Disposition of asciminib, a potent BCR-ABL1 tyrosine kinase inhibitor, in healthy male subjects

Article abstract of DOI:10.1080/00498254.2019.1594449

Asciminib is a potent, specific BCR-ABL1 inhibitor being developed for the treatment of patients with chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL). Here, we present the results of human oral absorption, distribution, metabolism, excretion (ADME) and in vitro studies that together provide an overall understanding of the metabolism, distribution and clearance of asciminib in humans. Asciminib was rapidly absorbed with a maximum plasma concentration at two hours post-dose. Total radioactivity and asciminib showed similar terminal half-lives in plasma. Oral asciminib absorption ranged between a minimum of 33%, and a maximum of 57% based on the metabolite profiles of late time-point feces collections. Asciminib was eliminated mainly through feces via unchanged asciminib excretion and metabolism. Direct glucuronidation and oxidation were major metabolic pathways in human that were catalyzed predominantly by UDP-glucuronosyltransferase (UGT)2B7 and cytochrome P450 (CYP)3A4, respectively. The relative contribution of the glucuronidation pathway to the total clearance of asciminib via metabolism is estimated to range ～28–58%, whereas the relative contribution of the oxidative pathway is estimated to range ～37–64%, based upon the maximum oral absorption in humans.

Full text of DOI:10.1080/00498254.2019.1594449

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: https://www.tandfonline.com/loi/ixen20
Disposition of asciminib, a potent BCR-ABL1
tyrosine kinase inhibitor, in healthy male subjects
Phi Tran, Imad Hanna, Fabian Kurt Eggimann, Joseph Schoepfer, Tapan Ray,
Bing Zhu, Lai Wang, Petra Priess, Xianbin Tian, Florence Hourcade-Potelleret
&
Heidi J. Einolf
To cite this article: Phi Tran, Imad Hanna, Fabian Kurt Eggimann, Joseph Schoepfer, Tapan Ray,
Bing Zhu, Lai Wang, Petra Priess, Xianbin Tian, Florence Hourcade-Potelleret & Heidi J. Einolf
(2019): Disposition of asciminib, a potent BCR-ABL1 tyrosine kinase inhibitor, in healthy male
subjects, Xenobiotica, DOI: 10.1080/00498254.2019.1594449
To link to this article: https://doi.org/10.1080/00498254.2019.1594449
Accepted author version posted online: 22
Apr 2019.
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Disposition of asciminib, a potent BCR-ABL1 tyrosine kinase inhibitor, in healthy
male subjects
1
1
2
2
1
Phi Tran , Imad Hanna , Fabian Kurt Eggimann , Joseph Schoepfer , Tapan Ray , Bing
1
1
2
1
2
Zhu , Lai Wang , Petra Priess , Xianbin Tian , Florence Hourcade-Potelleret , Heidi J.
1
Einolf
1
Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA.
2
Novartis Pharma AG, Basel, Switzerland
Corresponding Author: Phi Tran, Department of Pharmacokinetics and Sciences,
Novartis Institutes for Biomedical Research, One Health Plaza, East Hanover, New
Jersey, 07936, USA; phone: +1 862-778-3428; email: phi.tran@novartis.com

Abstract
1
. Asciminib is a potent, specific BCR-ABL1 inhibitor being developed for the
treatment of patients with chronic myelogenous leukemia (CML) and Philadelphia
chromosome positive acute lymphoblastic leukemia (Ph+ ALL).
2
. Here we present the results of human oral absorption, distribution, metabolism,
excretion (ADME) and in vitro studies that together provide an overall
understanding of the metabolism, distribution, and clearance of asciminib in
humans.
3
. Asciminib was rapidly absorbed with a maximum plasma concentration at 2 hours
post-dose. Total radioactivity and asciminib showed similar terminal half-lives in
plasma.
4
5
6
. Oral asciminib absorption ranged between a minimum of 33%, and a maximum of
57% based on the metabolite profiles of late time-point feces collections.
. Asciminib was eliminated mainly through feces via unchanged asciminib
excretion and metabolism.
. Direct glucuronidation and oxidation were major metabolic pathways in human
that were catalyzed predominantly by UDP-glucuronosyltransferase (UGT)2B7
and cytochrome P450 (CYP)3A4, respectively.
7
. The relative contribution of the glucuronidation pathway to the total clearance of
asciminib via metabolism is estimated to range ~28-58%, whereas the relative
contribution of the oxidative pathway is estimated to range ~37-64%, based upon
the maximum oral absorption in humans.

Key words
asciminib, CML, BCR-ABL1 inhibitor, tyrosine kinase inhibitor, AME, UGT,
glucuronide, cytochrome P450, CYP
Introduction
Chronic myeloid leukemia (CML) and the Philadelphia-positive (Ph+) subset of acute
lymphoblastic leukemia (ALL) are characterized by the presence of a reciprocal
translocation between chromosomes 9 and 22, leading to the generation of the BCR-
ABL1 fusion protein. When fused to BCR, the tyrosine kinase activity of ABL1 becomes
constitutively activated, leading to the aberrant activation of numerous downstream
signaling pathways that converge to promote the growth and survival of the cell (Faderl
et al., 1999).
Asciminib is a BCR-ABL1 inhibitor which differs from other currently available tyrosine
kinase inhibitors (TKIs) such as bosutinib (Bosulif®), dasatinib (Sprycel®), imatinib
(Gleevec®), nilotinib (Tasigna®), and ponatinib (Iclusig®) in that it is not an ATP-
competitive inhibitor of BCR-ABL1. Asciminib was designed to inhibit BCR-ABL1 by
targeting a distinct and separate allosteric site within the kinase domain (myristate-
binding pocket) from that in which ATP-competitive TKIs bind(Adrian et al., 2006,
Schoepfer et al., 2018). Asciminib mimics the myristate group to lock BCR-ABL1 in an
inactive conformation and thereby inhibit the kinase activity (Skora et al., 2013, Wylie et

al., 2014). By virtue of its potent BCR-ABL1 tyrosine kinase inhibitory activity,
asciminib has potential for the treatment CML and Ph+ALL patients. Furthermore,
asciminib when administered in combination with an ATP-site inhibitor, has the potential
to prevent the emergence of resistance due to point mutations being acquired in one of the
binding sites (Wylie et al., 2014).
Asciminib is undergoing evaluation in a first-in-human (FIH) phase I clinical study as
monotherapy and in combination with imatinib, nilotinib, or dasatinib for the treatment of
patients with refractory CML or Ph+ ALL (study CABL001X2101). The drug has been
shown to be well tolerated with the most common reported adverse events (AEs) (all
grades) as headache, fatigue and increased lipase. In the same study asciminib has
demonstrated anti-tumor activity at doses greater or equal to 10 mg twice daily (BID)
(Novartis data on file). Based on the available preclinical and clinical data, the overall
benefit-risk ratio for asciminib supports development of asciminib for patients with CML
and Ph+ ALL. The effects of asciminib are currently being assessed in a phase 3 pivotal
study as a single agent in patients with CML in chronic phase (CML-CP), previously
treated with 2 or more TKIs (study NCT03106779). In parallel, asciminib is being
evaluated in combination with imatinib in patients with CML-CP who have been
previously treated with imatinib and have not achieved deep molecular response (study
NCT03578367). We report here the results from a study that evaluated the absorption,
1
4
distribution, metabolism and excretion (ADME) of [ C]-asciminib in healthy male
subjects after a single oral dose of 80 mg. The primary objectives of the study were (1) to
1
4
determine the routes of excretion and mass balance of total radioactivity of [ C]-
asciminib in urine and feces. (2) To determine the pharmacokinetics of asciminib and

total radioactivity in blood/plasma. (3) To identify and quantify the metabolites of
asciminib in plasma, urine and feces. (4) To estimate the absorption of asciminib from the
urinary and fecal excretion profiles. (5) To elucidate key biotransformation pathways and
clearance mechanism of asciminib in humans. Additional in vitro experiments were
conducted to identify and characterize the enzymes [cytochrome P450 (P450s) and UDP
glucuronosyltransferases (UGTs)] involved in the metabolism of asciminib. These
studies were also followed up with in vitro studies to estimate the relative contributions
of glucuronidation and oxidative metabolism pathways to overall biotransformation of
asciminib. Furthermore, metabolism/transport studies examined the potential for
asciminib and its primary glucuronide metabolite to undergo active biliary transport in
the human hepatocyte co-culture model. The results from this human ADME study,
together with results from the in vitro studies, provide a better understanding of the
clearance mechanisms of asciminib and provide valuable information about any potential
drug-drug interactions that may need to be considered when co-administering with other
drugs.

Materials and Methods
14
Study drug: [ C]-Asciminib; specific activity 1 Ci/mg, radiochemical purity ≥98%)
was synthesized by the Isotope Laboratory of Novartis Pharmaceuticals Corporation
1
4
(
East Hanover, USA). The synthesis scheme for [ C]-asciminib is provided in
Supplemental information. Metabolite standards for M10, M27.5, M29.5, M30.5, M37,
2
M43.3, and [ H ]-asciminib, used as internal standard for the analysis of unchanged
5
asciminib, were synthesized by Novartis Pharmaceuticals Corporation. The preparation of
the asciminib and its metabolite standards is provided in Supplemental information, or
described in a patent submitted by Dodd et al (WO 2013171639). The chemical structures
of radiolabeled asciminib, the position of the radiolabel along with related compounds are
shown in Figure 1.
Human studies: The study protocol and the informed consent document were approved
by an independent institutional review board. The written informed consent was obtained
from the subjects before enrollment.
Four healthy male subjects from 19 to 29 years of age, with weights ranging from 72.8-
8
3.4 kg, participated in the study. Subjects were housed in the study center for at least 24
hours (h) before administration of study drug and 168 h (7 days) post-dose. After an
overnight fast of at least 10 h, the subjects were given a single oral dose of 80 mg of
14
[
C]-asciminib formulated as a suspension in water. To ensure the complete dosing, 80
14
mL of water was added to the bottle containing [ C]-asciminib to suspend all of the
powder in the bottle followed by three subsequent 50, 50, and 60 mL of water rinses (for
a total of 240 mL). The rinsed volumes were also administered to the same subject. After
the dose administration, the empty bottle was rinsed with organic solvent and the

1
4
remaining radioactivity was counted to verify the dose of [ C]-asciminib given to the
healthy subjects. The radioactive dose given per subject was 80 Ci based on dosimetry
assessments and approved by the radiation safety committee. After administration, the
volunteers continued to abstain from food for an additional 4 h.
For the quantitation of asciminib in plasma, blood was collected pre-dose and at 0.5, 1, 2,
3
, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post-dose by an indwelling cannula
inserted in a forearm vein. Venous blood was collected at each time point into a K2-
EDTA containing heparinized tubes. Plasma was separated from whole blood by
centrifugation, transferred to a screw-top polypropylene tube, and immediately frozen.
For the determination of radioactivity in blood and plasma and for metabolite profiling,
blood was collected for analysis at the same time points out to 168 h post-dose. Urine and
faecal samples were collected until the majority of radioactivity was recovered up to a
maximum period of 14 days post-dose. Subjects were discharged when either total
recovery wasꢀ≥ 90% or if the combined urinary and fecal excretion in the preceding 48-
hour period is ≤1%.
Radioactivity analysis of blood, plasma, urine, and feces samples: Analysis of total
radioactivity data in plasma, blood, feces, and urine was performed by Covance
Laboratories Inc., Madison, WI. All sample combustions were performed in a Model 307
Sample Oxidizer (Packard Instrument Company, Downers Grove, IL) and the resulting
14
[
C]-CO was trapped in a mixture of Perma Fluor and Carbo Sorb (PerkinElmer,
2
Downers Grove, IL). All samples were analyzed for radioactivity in Model 2900TR
liquid scintillation counters (Packard Instrument Company). Plasma and urine were
mixed with Ultima Gold XR (PerkinElmer, Downers Grove, IL) scintillation cocktail and

counted directly. Whole blood and fecal samples were combusted prior to liquid
scintillation counting.
Safety assessments
The safety assessments included the monitoring and recording of all adverse events
(AEs), serious adverse events (SAEs), laboratory tests (hematology, blood chemistry and
urinalysis), vital signs, ECG, cardiac monitoring (telemetry) and physical examination.
Analysis of unchanged asciminib
A previously validated liquid chromatography tandem mass-spectrometry (LC-MS/MS)
assay was slightly modified and used for the analysis of unchanged asciminib in plasma
(Menssen et al 2018). The method consists of protein precipitation, evaporation of the
resulting supernatant to dryness and analysis of the reconstituted sample residue by LC-
MS/MS in multiple reaction monitoring (MRM) mode using ESI as the ionization
technique. The method is suitable for the determination of asciminib in human plasma
(
K -EDTA) over the range of 1.00 (lower limit of quantitation, LLOQ) to 5000 ng/mL
2
using 25.0 µL of human plasma. Briefly, plasma protein precipitation was performed in a
6-well plate. A 25 µL aliquot of the internal standard (IS) working solution was added to
9
wells containing 25 µL of plasma. This was followed by vortex-mixing. A 200 µL aliquot
of acetonitrile was added to each well to precipitate the plasma protein. The 96-well was
then sealed, vortex-mixed, and centrifuged (1000 × g, 5 min, at room temperature). The
o
supernatant was evaporated to dryness under nitrogen at 45 C. The sample residues were
reconstituted in 400 µL of 0.1% formic acid in 20% acetonitrile. For analysis, aliquots
(20 μL) of the extracts were injected onto the high-performance liquid chromatography
(HPLC) system.

The HPLC system consisted of a Shimadzu model LC-300AD pumps and Shimadzu
model SIL-30ACMP autosampler. The column was a Mac-Mod ACE5 C8 (50 x 2.1 mm,
o
5
µm) maintained at 35 C. A gradient elution was used with flow rate set at 0.4 mL/min.
The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic
acid in acetonitrile (solvent B). The mobile phase was initially composed of solvent
A/solvent B (80:20) and held for 0.5 min; the gradient was then linearly programmed to
solvent A/solvent B (20:80) over 2 min and held for 0.5 min; back to original condition
over 0.1 min; the column was equilibrated for 0.9 min; total run time was 4 min. The
HPLC system was interfaced to a Sciex QTrap 6500 mass spectrometer (Applied
Biosystems/MDS Sciex, Concord, ON, Canada) that was operated in the positive ion
electrospray ionization mode. For multiple reaction monitoring, the transitions monitored
2
were m/z 450 → m/z 239 for asciminib, m/z 455 → m/z 244 for the IS ([ H ]
5
o
asciminib),). MS source temperature: 550 C; dwell time: 250 msec; collision energy:
4
7eV.
Pharmacokinetic analyses: The following pharmacokinetic parameters were determined
by non-compartmental analysis (Phoenix WinNonlin 6.3, Pharsight, Mountain View,
CA): area under the blood/plasma drug concentration-time curve between time 0 and time
last (AUC ); AUC until time infinity (AUC ); highest observed blood/plasma drug
last
0-∞
concentration (Cmax); time to highest observed drug concentration (tmax); apparent
terminal half-life (t ); apparent volume of distribution of unchanged asciminib (V /F)
1
/2
z
calculated as Dose/(AUC • λ ), where F is the oral bioavailability and λ is the terminal
z
z
rate constant; and the apparent oral clearance (CL/F) was calculated as Dose/AUC0-∞.

Sample preparation of plasma, urine, and feces for metabolite investigation. Semi-
quantitative determination of main and trace metabolites was performed by radio-HPLC
with off-line microplate solid scintillation counting and structural characterization by LC-
MS.
Plasma samples from each subject per time point were protein precipitated with 2
equivalent volumes of acetonitrile:ethanol: acetic acid (90:10:0.1 v/v) and removed by
centrifugation. Recoveries of radioactivity averaged 89%. The supernatant was
evaporated to near dryness under a stream of nitrogen using the Zymark turbo-vap LV
(Zymark Corp., Hopkinton, MA), and the residues were reconstituted in 110 μL of
methanol: water: formic acid (50:50:0.1, v/v) and 45 µL were injected onto the HPLC
system with off-line radioactivity detection. Urine was pooled from 0 to 48 h, accounting
for >90% of the dose recovered in urine and was proportional to the volume of urine
collected at each time point. An aliquot of the pooled urine was concentrated
approximately 8-fold by reducing the sample volume under a gentle stream of nitrogen
then combined with 50 L methanol: water: formic acid (50:50:0.1; v/v) followed by
centrifugation at 3500 rpm for 10 min. The aliquot was then injected onto the HPLC
system with off-line radioactivity detection. Feces homogenates were pooled (from 0 to
9
6 h or 0-168 h) on a weight basis to account for >90% of the dose recovered in feces.
Each pooled fecal samples was extracted three times with acetonitrile; by vortex-mixing
and centrifugation. The average recovery of sample radioactivity in the organic extracts
was 95%. Aliquots of the combined supernatant were evaporated to dryness under
nitrogen, reconstituted in methanol: water: formic acid (50:50:0.1, v/v), and aliquots

(45 µL) of the concentrated, reconstituted samples were injected onto the HPLC system
with off-line radioactivity detection.
HPLC instrumentation for metabolite pattern analysis. Asciminib and its metabolites
in plasma and excreta were analyzed by ultra-performance liquid chromatography
(UPLC) with off-line radioactivity detection using a Waters Acquity UPLC system
(
Waters, Milford, MA), equipped with a Phenomenex Gemini C column (3 µm, 4.6 ×
18
1
3
50 mm) and a guard column of the same phase, both maintained at a temperature of
°
0 C. Asciminib and its metabolites were resolved with gradient elution consisting of 4
mM ammonium acetate containing 0.15% formic acid (v/v) (solvent A) and acetonitrile
solvent B), at a flow rate of 1 mL/min. The gradient elution program was as follows (all
(
step were linear): 0-2 min, 3% solvent B; 2-10 min, 25% solvent B; 10-39 min, 25-34%
solvent B; 39-42 min, 34-44% solvent B; 42-45 min, 44-95% solvent B, hold at 95%
solvent B for 5 min. The mobile phase condition was returned to the starting solvent
mixture over 0.5 min. Then system was allowed to equilibrate for 10 min prior to the next
injection.
TM
The UPLC eluent was fractionated into a 96 deep-well Lumaplate (Packard Instrument
Company) microplates using a Collect Pal fraction collector (Leap Technologies,
Carrboro, NC) with a collection time of 7.5 sec per well. Samples were dried under a
®
stream of nitrogen, sealed, and counted for 15–40 min per well on a TopCount
microplate scintillation counter (Packard Instrument Company).
The amounts of unchanged asciminib and metabolites in plasma or excreta were derived
from the radio-chromatograms (metabolite patterns) by dividing the radioactivity in
original sample in proportion to the relative peak areas. Unchanged asciminib or

metabolites were expressed as concentrations (ngEq/mL (ngEquivalent/mL)) in plasma or
as percentage of dose in excreta. These values are to be considered as semi-quantitative
only, as opposed to that determined for the unchanged asciminib using the validated
quantitative LC/MS/MS assay.
Structural characterization of metabolites by LC-MS/MS.
Metabolite characterization was conducted with a Waters two-channel Z-spray
TM
(
LockSpray ) time-of-flight mass spectrometer (Synapt MS, Manchester, UK). Leucine
enkephalin was used as the mass reference standard for exact mass measurement and was
delivered via the second spray channel at a flow rate of 10 µL/min. The eluent from the
HPLC column was split, and about 200 µL/min was introduced into the atmospheric
ionization source after diverting to waste during the first 4 min of each run to protect the
mass spectrometer from nonvolatile salts. The mass spectrometer was operated at a
resolution of ~10000 m/ΔmFWHM with spectra being collected from 100 to 1000 amu. The
ionization technique employed was positive electrospray. The sprayer voltage was kept at
at 3100 V and the sampling cone voltage was kept at a potential of 20 V. For the TOF
MS/MS experiments, trap energies of 25 to 45 eV and transfer energy of 15 eV were used
with argon as the collision gas.
In vitro experiments
Kinetic analysis of asciminib metabolism by human liver microsomes.
1
4
The kinetics of [ C]-asciminib metabolism (specific activity of 53.2 mCi/mmol and
radiochemical purity 99%) was evaluated using pooled human liver microsomes (HLM, n
=
150 donors, mixed gender; Corning, Gentest, Woburn, Massachusetts) in the presence
of NADPH or UDPGA. HLM (1.5 mg protein/mL) in 100 mM potassium phosphate

buffer (pH 7.4) was preincubated with alamethicin (60 μg alamethicin/mg protein, final
concentration) for 15 min on ice (for glucuronidation reactions only). MgCl (5 mM,
2
14
final concentration) was added as well as various concentrations of [ C]asciminib (0.5%
DMSO v/v, final concentration). The samples were thermally equilibrated for 3 min at
3
7°C and were initiated by the addition of the cofactor (1 mM ß-NADPH or 4 mM
UDPGA, final concentration). The duplicate samples were incubated for 30 min at 37°C.
The samples were quenched with half volume of cold acetonitrile and the precipitated
protein was removed by centrifugation at 39,000 × g for 10 min at ~4°C in an Avante 30
1
4
high speed microcentrifuge (Beckman Coulter, Fullerton, CA). [ C]-Asciminib and its
metabolite(s) were analyzed by HPLC with off-line radioactivity detection, as described
below. The percentage of impurity co-eluting with metabolites was accounted for in the
reactions containing co-factor by subtraction of the percent impurity from the control
samples (no cofactor). The percent of radiochemical impurity in these reactions was <
4
%.
Metabolism of asciminib by human hepatocytes.
Primary human hepatocyte suspensions (single female donor, Triangle Research Labs,
Lonza, Research Triangle Park, NC) were pelleted (100 × g for 10 min at 4°C) and re-
suspended in Krebs-Henseleit maintenance medium (KHB) supplemented with sodium
biocarbonate, fructose, and glycine (final concentrations of 25, 10, and 3 mM,
respectively). The cells were pelleted as described above and re-suspended in the above
medium supplemented with 2% (v/v) FBS (mKHB) to achieve a final concentration of ~2
6
-1
6
×
10 cells·mL . Five hundred microliters (~1 × 10 cells) of the final cell suspension
was added to each well of a 12-well plate pre-dispensed with 0.5 mL of mKHB

1
4
containing [ C]asciminib (2.5 μM nominal concentrations) and incubated at 37°C in a
humidified cell culture incubator (5% CO , 95% air) without shaking. At various time
2
points (0.25-24 h) the contents of individual wells were collected and the empty well
washed with an additional 1 mL of acetonitrile, and stored at -80°C until the time of
analysis. Positive metabolism control incubations using terfenadine and 7-
ethoxycoumarin confirmed the metabolic activity of the hepatocyte incubations (data not
shown). Acetonitrile extracts from hepatocyte samples were concentrated under a stream
of N to approximately one-third of their original volume. The samples were centrifuged
2
and the supernatant was analyzed by HPLC with off-line radioactivity detection as
described below.
Metabolism of asciminib by recombinant enzymes.
To identify the enzyme(s) involved in the metabolism of asciminib in humans, several
recombinant CYP, flavin monooxygenase (FMO) and UGT enzymes (Corning, Gentest)
were examined for asciminib metabolizing activity. Recombinant human (rh) UGT1A1,
UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4,
UGT2B7, UGT2B10, UGT2B15, UGT2B17, or UGT control microsomes (1 mg
protein/mL, final concentration) in 100 mM potassium phosphate buffer (pH 7.4) were
preincubated with alamethicin (60 μg alamethicin/mg protein, final concentration) for 15
1
4
min on ice. MgCl (5 mM, final concentration) and [ C]-asciminib (43 μM, final
2
concentration) were then added and the samples were thermally equilibrated at 37ºC for 3
min in an Eppendorf Thermomixer prior to initiation with UDPGA (4 mM, final
concentration). The samples were incubated for 30 min at 37ºC then quenched by the
addition of a half volume of cold acetonitrile. The precipitated protein was removed by

centrifugation and an aliquot of each sample was analyzed by HPLC with off-line
radioactivity.
Recombinant human CYP enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4,
CYP3A5, CYP4A11, CYP4F2, CYP4F3A, CYP4F3B, CYP4F12 (100 pmol CYP/mL),
the recombinant human FMO enzymes FMO1, FMO3, or FMO5 (1 mg microsomal
14
protein/mL), or control microsomes were incubated with [ C]-asciminib and samples
prepared as described above without alamethecin pretreatment and the reaction was
initiated by the addition of β-NADPH (1 mM, final concentration) for the CYP reactions
1
4
or by [ C]-asciminib for the FMO reactions.
Kinetic analysis of asciminib metabolism by rhCYP enzymes
Kinetic parameters of asciminib metabolism by rhCYP enzymes, CYP2C8, CYP2D6,
CYP3A4, CYP2J2 and CYP4F12 were determined to estimate the relative contribution of
these enzymes to the total oxidative metabolism of asciminib in human liver.
Recombinant human CYP2C8 (200 pmol CYP/mL; 1.6 mg microsomal protein/mL),
CYP2D6 (200 pmol CYP/mL; 1.9 mg microsomal protein/mL), CYP3A4 (150 pmol
CYP/mL; 0.81 mg microsomal protein/mL), CYP2J2 (100 pmol CYP/mL; 1.4 mg
microsomal protein/mL), CYP4F12 (200 pmol CYP/mL; 0.50 mg microsomal
1
4
protein/mL) were thermally equilibrated with varying concentrations of [ C]-asciminib
(in duplicate) in 100 mM potassium phosphate buffer (pH 7.4) containing 5 mM MgCl2,
final concentrations, for 3 min at 37°C in an Eppendorf Thermomixer. The reactions were
initiated by the addition of 1 mM NADPH (final concentration) and were incubated for
3
0 min at 37°C. The reactions were quenched by the addition of a half volume of cold

acetonitrile and the precipitated protein was removed by centrifugation at 39,000 × g for
0 min at ~4°C in an Avante 30 high speed microcentrifuge (Beckman Coulter, Fullerton,
1
CA). An aliquot (50 μL) of the supernatant from each sample was analyzed by UPLC
with off-line radioactivity detection. The percentage of impurity co-eluting with
metabolites was accounted for in the reactions containing NADPH by subtraction of the
1
4
percent impurity from the control samples (no NADPH). [ C]-Asciminib metabolism
activity was plotted against substrate concentration and the kinetic parameters, K and
m
Vmax values, were determined by non-linear regression analysis using a hyperbolic
function v = Vmax·[S]/(K +[S]), or an equation for substrate inhibition (uncompetitive): v
m
=
Vmax/(1+K /[S]+[S]/K ) (for certain metabolite(s) formed by rhCYP2C8 or rhCYP3A4).
m i
The K values were corrected for microsomal protein binding of asciminib in the in vitro
m
incubation. The fraction of unbound asciminib in the microsomal incubations (fu ) was
mic
determined by ultracentrifugation (Supplemental Information and Supplemental Figure
1
).
Disposition following heptocyte co-culture incubations
1
4
The disposition of [ C]-asciminib (25 µM) following 30 h incubations with human
hepatocytes co-cultured with human fibroblasts (Novik et al, 2017) was investigated
using a modification of the supplier’s protocol (Hµrel Corporation, New Brunswick, NJ).
14
[
C]-Asciminib was incubated with hepatocyte co-cultures at 37ºC in 24-well plates in a
volume of ~400 µL in a tissue culture incubator without shaking. At the end of the
14
incubation period supernatants were collected to examine the extent of [ C]-asciminib
biotransformation. The attached cells were rapidly washed with HBSS buffer (2×500

µL), followed by a 5 min incubation with the same buffer (300 µL) and the supernatants
collected. Finally, the attached cells were treated (5 min) with a tight-junction disrupting
reagent (300 µL, Hµrel Corporation) to allow molecules trapped in hepatocyte
canalicular spaces to diffuse into the media. The remaining cells were treated with 70%
methanol (500 µL) and the resulting suspension was centrifuged. All samples were
analyzed by LC-MS/MS and asciminib and its glucuronide conjugate in each sample
1
4
quantified using a standard curve constructed using a [ C]-asciminib hepatocyte
incubation mixture containing both parent and the glucuronide metabolite.
HPLC with radiodetection for in vitro experiments
The chromatographic separation was performed on a Waters Acquity Ultra Performance
LC system (Waters, Milford, MA), equipped with a Phenomenex Gemini C18 column (3
µm, 4.6 x 150 mm) and a guard column of the same phase, both maintained at a
°
temperature of 30 C. Elution was performed with a gradient of 0.1% formic acid in 1mM
ammonium acetate, pH 2.8 (mobile phase A), and acetonitrile (mobile phase B) at a flow
rate of 1.0 mL/min. The gradient elution program was as follows (all step were linear): 0-
2
min, 2% solvent B; 2-10 min, 25% solvent B; 10-39 min, 25-34.4% solvent B; 39-42
min, 34.4-43.4% solvent B; 42-43 min, 43.4-95% solvent B, hold at 95% solvent B for 4
min. The mobile phase condition was returned to the starting solvent mixture over 0.1
min. Then system was allowed to equilibrate for 13 min prior to the next injection. Off-
line radioactivity measurement was performed with TopCount low level radioactivity
detection. The UPLC eluate was collected with a fraction collector at 7 sec per fraction
into Deep-well 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 (Packard Instrument
Company) at a counting time of 10 min per well.
Data analysis
1
4
To determine the metabolism activity of [ C]-asciminib in the HLM kinetic assay and
human hepatocyte incubations, the percent of radioactivity of each peak in the HPLC
chromatogram (parent and metabolites) were quantified (totaling 100%). The amount of
individual metabolites formed in the reaction was based upon the percentage of
14
radioactivity in the product peak with respect to the total amount of [ C]-asciminib in the
starting reaction. For HLM, the metabolism activity was calculated as the amount of
product formed per total amount of microsomal protein in the reaction per reaction time
(i.e. nmol metabolite formed/mg microsomal protein/h). Enzyme kinetic parameters,
Vmax and K , for the biotransformation by HLM and major metabolizing enzymes were
m
calculated using GraphPad PRISM (version 6.0, San Diego, CA). The parameters Vmax
and K were determined using the Michaelis-Menten or substrate inhibition model.
m
Intrinsic clearance (CL ) was calculated as V /K .
int
max
m
To quantify the contribution of the different metabolic pathways to asciminib clearance in
1
4
human hepatocytes, the clearance of [ C]-asciminib by formation of each metabolite or
pathway was determined. The intrinsic clearance by a specific metabolic pathway (e.g.
6
CYP- or UGT-mediated) in hepatocytes (CLint,hep, units of μL/h/10 cells) was calculated
based on the disappearance of parent compound to the formed metabolite(s) as a function
of time (measured in h) using the integrated Michaelis–Menten equation, as described by
Equation 1 (Obach et al 1997).

0
.693
푉
퐶퐿ꢀ푛푡,ℎ푒푝
=
×
Eq. 1
푡½
푁
Where t is the half-life that was derived from the disappearance of parent by the
½
formation of the specific metabolite(s) as a function of time (measured in h), (0.693/t ) is
½
the apparent first-order rate constant for disappearance of parent as a function of time, V
is the final incubation volume (1000 μL) and N is the number of cells (in millions) per
incubation. Only those data within the linear range on a plot of log concentration
remaining vs. time were included to determine the disappearance rate constants (in this
case was up to and including the 24-h time-point).

Results:
Dose administration: The doses administered to subjects ranged from 79.4 to 81.4 mg
(mean of 80.3 mg) (77.0 to 78.9 μCi, mean value of 77.9 μCi), and were close to the
target dose of 80 mg (80 μCi).
1
4
Safety and tolerability data: A single oral dose of 80 mg [ C]-asciminib was generally
safe and well tolerated by healthy male subjects enrolled in this study. Three subjects
(75%) reported at least one AE during the study. All the AEs reported were of grade 1
severity. Headache, photophobia, blurred vision, head discomfort and dizziness were the
reported AEs suspected by the investigator to be related to the study drug. There were no
severe adverse events (SAEs) or other clinical relevant AEs reported in the study. No
subjects discontinued from the study due to an AE. No subjects had clinically significant
changes in laboratory measurements, vital signs, and ECG test results in the study
Pharmacokinetics of asciminib and total radioactivity in healthy volunteers: The
mean plasma concentration-time profiles of unchanged asciminib and total radioactivity,
1
4
after a single oral administration of [ C]-asciminib after a dose of 80 mg are presented in
Figure 2. The pharmacokinetic parameters of unchanged asciminib and total radioactivity
are reported in Table 1.
14
Following a single oral administration of 80 mg of [ C]-asciminib, the geometric mean
of the peak concentration (Cmax) of unchanged asciminib in plasma was 1790 ng/mL and
Tmax was reached at a median time of 2 h (range: 2-3 h). The geometric mean terminal
T1/2 was 14.2 h. The geometric mean systemic exposure (AUC ) was 18400 ng.h/mL,
inf
with apparent oral clearance (CL/F) of 4.34 L/h and apparent volume of distribution

(
z
V /F) of 89.0 L. The inter-subject variability is low, with CV% of ~11% for both Cmax
and AUCinf.
1
4
Following a single oral dose of 80 mg of [ C]-asciminib, the geometric mean total
radioactivity peak concentration (Cmax) in blood was 950 ngEq/mL and Tmax was reached
at a median time of 2 h (range: 2-3 h). In plasma, the geometric mean Cmax was
1
720 ngEq/mL and Tmax was reached at a median time of 2.5 h (range: 2-3 h). The mean
blood: plasma AUC ratio for total radioactivity was 0.58, suggesting that the drug related
radioactivity has a preferential distribution to plasma. The geometric mean terminal half-
lives of radioactivity in blood and plasma were similar, 11.7 h and 11.6 h, respectively.
Total radioactivity and asciminib showed similar terminal half-lives in plasma (geometric
mean of 11.6 and 14.2 h, respectively), indicating an approximately parallel decrease of
parent compound and metabolites. This finding is consistent with asciminib being the
predominant drug-related component in circulation.
Excretion and mass balance in urine and feces: After a single oral dose of 80 mg of
14
[
C]-asciminib, the excretion of radioactivity from all four subjects in urine and feces is
presented in Table 2. Overall, good mass balance was achieved in all four subjects. The
mean recovery of radioactivity in urine and feces was 91.0% (± 2.25% dose) over the
2
16 h study, with recovery in individual subjects ranging from 88.5 to 93.9%. The
radioactivity was excreted predominantly in the feces, averaging 80.0% of the total dose.
Renal excretion was minor, accounting for 11.0% of the total dose.
Metabolite Profiling: The metabolite profile summarizing the pharmacokinetics of
asciminib and its metabolites in plasma and percentage of asciminib and its metabolites in

excreta are presented in Table 3 and Table 4, respectively. A representative HPLC radio-
chromatogram of circulating metabolites is shown in Figure 3A.
In plasma, in each of the subjects, parent asciminib was the predominant drug-related
component at all time points analyzed, ranging from 91.9 to 94.2% of the total
radioactivity AUC0-24h , with an average value of 92.7% (Table 3). Metabolites detected
involved glucuronidation and oxidation at the pyrrolidinol ring (ketone formation,
oxidative ring opening). Metabolite M30.5 (direct O-glucuronide of asciminib) accounted
for an average of 4.93% of total AUC. Minor metabolite M44 (oxidation of alcohol to
ketone) accounted for 1.88% of total AUC. Trace amount of M29.5 (alcohol formed from
oxidative opening of the pyrrolidinol ring; 0.39% of total AUC) was also detected.
Representative HPLC radio-chromatograms for metabolites in urine and feces
(cumulative profile) are shown in Figure 3B and Figure 3C, respectively. After the oral
administration of asciminib to humans, a total of 10 radiolabeled metabolites of asciminib
were characterized and quantified in the excreta. In urine, unchanged asciminib averaged
2
.5% of the dose (Table 4). The mean renal clearance (CLr) for asciminib was estimated
6
by dividing the mean amount of asciminib excreted in the urine (2.0 × 10 ng) by the
mean plasma AUC0-inf of asciminib (18500 ng*h/mL). The estimated mean CLr of
asciminib (0.108 L/h) was 1.4% of the glomerular filtration rate (7.5 L/h). The most
abundant metabolite detected in the urine was M30.5 (averaged 7.0% of dose). Other
metabolites included M8 (amide hydrolysis), M10 (O-dealkylation), M27.5 (N-
glucuronide of asciminib), M29.5, M37 (carboxylic acid metabolite derived from
oxidative opening of the pyrrolidinol ring), M39 (another carboxylic acid metabolite

derived from oxidative opening of the pyrrolidinol ring) and M43.3 (carboxylic acid
metabolite). These minor metabolites were present at ≤ 0.4% of the dose (Table 4).
In the cumulative feces (pooled from 0-96 h or 0-168 h, a representative chromatogram is
shown in Figure 3C), asciminib averaged 56.7% of the administered radioactive dose.
The fecal profiles showed M39 and M43.3 as the most prominent metabolites, accounting
for approximately 7.8% (M39) andapproximately 34.6% (M43.3) of the administered
dose. Minor metabolites detected included M10, M27.5, M29.5, M37, M44 and M45.
These minor metabolites were present at ≤2.2% of the dose. Metabolite M30.5 (O-
glucuronide of asciminib) although detected in urine as the dominant component, was
absent in the feces. Metabolite M30.5 (direct glucuronide), presumably, was converted
back to parent asciminib while residing in the luminal tract, possibly catalyzed by
glucuronidase within the gastro-intestinal tract (Aura AM et al, 2002). The absence of
M30.5 in the feces was consistent with an in vitro study investigating the stability of
metabolite M30.5 in human feces, which demonstrated near complete hydrolysis of
M30.5 resulting in the formation of asciminib (data not shown). Due to the unstable
nature of the glucuronide in the gastrointestinal tract, the extent to which asciminib was
eliminated via metabolism to the glucuronide vs. excretion intact could not be readily
assessed. Hence, the percentage of intact asciminib (56.7% of the dose) that is detected in
the feces is likely to be over-estimated. Furthermore, the extent to which M30.5
contributed to the disposition of asciminib could not be easily assessed in this in vivo
study.

The metabolite profiles were also obtained from the late time point feces (pooled from
6-144 or 144-168 h, a representative chromatogram is shown in Figure 3D). With regard
9
to metabolites, the radiochromatograms from the cumulative feces and late feces were
similar. However, asciminib was more prominent in the cumulative feces profiles,
representing 68-77% of the radiochromatographic area compared to 52-55% in the late-
time point feces profiles (data not shown). The late-time point profile is assumed to
represent the drug-related material that has been absorbed and subsequently excreted (no
unabsorbed, unchanged asciminib present). On the other hand, the cumulative profile
represents a combination of drug-related material eliminated in the feces from absorbed
and unabsorbed portions. These assumptions were used to estimate the maximum
absorption fraction of asciminib as detailed below.
Estimation of minimum and maximum absorption fraction of asciminib
The minimum absorption was estimated using the following equation:
Minimum absorption = dose recovered in urine + dose recovered in feces as metabolites.
The mean radioactive dose recovered in the urine was 11.0% (Table 2), the mean
radioactive dose recovered in the feces in the form of metabolites was 21.8% (sum of all
metabolites detected in feces, Table 4). Based on these values, the estimated minimum
level of oral absorption was 33%.
The maximum extent of oral absorption for asciminib in human was estimated using the
late feces profiles and was based on the following assumptions: (1) The cumulative feces
profile (Figure 3C) represents both absorbed and unabsorbed asciminib-related material.
The absorbed portion comprised both unstable glucuronide metabolite(s) that were

converted back to parent asciminib, and asciminib which was either excreted via bile and/
or direct intestinal secretion. (2) The late time point feces profile (Figure 3D) represents
asciminib-related material that has been absorbed and subsequently excreted (no
unabsorbed asciminib present). Once the contribution from unabsorbed drug is subtracted
out, the ratio of asciminib to metabolites is assumed to be constant over time. The
absorbed asciminib-related fraction was calculated using the following equation:
Fraction asciminib absorbed = ratio of asciminib/metabolites from late feces profiles ×
fraction of dose detected as metabolites in cumulative feces profiles.
For this study, a mean ratio of asciminib/metabolites of 1.1 was obtained from the late
feces profiles, and the mean fraction of dose detected as metabolites in cumulative feces
profiles was 21.8%. Thus, the estimated absorbed asciminib-related fraction was 24%. A
maximum absorption of approximately 57% was obtained by adding the 24% absorbed
asciminib -related obtained above to the minimum estimated absorption of 33%.
Metabolite characterization by mass spectrometry: Metabolite structures were
characterized by LC-MS/MS using a combination of full and product ion scanning
techniques and elemental composition by exact mass measurement. The structure of
major metabolites, where possible, was supported by comparisons of their
chromatographic retention times and mass spectral fragmentation patterns with those of
synthetic standards.
+
Under positive ESI, asciminib had a MH ion occurred at m/z 450 with characteristic
fragment ions observed at m/z 211, 239, 257, 330, 390, 404, 422 and 432 (Figure 4).
Cleavage at the amide bond with charge retention on the carbonyl site generated the

fragment at m/z 257, subsequent loss of CO and of H O gave fragment at m/z 211.
2
Neutral loss of H O from asciminib produced the major fragment at m/z 432, while loss
2
of the chlorodifluoromethanol side chain plus one molecule of H O produced the
2
fragment at m/z 330. Cleavage across the pyrrolidinol ring produced fragments at m/z
3
90, 404 and 422. High resolution mass spectral information for each of these major
fragment ions supported the proposed fragmentation. Analogous fragment ions were
observed in the mass spectra of the metabolites, which allowed the assignment of regions
of biotransformation (Table 5).
The proposed biotransformation pathways and partial or complete structures of the
metabolites are given in Figure 5.
Metabolite pathways of asciminib in human: The metabolism of asciminib involved
the following primary biotransformation pathways: oxidation at the pyrrolidinol ring,
direct glucuronidation, O-dealkylation, and amide hydrolysis (Figure 5). Oxidative
opening of the pyrrolidinol ring formed the alcohol metabolite M29.5 and the carboxylic
acid metabolites M37, M39 and M43.3. Together, these four metabolites accounted for
approximately 17% of the dose elimination in human excreta (Table 4). Oxidation of the
pyrrolidinol ring led to the formation of metabolites M44 and M45, together accounted
for a total of 3.0% of dose. Direct glucuronidation pathway formed metabolites M27.5
(N-glucuronide of asciminib) and M30.5 (O-glucuronide of asciminib). Metabolite M27.5
was detected in both urine and feces (2% of dose), while M30.5 was observed in the urine
but not the feces, and was the major component in the urine radiochromatogram (7% of
dose). The amide hydrolysis and O-dealkylation pathways led to the formation of M8 and
M10, respectively. Metabolite M8 accounted for 0.4% of the dose while metabolite M10

accounted for 0.8% of the dose. All metabolites detected in this human study were also
observed in the rats, and/or monkeys in vivo (Supplemental Figure 2).
In vitro metabolism experiments
Metabolite profile in HLM
14
[
C]-Asciminib was metabolized in HLM in the presence of NADPH to form the
oxidative metabolites M10, M28.5, M29.5, M32, M39, M43.3, and M44. Direct
glucuronide metabolites M27.5, M30.5, and M36 were detected in HLM incubations
14
that contained UDPGA. The metabolic pathways of [ C]-asciminib in HLM is provided
in Supplemental Figure 3. Metabolites M27.5 and M36 are both N-glucuronides of
asciminib, their synthesis were given in Supplemental information. Metabolites M28.5,
M32 and M36 were not detected (or below the detection limit) in human in vivo.
Enzyme kinetics of asciminib metabolism in HLM
1
4
Kinetic parameters (K , Vmax) of [ C]-asciminib metabolism in pooled HLM in the
m
14
presence of NADPH or UDPGA were determined by the analysis of [ C]-asciminib
concentration dependence on the rate of metabolite formation and non-linear regression
analysis of the steady-state data. The unbound K value (corrected for microsomal
m
1
4
protein binding, see Supplemental data) for total [ C]-asciminib oxidative metabolism
formation of M10, M28.5, M29.5, M32, M39, M43.3, and M44) was estimated to be
2.6 ± 0.81 μM and the Vmax value was 1.56 ± 0.055 nmol/h/mg protein. The unbound
(
1
intrinsic hepatic microsomal oxidative clearance (CLint,u was estimated to be 0.124
mL/h/mg protein (Table 6).
14
The unbound K value for total [ C]-asciminib direct glucuronide conjugate formation
m
(formation of M27.5, M30.5, and M36) was estimated to be 12.4 ± 1.4 μM and the Vmax

value was 2.84 ± 0.16 nmol/h/mg protein. The unbound intrinsic hepatic microsomal
conjugative clearance was estimated to be 0.229 mL/h/mg protein. Consequently, the
total unbound hepatic intrinsic clearance (CLint,u total), which represents the sum of the
oxidative and conjugative intrinsic clearances is estimated to be 0.353 mL/h/mg protein.
The oxidative and glucuronidation pathways constituted ~35% and ~65% of overall
clearance, respectively.
Contributions of oxidative vs. glucuronidation pathways in human hepatocytes
Direct glucuronidation was the major biotransformation pathway in human hepatocytes
(
consistent with the HLM CL assessment), leading to the formation of the major direct
int
glucuronides M27.5, M30.5, and M36. The other major metabolites included the
oxidation products M39 and M44. To better quantify the contribution of the different
metabolic pathways to asciminib clearance in human hepatocytes, the clearance of
asciminib by formation of each metabolite or pathway was determined. As shown in
Supplemental Table 4, formation of each metabolite (as a percent of the total
radioactivity of the integrated metabolite peaks with time) is listed with the proposed
metabolic reaction and enzyme involved in its formation. The intrinsic clearance by a
specific metabolic pathway (e.g. CYP-, UGT2B17-, UGT2B7-, or UGT1A3/UGT1A4-
mediated) in hepatocytes was calculated based on the disappearance of parent compound
to the formed metabolite(s) as a function of time as described in the Materials and
Methods section. The calculated intrinsic clearance of asciminib by formation of specific
metabolites in human hepatocytes can be found in Table 8. The percent contribution of
CYP-mediated oxidative clearance was estimated to be 35% and the glucuronidation
pathway was 60% (5% as uncharacterized P20). This result was in-line with the

contributions determined from HLM, vide supra. Assuming UGT2B17 contributed
insignificantly to the formation of M30.5 (as mentioned above) the contribution of
UGT2B17 (formation of M27.5), UGT2B7 (formation of M30.5), and UGT1A3/4
(formation of M36) to total glucuronidation clearance of asciminib in hepatocytes was
estimated to be 27.9, 47.9, and 24.2%, respectively (Table 8). This higher contribution of
UGT2B7 to the total glucuronidation clearance of asciminib was in-line with the
chemical inhibition results (Supplemental Table 1).
Biotransformation of asciminib by recombinant human enzymes
Microsomes prepared from baculovirus-infected insect cells expressing single human
CYP, UGT, and FMO enzymes were used to examine the ability of specific enzymes to
14
14
metabolize [ C]-asciminib. There was no metabolism of [ C]-asciminib by FMO1,
FMO3, or FMO5 observed in this study. The human CYP enzymes found capable of
asciminib oxidative metabolism were CYP1A1, CYP1B1, CYP2C8, CYP2C9, CYP2D6,
CYP2J2, CYP3A4, and CYP4F12 determined from incubations with 19 individual
recombinant human CYP enzymes. Due to the lack of endogenous expression of
CYP1A1 and CYP1B1 in liver and very low metabolism by recombinant CYP2C9,
further evaluation of these enzymes for asciminib clearance contribution was not
performed. Relative contributions of CYP2C8, CYP2D6, CYP2J2, CYP3A4, and
CYP4F12 to total asciminib oxidative clearance in HLM were assessed by individual
CYP enzyme kinetics using recombinant CYP enzymes and scaling of the clearance by
the CYP abundance in HLM. As shown in Table 7, the relative contributions of
individual CYP enzymes to asciminib hepatic oxidative clearance in HLM were 96% by
CYP3A4, 2.08% by CYP2J2, 1.36% by CYP2C8, and 0.574% by CYP2D6. Due to the