRUG DISPOSITION
Biopharm. Drug Dispos. (2014)
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/bdd.1889
Nonclinical pharmacokinetics and metabolism of EPZ-5676, a
novel DOT1L histone methyltransferase inhibitor
Aravind Basavapathruni, Edward J. Olhava, Scott R. Daigle, Carly A. Therkelsen, Lei Jin, P. Ann Boriack-Sjodin,
Christina J. Allain, Christine R. Klaus, Alejandra Raimondi, Margaret Porter Scott, Angelos Dovletoglou,
Victoria M. Richon†, Roy M. Pollock, Robert A. Copeland, Mikel P. Moyer, Richard Chesworth,
Paul G. Pearson, and Nigel J. Waters*
Epizyme Inc., 400 Technology Square, Cambridge, MA, USA
ABSTRACT: (2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]
imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a
novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment
of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and
metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity
that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo
pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration;
EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of
distribution 2–3 fold higher than total body water. EPZ-5676 showed biexponential kinetics
following i.v. administration, giving rise to a terminal elimination half-life (t1/2) of 1.1, 3.7 and
13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also
studied and utilized for in vitro–in vivo extrapolation purposes. There was good agreement between
the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the
predominant elimination route in preclinical species. Furthermore, low renal clearance was observed
in mouse, approximating to fu-corrected glomerular filtration rate (GFR) and thus passive glomerular
filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated
product (M4) as well as an N-oxide (M6). Copyright © 2014 John Wiley & Sons, Ltd.
Key words:
MLL-rearranged leukemia; nonclinical pharmacokinetics; in vitro–in vivo extrapolation;
metabolite identification
Introduction
Recent advances in the understanding of cancer
incidence have implicated epigenetics and epigenetic targets as potential avenues for therapeutic
*Correspondence to: Epizyme Inc., 400 Technology Square, 4th
Floor, Cambridge, MA 02139, USA.
E-mail: [email protected]
†
Present address: Sanofi, 270 Albany Street, Cambridge, MA,
02139, USA
Copyright © 2014 John Wiley & Sons, Ltd.
intervention. Epigenetic modifications that may
play a hand in cancer development range from
changes in chromatin remodeling, DNA methylation or post-translational modifications of histones
[1]. One such histone modification is strongly tied
to a specific form of leukemia, in which translocation of the mixed lineage leukemia (MLL) gene
results in MLL-fusion proteins that can aberrantly
associate with the histone methyltransferase
DOT1L (disruptor of telomeric silencing-1 like),
resulting in ectopic DOT1L-catalysed methylation
Received 5 August 2013
Revised 27 November 2013
Accepted 31 December 2013
A. BASAVAPATHRUNI ET AL.
of lysine 79 of histone H3 (H3K79) [2–8]. Aberrant
H3K79 methylation serves to drive the expression
of MLL target genes and an oncogenic phenotype.
The strong causality between the H3K79 methylation mark and a cancer phenotype provides
an opportunity for small molecule intervention
of DOT1L catalytic activity. We have reported
previously structure-guided medicinal chemistry
efforts that yielded a potent DOT1L inhibitor,
EPZ004777, demonstrating the first meaningful
proof of concept in histone methyltransferase (HMT)
inhibition [9,10]. Further expansion of our medicinal
chemistry efforts generated the potent molecule
EPZ-5676 ((2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)
ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol), an aminonucleoside analog with
improved inhibition versus DOT1L in in vitro
biochemical and cellular assays [11]. EPZ-5676
inhibits DOT1L with a Ki of ≤ 80 pM and displays
37000-fold selectivity over a panel of other HMTs.
The potency is further exemplified by treatment
in a rat xenograft model of MLL-rearranged
leukemia with EPZ-5676, in which continuous
intravenous (i.v.) infusion of EPZ-5676 caused
complete tumor regressions that were sustained
beyond the compound infusion period with no
significant weight loss or signs of toxicity [11].
This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, a novel
DOT1L inhibitor and the first member of the novel
HMTi class to enter clinical development as a potential therapeutic agent in MLL-rearranged
leukemia. The objectives of this work were to
characterize the pharmacokinetics following i.v.
and p.o. administration in mouse, rat and dog, to
assess the cross-species in vitro–in vivo correlation
and to identify the primary metabolic and elimination pathways involved in the clearance of
EPZ-5676. Understanding the pharmacokinetic
properties along with the remarkable potency
of EPZ-5676 both in vitro and in vivo promoted
the development of this molecule for acute leukemias bearing MLL-rearrangements. EPZ-5676
is currently in Phase I evaluation and represents
not
only
the
first
reported
histone
methyltransferase inhibitor to enter human clinical trials, but a further step towards understanding the link between epigenetic processes
and the pathophysiology of cancer.
Copyright © 2014 John Wiley & Sons, Ltd.
Materials and Methods
Chemicals and reagents
EPZ-5676 was synthesized by Epizyme [11]. All
other reagents were purchased from sources as
described below.
In vivo pharmacokinetics
All animal studies were conducted as per
approved IACUC protocols.
Pharmacokinetic study in mouse. The pharmacokinetics of EPZ-5676 was evaluated in male CD1mice (28–29 g, male, n = 21, purchased from BK
Laboratory Animal Co. Ltd) following i.v. bolus
administration of doses of 5 mg/kg and oral
administration at doses of 20 mg/kg. Oral gavage
and i.v. tail vein injection doses were administered
in a 10% ethanol and 90% saline vehicle. For i.v.
administration, blood samples were taken (n = 3
per time-point; two time-points per mouse) at
0.05, 0.167, 0.5, 1, 2, 4, 6 and 24 h post-dose into
pre-chilled K2-EDTA tubes. For p.o. dosing, blood
samples were taken (n = 3 per time-point; two
time-points per mouse) at 0.167, 0.5, 1, 2, 4 and
6 h post-dose into pre-chilled K2-EDTA tubes.
Blood samples were put on wet ice and
centrifuged at 4°C (2000 × g for 5 min) to obtain
plasma within 15 min of sample collection. Plasma
samples were stored at 20 °C prior to LC-MS/
MS analysis. CD-1 mice (n = 3) also received a single 5 mg/kg i.v. administration of EPZ-5676
followed by urine collection in metabolism cages
for 240 min post-dose. The urine aliquots were
pooled, the total volume recorded and stored frozen at 20 °C prior to LC-MS/MS analysis.
Pharmacokinetic study in rat. The pharmacokinetics
of EPZ-5676 was evaluated in male SpragueDawley rats (n = 3 per dose route, 245–265 g,
purchased from SLAC Laboratory Animal Co.
Ltd). For the i.v. bolus, 1 mg/kg doses prepared in
0.4% hydroxypropyl-beta-cyclodextrin (HPBCD)
in saline were administered via foot dorsal vein
injection. For p.o. administration, 10 mg/kg doses
prepared in 10% ethanol: 5% Solutol HS15: 85%
(5% of dextrose in water) were administered by oral
gavage. Serial blood sampling was employed in
each animal at each time-point, 0.05, 0.217, 0.5, 1,
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
2, 4, 8 and 24 h following i.v. administration and
0.25, 0.5, 1, 2, 4, 6, 8 and 24 h following p.o. administration, with 150 μl of blood collected via the tail vein
into pre-chilled K2-EDTA tubes. Blood samples were
put on wet ice and centrifuged at 4 °C (2000 × g for
5 min) to obtain plasma within 15 min of sample
collection. Plasma samples were stored at 20 °C
prior to LC-MS/MS analysis.
Pharmacokinetic study in dog. The intravenous (i.v.)
pharmacokinetics of EPZ-5676 was evaluated in
beagle dogs (male, n = 3, 7.5–8 kg purchased from
Beijing Marshall Biotechnology Co. Ltd) following
a single i.v. administration at a dose of 1 mg/kg.
The i.v. doses were administered by a single
intravenous infusion over 1 min into the cephalic
vein in a 10% ethanol and 90% saline vehicle. At
designated time-points (pre-dose, 0.083, 0.25, 0.5,
1, 2, 4, 8 and 24 h post-dose), the animals were
restrained manually, and approximately 0.5 ml
blood per time point was collected from the noninjected cephalic vein into pre-chilled K2-EDTA
tubes. Blood samples were put on wet ice and
centrifuged at 4 °C (2000 × g for 5 min) to obtain
plasma within 15 min of sample collection. Plasma
samples were stored at 20 °C prior to LC-MS/
MS analysis.
LC-MS/MS bioanalysis and pharmacokinetic data
analysis
EPZ-5676 was extracted from K2-EDTA plasma or
urine by protein precipitation using an acetonitrile-containing internal standard (a structural analog of EPZ-5676 at a concentration of 5 ng/ml).
Typically, samples were injected onto an LC-MS/
MS system using a Waters BEH phenyl column.
The aqueous mobile phase was water with 0.1%
NH4OH (A), and the organic mobile phase was
acetonitrile with 0.1% NH4OH (B). The gradient
was as follows: 37% B for the first 0.2 min,
increased to 44% B from 0.2 to 0.6 min, maintained
at 44% B for 0.5 min, and decreased to 37% B
within 0.05 min. The injection volume was 2 μl,
and the total run time was 1.5 min with a flow rate
of 0.6 ml/min. The retention time of EPZ-5676
was 0.85 min. The ionization was conducted in
the positive ion mode using the multiple reaction
monitoring (MRM) transition [M + H]+ m/z 563.5
parent ion to m/z 326.3 daughter ion, incorporating
Copyright © 2014 John Wiley & Sons, Ltd.
a turbo-ionspray interface. Eight to ten calibration
standards were prepared in blank plasma or urine
of the relevant species providing a typical standard
curve concentration range of 0.5–1000 ng/ml.
Calibration curves were performed in duplicate in
each analytical run together with low, mid and high
concentration QCs in duplicate. All standard and
QC measured concentrations fell within 85–115%
of the nominal concentration.
Pharmacokinetic parameters were calculated by
noncompartmental methods using WinNonlin
(version 5.3; Pharsight, St Louis, Missouri). Terminal t1/2 values were determined by regression of
at least three data-points in the later phase of the
time–concentration profile. The volume of distribution at steady state was calculated as below:
VDss¼
Dose · AUMC0 inf
ðAUC0 inf Þ2
Parameters are presented as mean ± SD where applicable. Parent excretion in urine was calculated as
the % dose excreted = (urine concentration * urine
volume)/dose, accounting for the sample pooling
across three animals. The renal clearance, CLr, was
calculated as the amount in urine to time t/AUC0-t.
In vitro stability assays in liver microsomes and
hepatocytes
Liver microsomes (final protein concentration
0.5 mg/ml), 0.1 M phosphate buffer at pH 7.4 and
EPZ-5676 (final concentration of 3 μM; final
dimethylsulfoxide (DMSO) concentration of
0.25%) were pre-incubated at 37 °C prior to the
addition of NADPH (final concentration of 1 mM)
to initiate the reaction. The final incubation
volume was 50 μl. Control incubations were
included for each species where 0.1 M phosphate
buffer pH 7.4 was added instead of NADPH
(minus NADPH). Positive control compounds
(diazepam and diphenhydramine for rodent,
verapamil and dextromethorphan for human,
testosterone in all species) were incubated in
parallel to confirm microsomal activity. The
intrinsic clearance values obtained were within
the range of historical data. EPZ-5676 and controls
were incubated for 0, 5, 15, 30 and 45 min. The control (minus NADPH) was incubated for 45 min
only. The reactions were stopped by transferring
Biopharm. Drug Dispos. (2014)
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A. BASAVAPATHRUNI ET AL.
25 μl of incubate to 50 μl methanol at the appropriate
time points. The incubation plates were centrifuged
at 1640 × g for 20 min at 4 °C to aid protein
precipitation.
Human, Beagle dog, Sprague-Dawley rat and
CD-1 mouse cryopreserved hepatocytes were
obtained from XenoTech and stored at 150 °C
until use. The hepatocytes were thawed and prepared according to the vendor’s instructions,
pooled into Krebs Henseleit buffer (KHB,
pH 7.4), and kept on ice prior to initiating the
experiment. The hepatocyte suspensions were
pre-incubated in a shaking water bath at 37 °C
for 3 min, and then the reaction was initiated by
the addition of EPZ-5676 into the hepatocyte
suspensions (1.5 × 106 cells/ml) at a final concentration of 3 μM, and a DMSO content of 0.1%.
The reaction mixture was incubated in a shaking
water bath at 37 °C. Aliquots of the incubation solutions were sampled at 0, 15, 30, 60 and 120 min.
The reaction was immediately terminated by the
addition of three volumes of ice-cold acetonitrile
containing 0.1% formic acid and internal
standards. After centrifugation at 1640 × g for
10 min, the supernatants were transferred into
HPLC vials, and the test compound was analysed
by LC-MS/MS. Testosterone (20 μM) and
7-hydroxycoumarin (100 μM), were performed in
parallel to confirm the enzyme activities of the
hepatocytes used.
The in vitro t1/2 values were determined by plotting the natural logarithm of the analyte/IS peak
area ratios versus time, with the slope of the linear
regression (k) converted to in vitro t1/2 values by
in vitro t1/2 = 0.693/k. Experimental half-lives
were transformed to the corresponding scaled intrinsic clearance values (in units of ml/min/kg)
as below:
CL int ¼
0:693
mL incubation
·
· MPPGL · LWPBW
in vitro t1=2 mg microsones
CL int ¼
0:693
mL incubation
·
· HPGL · LWPBW
in vitro t1=2
millioncells
where MPPGL is microsomal protein per gram
liver, HPGL is hepatocellularity (million cells per
gram liver) and LWPBW is grams liver per kg
body weight.
The scaled intrinsic clearance values were then
subsequently scaled to predicted in vivo clearance
values, using the well-stirred venous equilibration
Copyright © 2014 John Wiley & Sons, Ltd.
model as described previously [12–14] and as
shown below;
CLH ¼
QH CL int
QH þ CL int
CLH ¼
QH f up CL int
QH þ f up CL int
where QH is the blood flow, fup is the fraction
unbound in plasma and CLint is the scaled intrinsic
clearance. The appropriate species-specific scaling
factors including MPPGL, HPGL, LWPBW and
hepatic blood flows were used throughout [15–17].
Since the microsomal incubational binding of EPZ5676 was measured close to unity (Table 2), the two
versions of the well-stirred model that were applied
to the data are as shown above; (i) scaled CL with no
correction for binding parameters and (ii) scaled CL
with correction for fraction unbound in plasma
(fup) only. These data are presented in Table 2.
Plasma protein binding, blood partitioning and
plasma stability assays
Plasma protein binding was assessed by equilibrium dialysis, utilizing the HT-dialysis cell format
with a cellulose semi permeable membrane
(molecular weight cut-off of 5000 Da). Plasma was
warmed to 37 °C and adjusted to pH 7.4 before
use. Male Sprague-Dawley rat, male Beagle dog,
male CD-1 mouse and mixed sex human plasma
(Harlan Sera-Lab Ltd, Loughborough, UK) were
used for the studies. A 5 μM test compound solution
was prepared in isotonic phosphate buffer and rat,
dog, mouse and human plasma (final DMSO
concentration of 0.5%). The plasma-containing
solution was introduced to one side of the
membrane, and the plasma-free on the other.
Incubations were performed for 16 h in duplicate
in order to allow the compound to reach equilibrium. Mass balance and recovery were assessed
post-incubation and were > 85% in all cases.
Haloperidol was incubated in parallel as the control
compound for each species. At the end of the equilibration time the cells were emptied. Following
protein precipitation, the samples were centrifuged
and analysed by LC-MS/MS. The samples from the
protein-containing compartment were quantified
using calibration standards prepared in plasma
and the protein-free compartments were quantified
using calibration standards prepared in dialysis
buffer. Using a similar methodology, the incubational
binding of 3 μM EPZ-5676 to liver microsomes
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NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
(0.5 mg/ml) from mouse, rat, dog and human was
assessed, with amitriptyline as a positive control
compound (fuinc 0.35–0.4).
For blood partitioning, male Sprague Dawley
rat, male Beagle dog and male CD-1 mouse blood
was sourced from Harlan Sera-Lab Ltd, Loughborough, UK. Mixed sex human blood was obtained
from in-house healthy donors. The hematocrit
was measured using a Hettich Hematokrit 210
and calculated as the percentage of packed cell
volume compared with the total volume of whole
blood. EPZ-5676 (final test compound concentration 0.5 μM, final DMSO concentration 0.05%) was
incubated separately with fresh heparinized whole
blood, reference red blood cells and reference
plasma for 60 min at 37 °C in triplicate. Following
incubation, the whole blood cell samples were
centrifuged for 5 min at 5000 × g at 4 °C. The spiked
reference plasma was stored on ice during this
period. The spiked reference red blood cells were
freeze-thawed quickly three times to assist in
lysing the red blood cells. Following centrifugation
of the whole blood experimental sample, an aliquot
was sampled from the plasma and red blood cell
layers for analysis. As before, the red blood cell
layer was freeze-thawed quickly three times to lyse
the red blood cells. After protein precipitation and
centrifugation, the supernatants for the experimental samples and reference samples were
analysed by LC-MS/MS. Blood-to-plasma ratios
were calculated as described previously [18].
Chlorthalidone was used as a positive control
in this assay (rat B:P ratio of 73).
For plasma stability, EPZ-5676 (1 μM) was incubated with pooled lots of human, Beagle dog,
Sprague Dawley rat and CD-1 mouse plasma for
0, 15, 30, 60 and 120 min at 37 °C. Samples were
quenched in methanol and analysed by LC-MS/
MS analysis.
from the donor and receiver chambers at 120 min.
Each determination was performed in duplicate.
The co-dosed lucifer yellow flux was also measured
for each monolayer to ensure cell monolayers
remained intact during the incubation. The recovery of EPZ-5676 in donor and recipient wells postincubation was > 90% for all replicates. All samples
were assayed by LC-MS/MS.
Metabolite profiling and identification
EPZ-5676 was incubated with liver microsomes
of various species (mouse, rat, dog or human).
In vitro metabolite profiling and identification were
conducted after incubating EPZ-5676 (final concentration of 10 μM) with mouse, rat, dog or human
liver microsomes (final protein concentration of
0.5 mg/ml) at 37 °C in 100 mM potassium
phosphate buffer containing 2 mM Mg2+ in the
presence of 2 mM NADPH and 2 mM uridine
diphosphoglucuronic acid (UDPGA) (with the addition of 0.1 mg/ml alamethacin to human and rat
microsomes). For all liver microsomal incubations,
samples were taken at 0 and 20 min. All samples
were quenched by using the acetonitrile/methanol
solution and analysed using an LC-MS/MS Q-Trap
system (AB Sciex, Framingham, MA).
The major metabolites of EPZ-5676 in terms of
the mass spectrometry response were identified
by comparison of the LC-MS total ion chromatograms (TIC) of 0 min and 20 min samples in full
scan mode using LightSight™ 2.0 software. The
corresponding product ion tandem mass spectra
of EPZ-5676 and its metabolites were obtained
by using enhanced product ion (EPI) scans during
positive ion electrospray. The possible chemical
structures of the metabolites were deduced based
on their MS1 and MS2 spectra. In addition, the
hydroxylated t-butyl analog of EPZ-5676 was
synthesized to aid metabolite structure elucidation.
MDCK cell permeability assays
Confluent monolayers of Madin-Darby canine
kidney (MDCK) or MDCK-MDR1 (P-glycoprotein)
cells, 7–14 days old, in Transwell® dual-chamber
plates, with apical and basolateral compartments
buffered at pH 7.4, were dosed on the apical side
(A-to-B) or basolateral side (B-to-A) with EPZ5676 (10 μM) and incubated at 37 °C with 5% CO2
in a humidified incubator. Samples were taken
Copyright © 2014 John Wiley & Sons, Ltd.
Results
In vivo pharmacokinetics
The pharmacokinetics of EPZ-5676 was studied
following i.v. bolus administration to mouse, rat
and dog as well as following p.o. administration
to mouse and rat. The time–concentration data
are shown in Figure 1 and the parameters derived
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
A. BASAVAPATHRUNI ET AL.
from non-compartmental analysis are displayed
in Table 1. In mouse, rat and dog the plasma clearance was 77, 68 and 19 ml/min/kg, respectively,
which equates to an extraction ratio of 0.86, 0.97
and 0.61, respectively (based on the total CL being
entirely hepatic and using species-specific liver
blood flows of 90, 70 and 31 ml/min/kg,
respectively). Volumes of distribution at steady
state were determined to be 1.58, 1.66 and 2.44 l/
kg in mouse, rat and dog, respectively. In
physiological terms, this corresponds to about
2.2-, 2.4- and 3.5-fold greater than the total
body water (0.7 l/kg), respectively, indicating
partitioning into the peripheral tissue compartments. The kinetics following i.v. bolus administration in all three species showed bi-exponential
decline, as evidenced by a terminal elimination
half-life that was greater than the mean residence
time (Table 1). In mouse and rat, following p.o.
administration the exposure in terms of Cmax,
AUC and oral bioavailability was low. Following
i.v. administration in mouse, the parent excreted
in urine equated to a CLr of 4.4 ml/min/kg.
Plasma protein binding and blood:plasma
partitioning
The in vitro binding and partitioning data are
shown in Table 2. The free fraction in plasma for
EPZ-5676 did not show any marked species differences with values of 0.138, 0.272, 0.234 and 0.125
in mouse, rat, dog and human, respectively. The
blood-to-plasma partitioning data across species
did not suggest any significant binding of EPZ5676 to erythrocytes with values suggesting a
fairly equal distribution between plasma and
blood components. Based on these data, plasma
clearance, rather than blood clearance, was used
in all further data analysis.
Figure 1. The preclinical pharmacokinetics of EPZ-5676 determined in mouse, rat and dog. Data are shown graphically as
(A) concentration vs. time profile of mean ± SD (n = 3) plasma
concentrations following i.v. bolus (5 mg/kg) administration to
CD-1 mouse (formulated in 10% ethanol: 90% saline); (B)
concentration vs. time profile of mean ± SD (n = 3) plasma
concentrations following i.v. bolus (1 mg/kg formulated in 0.4%
HPBCD in saline) administration to SD rat; (C) concentration
vs. time profile of mean ± SD (n = 3) plasma concentrations
following i.v. bolus (1 mg/kg formulated in 10% ethanol: 90%
saline) administration to male Beagle dog
Copyright © 2014 John Wiley & Sons, Ltd.
In vitro metabolic stability
A summary of the metabolic stability data across
species is shown in Table 2. Representative plots
of the depletion of EPZ-5676 over time in liver microsome incubations are shown in Figure 2. EPZ5676 did not show any instability in mouse, rat,
dog and human plasma in vitro. Liver microsomal
incubations supplemented with NADPH showed
moderate turnover in mouse, rat, dog and human
which, when scaled by the well-stirred venous
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
Table 1. Pharmacokinetic parameters for EPZ-5676 in mouse, rat and dog after i.v. administration and in mouse and rat following
p.o. administration. Expressed as mean ± SD where applicable. n.d., not determined
CD-1 mouse
SD rat
Parameter
i.v. bolus
p.o.
i.v. bolus
p.o.
n
Dose (mg/kg)
Cmax (μM)
tmax (h)
AUC0-t (μM.h)
AUC0-inf (μM.h)
t1/2 (h)
MRT (h)
CL (ml/min/kg)
CLr (ml/min/kg)
VDss (l/kg)
F (%)
3/tpt
5
7.99 ± 1.90
3/tpt
20
0.0019
0.5
0.0014
n.d.
n.d.
n.d.
3
1
2.04 ± 0.23
3
10
0.0007a
0.25
n.d.
n.d.
n.d.
n.d.
1.95 ± 0.28
1.96 ± 0.30
1.14 ± 0.35
0.35 ± 0.06
76.7 ± 11.5
4.4
1.58 ± 0.23
0.43 ± 0.03
0.44 ± 0.03
3.73 ± 1.03
0.41 ± 0.10
67.8 ± 5.3
n.d.
1.66 ± 0.42
n.d.
Beagle dog
i.v. bolus
3
1
5.06 ± 0.60
1.55 ± 0.16
1.60 ± 0.15
13.6 ± 2.8
2.17 ± 0.89
18.7 ± 1.7
n.d.
2.44 ± 1.11
n.d.
a
Corresponds to LLOQ of bioanalytical assay.
Table 2. Liver microsome stability, hepatocyte stability, scaled hepatic CL and blood and plasma binding across species for EPZ-5676
Species
Mouse
Rat
Dog
Human
Scaled hepatic CL from liver microsomes with no binding correction (ml/min/kg)
Scaled hepatic CL from liver microsomes with fup correction (ml/min/kg)
Scaled hepatic CL from hepatocytes with no binding correction (ml/min/kg)
Fraction unbound in plasma (fup)
Fraction unbound in liver microsome incubation (fuinc)
Blood: plasma ratio
Half-life in plasma in vitro (min)
78
43
26
0.138
0.797
2.15
> 120
45
23
7
0.272
0.771
0.77
> 120
20
9
21
0.234
0.717
1.16
> 120
17
8
<3
0.125
0.772
0.65
> 120
equilibration liver model (with no correction for
binding), gave hepatic CL values of 78, 45, 20
and 17 ml/min/kg indicating moderate to high
hepatic extraction in mouse, rat, dog and human,
respectively. Incorporating the fraction unbound
in plasma into the microsomal scaling gave
hepatic CL values of 43, 23, 9 and 8 ml/min/kg
in mouse, rat, dog and human, respectively.
Incubational binding to liver microsomes across
species was shown to be low (fu > 0.7 in all cases)
and so was not considered a major contributing
factor in the in vitro–in vivo extrapolation (IVIVE)
for either liver microsomes or hepatocytes since
it is largely driven by non-specific membrane
partitioning and physicochemical properties. In
liver microsomal preparations supplemented with
UDPGA and alamethacin, no turnover was
observed indicating glucuronidation is not a
primary metabolic pathway for EPZ-5676 (data
not shown). In the hepatocyte suspensions, the
turnover of EPZ-5676 was very low giving rise to
Copyright © 2014 John Wiley & Sons, Ltd.
low CL estimates in all species tested, with the exception of dog where a hepatic CL value of 21 ml/min/
kg was observed.
Permeability in MDCK cell monolayers
The permeability of EPZ-5676 in mock and
MDR1-transfected MDCK cell monolayers is
shown in Table 3. EPZ-5676 showed low apicalto-basolateral permeability in both cell lines with
mean Papp values of less than 0.1 × 10-6 cm/s estimated over a 120 min incubation. The relative
efflux ratio between the transfected and native
cell lines suggests EPZ-5676 was not a substrate
for P-gp. However, both cell lines indicate an
efflux ratio of approximately 3, suggesting the
action of a native transporter protein in the
basolateral-to-apical efflux of EPZ-5676. Similar
observations were made in the transport assays
using the Caco-2 cell line (data not shown). This
is currently being investigated further.
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
A. BASAVAPATHRUNI ET AL.
Figure 2. Representative plots showing the depletion of EPZ-5676 in liver microsomes supplemented with NADPH. Parent depletion expressed as natural logarithm of the percent remaining as peak area ratio (parent/internal standard). (A) mouse, (B) rat, (C)
2
dog and (D) human. The r for all regressions was > 0.95
Table 3. Permeability of EPZ-5676 across MDCK cell monolayers over 2 h
Cell line
MDCK - mock
MDCK – MDR1 transfected
-6
Direction
Mean Papp (× 10 cm/s)
Apical-to-basolateral
Basolateral-to-apical
Apical-to-basolateral
Basolateral-to-apical
0.09
0.30
< 0.06
0.22
Structural elucidation of the major metabolites of
EPZ-5676 by LC-MS and LC-MS/MS
The metabolism of EPZ-5676 was studied in vitro in
liver microsomes supplemented with NADPH and
UDPGA, with several metabolites detected in
mouse, rat, dog and human. LC-MS and LC-MS/
MS were used for identification of EPZ-5676 and its
metabolites. A representative HPLC-MS chromatogram of the metabolite profile following a 20 min
incubation is shown in Figure 3. The molecular ions
and characteristic fragment ions are illustrated in
Figures 4–7. A summary of the metabolites
identified is presented in Table 4 and the proposed
metabolic pathway is shown in Figure 8.
EPZ-5676. The protonated molecular ion of EPZ5676 was m/z 563. The proposed fragmentation
Copyright © 2014 John Wiley & Sons, Ltd.
Efflux ratio
3.3
Relative efflux ratio
<2
> 3.8
pathway is shown in Figure 4. Loss of the adenine
ring gave m/z 428, with m/z 136 corresponding to
the protonated adenine ring itself. Loss of the
adenosine moiety gave m/z 326, due to the neutral
loss of both the ribose and adenine ring systems.
Cleavage of the N-cyclobutyl bond gave rise to m/
z 255 corresponding to the protonated t-butylbenzimidazole-cyclobutyl portion of EPZ-5676. Metabolites showed similar fragmentation pathways,
which allowed the elucidation and assignment of
metabolite structures.
M1. The protonated molecular ion of M1 was
m/z 579, indicating a mass shift of +16 Da and
a mono-hydroxylation of EPZ-5676. The
proposed fragmentation pathway is shown in
Figure 5. Comparison of the MS2 data of M1
Biopharm. Drug Dispos. (2014)
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NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
Figure 3. Representative HPLC-MS chromatograms for T0min and T20min incubations of EPZ-5676 in liver microsomes
supplemented with NADPH and UDPGA. This plot corresponds to a mouse liver microsome experiment
with that from the parent compound suggested
the mono-hydroxylation occurred on the t-buty
l-benzimidazole-cyclobutyl portion of the molecule. This was supported by the fragment ions
from M1 with m/z 444, 342, 330 and 271 all
retaining a +16 Da mass shift with corresponding ions from EPZ-5676 (m/z 428, 326, 314 and
255). The fragment ion m/z 136, corresponding
to the adenine ring, was present in MS2 spectra
for both parent and M1. The exact site of
hydroxylation was confirmed on the t-butyl
group with a synthesized authentic reference
standard, which showed an identical retention
time and MS fragmentation pattern to M1.
M3. The protonated molecular ion of M3 was
m/z 579, indicating a mass shift of +16 Da and
a mono-hydroxylation of EPZ-5676. The MS2
data for M3 gave fragment ions of m/z 444,
342 and 271, and was differentiated structurally
from M1 based on chromatographic separation.
M4. The protonated molecular ion of M4 was
m/z 521, indicating a mass shift of 42 Da
and dealkylation of the N-isopropyl group of
EPZ-5676. The proposed fragmentation pathway is shown in Figure 6. Comparing the MS2
data with that of parent revealed the fragment
ions m/z 255 and 136 were identical in both
Copyright © 2014 John Wiley & Sons, Ltd.
species, while the product ions m/z 386, 368,
350 and 284 supported N-dealkylation of the
isopropyl group.
M5. The protonated molecular ion of M5 was
m/z 579, indicating a mass shift of +16 Da and
a mono-hydroxylation of EPZ-5676. The MS2
data for M5 gave fragment ions of m/z 444,
342 and 271, and as such could not be differentiated structurally from M3.
M6. The protonated molecular ion of M6 was
m/z 579, indicating a mass shift of +16 Da and
based on retention time was tentatively assigned
as an N-oxidation product of EPZ-5676. The
proposed fragmentation pathway is shown in
Figure 7. The MS2 product ions of m/z 428, 326
and 255 were characteristic of the benzimidazole,
cyclobutyl and ribose moieties of the parent molecule suggesting oxidation of the adenine ring. The
rationale for the N-oxide assignment is further described in Discussion.
Discussion
EPZ-5676 is a novel DOT1L inhibitor and the first
member of the novel HMTi class to enter clinical
development as a potential therapeutic agent in
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
A. BASAVAPATHRUNI ET AL.
Figure 4. LC-MS chromatogram, MS1 and MS2 spectra (A) with proposed fragmentation pathways of EPZ-5676 (B)
MLL-rearranged leukemia. The discovery of
EPZ-5676 was facilitated by a structure-guided
medicinal chemistry approach [10] and has
shown superior efficacy in preclinical models
of MLL-rearranged leukemia [11]. The aims of
this work were to characterize the pharmacokinetics following i.v. and p.o. administration in
mouse, rat and dog, to assess the cross-species
in vitro–in vivo correlation to gain insight into
the primary elimination pathways involved in the
clearance of EPZ-5676, and to determine the major
metabolic pathways.
The in vivo time–concentration profiles in mouse,
rat and dog following i.v. bolus administration
showed biexponential kinetics that was more
apparent as the body size of the species increased.
Copyright © 2014 John Wiley & Sons, Ltd.
This resulted in terminal half-lives increasing from
1.1 h in mouse, 3.7 h in rat and 13.6 h in dog. In
addition, terminal t1/2 was longer than mean
residence time (MRT) (3–9 fold) further supporting
multi-exponential kinetics in the animal species
[19]. As for many drugs that exhibit multiphasic
concentration vs time profiles, the MRT will be a
better indicator than t1/2 of the potential dosing
frequency needed and expected accumulation ratio
that will occur with repeated administration. The
CL in all species was moderate to high with
estimated hepatic extraction ratios of 0.80, 0.97
and 0.61 in mouse (accounting for the measured
renal component), rat and dog, respectively.
Expressing CL in its unbound or blood form did
not change the interpretation of the cross-species
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
Figure 5. MS2 spectra of M1 (m/z 579) and EPZ-5676 (m/z 563) (A) with proposed fragmentation (B)
differences, since there was reasonable agreement
across species in the plasma-free fraction and with
blood partitioning values around unity. The
volume of distribution at steady state was consistent across species with values 2–3 fold greater
than the total body water indicating partitioning
into peripheral tissues. The unbound volume of
distribution at steady state (VDss) was also fairly
consistent across species at 11.4, 6.1 and 10.4 l/kg
in mouse, rat and dog, respectively. EPZ-5676
showed negligible oral bioavailability in mouse
and rat, which is in line with the physicochemical
property space that is generally regarded as
necessary for favorable gastrointestinal absorption,
e.g. PSA < 120 Å2, MW < 500 Da [20]. EPZ-5676
has a calculated logP of 3.26, a PSA of 144 Å2 and
a molecular weight of 563 Da. The oral absorption
Copyright © 2014 John Wiley & Sons, Ltd.
is permeability-limited based on the low passive
permeation observed in MDCK cell monolayers.
Additionally the data suggest that low intrinsic
permeation is the key driver rather than an active
efflux process as there was no indication of EPZ5676 as a P-gp substrate. The oral exposure may
also be perturbed by moderate-to-high first pass
extraction in rodents. Based on these data, an i.v.
dosing paradigm was pursued as the clinical route
of administration.
The scaled clearance from liver microsomes showed
excellent agreement with in vivo clearances in the
preclinical species, supporting perfusion-limited CL
and hepatic oxidative metabolism as the primary elimination pathway. Additional in vitro metabolism
studies confirmed no evidence of glucuronidation in
all species tested and no instability in blood plasma.
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
A. BASAVAPATHRUNI ET AL.
Figure 6. MS2 spectra of M4 (m/z 521) and EPZ-5676 (m/z 563) (A) with proposed fragmentation (B)
Moreover, low renal clearance was observed in mouse.
The estimated passive renal filtration (expressed as
GFR*fu) in mouse was ca. 2 ml/min/kg for EPZ-5676
which is slightly lower than the observed CLr of
4.4 ml/min/kg. This suggests largely a passive
glomerular filtration mechanism with perhaps a
marginal contribution from active tubular secretion in
mouse kidney. Notwithstanding, renal elimination of
parent is a quantitatively minor contribution to the
overall elimination of EPZ-5676, representing ca. 7%
of mouse renal blood flow.
Scaled clearance from liver microsomes
supplemented with NADPH provided good
agreement with in vivo clearance across all three
preclinical species. Even in the case of incorporating unbound fraction in plasma into the wellCopyright © 2014 John Wiley & Sons, Ltd.
stirred venous equilibration model, the CL
estimates remained within 2–3 fold of the
measured clearance. This case study highlights
one of the current challenges and limitations in
IVIVE in terms of whether to incorporate the
plasma-free fraction when the level of protein
binding is low to moderate and introduces a fold
change in CL coincident with the current practical
limit in predictive accuracy for IVIVE of 2–3 fold.
It is a common observation that liver microsomes
have a tendency to overpredict CL especially
for compounds with low passive membrane
permeability, although that was not apparent for
EPZ-5676. However, low scaled hepatocyte CL
values were obtained for EPZ-5676 in mouse, rat
and human (CLint < 4 μl/min/million cells). Dog
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
Figure 7. MS2 spectra of M6 (m/z 579) and EPZ-5676 (m/z 563) (A) with proposed fragmentation (B)
Table 4. Summary of metabolites of EPZ-5676 generated in liver microsomes supplemented with NADPH and UDPGA in various species
Abundance based on UV spectra (%)*
Metabolite
Parent
M1
M3
M4
M5
M6
Mass
shift (Da)
m/z
Retention
time (min)
0
+16
+16
42
+16
+16
563.2
579.3
579.3
521.3
579.3
579.3
18.5
16.3
17.2
17.4
17.8
18.0
Mouse
Rat
Human
25.4
4.5
6.7
59.4
11.3
59
2.6
3.5
3.8
10.3
9.3
*Not possible accurately to quantify the metabolites in dog by UV due to low signal.
was a clear outlier in terms of scaled hepatocyte
clearance and this may relate to a hepatic uptake
process well represented in dog. This is supported
by the slightly higher VDss observed in dog which
would correspond to greater tissue permeation
Copyright © 2014 John Wiley & Sons, Ltd.
and uptake. Interestingly, with the exception of
dog, the scaled hepatocyte data gave rise to much
lower values between 3- and 10-fold lower than
the observed CL, suggesting that permeation or
hepatocyte uptake was rate limiting. This has
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
A. BASAVAPATHRUNI ET AL.
Figure 8. The proposed major metabolic pathways of EPZ-5676 in mouse, rat, dog and human liver microsomes (supplemented
with NADPH and UDPGA)
been demonstrated for other compounds showing
a similar disparity between the liver microsome
and hepatocyte clearance [21].
Liver microsomes were selected for metabolite
identification and profiling, in light of the superior
cross-species IVIVE and much lower turnover
observed in hepatocytes. In liver microsomes
supplemented with NADPH and UDPGA, several
oxidative metabolites were observed. Metabolite
M1 was confirmed to be the product of hydroxylation on the t-butyl group based on the identical
LC-MS/MS characteristics of a synthesized
authentic standard. Metabolite M1 was observed
in all species including human. Metabolites M3
and M5 were distinct mono-hydroxylations on
the benzimidazole portion of the molecule. Due
to the poor MS fragmentation of the cyclobutylbenzimidazole moiety, LC-MS/MS alone was not
sufficient to elucidate the exact position of these
two hydroxylations. Metabolite M3 was only observed in rat, whilst M5 was present in all
preclinical species as well as human. Metabolite
Copyright © 2014 John Wiley & Sons, Ltd.
M4, N-dealkylation and loss of the isopropyl
group, was observed in all species tested whilst
M6, the N-oxidation of the adenine ring, was only
observed in rat. The most compelling evidence for
the assignment of M6 is based on previous
work on the oxidative metabolism of adenine
analogs. Lam and colleagues have demonstrated
that 9-substituted adenine analogs including 9-benz
yl adenine predominantly form the 1-N-oxide in
rodent microsomes whilst adenine itself and smaller
analogs such as 9-methyl adenine do not [22]. In
addition, none of the adenine analogs tested
underwent N-hydroxylation at the 6-amino group
[22]. No glucuronides of EPZ-5676 or its hydroxylated metabolites were detected and no metabolites
unique to human were present in this in vitro
metabolism study. Other groups have recently
reported on the in vitro metabolic stability of similar
nucleoside-analog inhibitors of DOT1L [23,24]. The
replacement of the ribose moiety with carbocycles,
such as cyclopentane, and to a lesser extent
cyclopentene, was advocated based on in vitro
Biopharm. Drug Dispos. (2014)
DOI: 10.1002/bdd
NONCLINICAL PK AND METABOLISM OF EPZ-5676, A DOT1L INHIBITOR
stability in human plasma and liver microsomes.
Differences in human liver microsome turnover
were observed between these two carbocyclic
analogs with the implication that the 5-membered
ring system was a metabolic liability. Our data do
not support the ribose moiety as being a major
metabolic soft-spot but rather suggest that P450mediated metabolism elsewhere on the molecule is
the major metabolic pathway for the DOT1L
nucleoside analog chemotype.
Conclusion
EPZ-5676 showed biexponential kinetics following
i.v. administration, giving rise to a terminal t1/2 of
1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. Steady state VD was 2–3-fold greater than
total body water with a high clearance in rodent
and moderate clearance in dog. EPZ-5676 exhibited
a low oral bioavailability in rodent. In vitro scaling
of liver microsome clearance data showed good
agreement with the in vivo clearance across species
indicating P450-mediated metabolism as a primary
elimination pathway. Moreover, low renal clearance was observed in mouse mediated largely
by passive glomerular filtration. Hepatocyte clearance suggested permeation- or hepatic uptakelimitations. The metabolic pathways for EPZ-5676
across species included three monohydroxylated
metabolites (M1, M3 and M5), one N-dealkylated
product (M4) as well as an N-oxide (M6). EPZ-5676
is a first-in-class DOT1L inhibitor and is currently under
clinical investigation for MLL-rearranged leukemias.
Further work is underway at present to characterize
the metabolism and disposition of EPZ-5676.
Conflict of Interest
The authors have declared that there is no conflict
of interest. All authors are employees of, and/or
hold equity in, Epizyme, Inc.
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