Pharmacokinetics, metabolism, and excretion of [14C]axitinib, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in humanss

Article abstract of DOI:10.1124/dmd.113.056531

The disposition of a single oral dose of 5 mg (100 mCi) of [14 ^C]axitinib was investigated in fasted healthy human subjects (N = 8). Axitinib was rapidly absorbed, with a median plasma Tmax of 2.2 hours and a geometric mean C_max and half-life of 29.2 ng/ml and 10.6 hours, respectively. The plasma total radioactivity-time profile was similar to that of axitinib but the AUC was greater, suggesting the presence of metabolites. The major metabolites in human plasma (0-12 hours), identified as axitinib N-glucuronide (M7) and axitinib sulfoxide (M12), were pharmacologically inactive, and with axitinib comprised 50.4%, 16.2%, and 22.5% of the radioactivity, respectively. In excreta, the majority of radioactivity was recovered in most subjects by 48 hours postdose. The median radioactivity excreted in urine, feces, and total recovery was 22.7%, 37.0%, and 59.7%, respectively. The recovery from feces was variable across subjects (range, 2.5%-60.2%). The metabolites identified in urine were M5 (carboxylic acid), M12 (sulfoxide), M7 (N-glucuronide), M9 (sulfoxide/N-oxide), and M8a (methylhydroxy glucuronide), accounting for 5.7%, 3.5%, 2.6%, 1.7%, and 1.3% of the dose, respectively. The drug-related products identified in feces were unchanged axitinib, M14/15 (mono-oxidation/sulfone), M12a (epoxide), and an unidentified metabolite, comprising 12%, 5.7%, 5.1%, and 5.0% of the dose, respectively. The proposed mechanism to form M5 involved a carbon-carbon bond cleavage via M12a, followed by rearrangement to a ketone intermediate and subsequent Baeyer-Villiger rearrangement, possibly through a peroxide intermediate. In summary, the study characterized axitinib metabolites in circulation and primary elimination pathways of the drug, which were mainly oxidative in nature.

Full text of DOI:10.1124/dmd.113.056531

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2014/03/07/dmd.113.056531.DC1.html
1521-009X/42/5/918–931$25.00
http://dx.doi.org/10.1124/dmd.113.056531
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 42:918–931, May 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Pharmacokinetics, Metabolism, and Excretion of [14C]Axitinib,
a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase
Inhibitor, in Humanss
Bill J. Smith, Yazdi Pithavala, Hai-Zhi Bu,1 Ping Kang,2 Brian Hee, Alan J. Deese,3 William F. Pool,
Karen J. Klamerus, Ellen Y. Wu, and Deepak K. Dalvie
Pharmacokinetics, Dynamics and Metabolism (B.J.S., H.-Z.B., P.K., W.F.P., E.Y.W., D.K.D.), Pfizer Oncology-Clinical
Pharmacology (Y.P., B.H., K.J.K.), and Pharmaceutical Sciences (A.J.D.), Pfizer Inc., Worldwide Research and Development,
La Jolla Laboratories, San Diego, CA
Received December 16, 2013; accepted March 7, 2014
ABSTRACT
The disposition of a single oral dose of 5 mg (100 mCi) of
[
subjects (range, 2.5%–60.2%). The metabolites identified in urine
14C]axitinib was investigated in fasted healthy human subjects were M5 (carboxylic acid), M12 (sulfoxide), M7 (N-glucuronide), M9
(N = 8). Axitinib was rapidly absorbed, with a median plasma Tmax of (sulfoxide/N-oxide), and M8a (methylhydroxy glucuronide), ac-
2.2 hours and a geometric mean Cmax and half-life of 29.2 ng/ml and counting for 5.7%, 3.5%, 2.6%, 1.7%, and 1.3% of the dose,
10.6 hours, respectively. The plasma total radioactivity-time profile respectively. The drug-related products identified in feces were
was similar to that of axitinib but the AUC was greater, suggesting unchanged axitinib, M14/15 (mono-oxidation/sulfone), M12a (epox-
the presence of metabolites. The major metabolites in human ide), and an unidentified metabolite, comprising 12%, 5.7%, 5.1%,
plasma (0–12 hours), identified as axitinib N-glucuronide (M7) and and 5.0% of the dose, respectively. The proposed mechanism to
axitinib sulfoxide (M12), were pharmacologically inactive, and with
axitinib comprised 50.4%, 16.2%, and 22.5% of the radioactivity,
respectively. In excreta, the majority of radioactivity was recovered
in most subjects by 48 hours postdose. The median radioactivity
excreted in urine, feces, and total recovery was 22.7%, 37.0%, and
form M5 involved a carbon-carbon bond cleavage via M12a,
followed by rearrangement to a ketone intermediate and sub-
sequent Baeyer-Villiger rearrangement, possibly through a peroxide
intermediate. In summary, the study characterized axitinib metab-
olites in circulation and primary elimination pathways of the drug,
59.7%, respectively. The recovery from feces was variable across which were mainly oxidative in nature.
Introduction
of rapid growth (Folkman et al., 1971). Solid tumors in many organ
systems have high expression of VEGFR compared with normal
tissues (Hanahan and Folkman, 1996).
Axitinib (AG-013736) (Fig. 1) is an oral medication that has been
approved in the United States and multiple countries as a treatment
of renal cancer. In the United States, axitinib is indicated for the
treatment of advanced renal cell carcinoma in patients who have failed
one prior systemic therapy (Inlyta, 2012). Axitinib is an inhibitor of
ATP binding to the kinase domain of vascular endothelial growth
factor receptors (VEGFRs) 1, 2, and 3 with subnanomolar affinity
(Bender et al., 2004; Hu-Lowe et al., 2008; McTigue et al., 2012). The
VEGFR signaling network is a regulator of angiogenesis, which is
necessary to supply tumors with oxygen and nutrients during phases
During phase 1 clinical trials the pharmacokinetics of axitinib were
evaluated over a dose range of 2–20 mg twice daily in cancer patients
(Rugo et al., 2005). Time to maximal plasma concentration (Tmax) and
half-life (t1/2) ranged from 1.7 to 6.0 hours and 1.7 to 4.8 hours,
respectively. Exposure increased linearly as assessed by Cmax and area
under the plasma concentration–time curve (AUC). In vitro studies
suggested that CYP3A was responsible for substrate consumption of
axitinib in human liver microsomes (Zientek et al., 2010). The role of
CYP3A in the metabolism of axitinib was supported by results from
clinical drug interaction studies where ketoconazole increased the
axitinib plasma AUC ratio relative to a control arm by 2-fold and
rifampin pretreatment decreased this ratio to 0.2 (Pithavala et al., 2010,
2012b).
This study was sponsored by Pfizer Inc.
1Current affiliation: 3D BioOptima Co., Ltd., Suzhou, China.
2Current affiliation: Vertex Pharmaceuticals Inc., San Diego, California.
3Current affiliation: Genentech, South San Francisco, California.
dx.doi.org/10.1124/dmd.113.056531.
Human radiolabeled mass balance studies are usually performed
during drug development to identify and quantify the exposure to
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: ADME, absorption, distribution, metabolism, and excretion; AUC, area under the plasma concentration–time curve; AUCinf, area
under the plasma concentration–time curve from time zero to infinity; b-RAM, radioactivity monitor; 1D, one-dimensional; DMSO, dimethylsulfoxide;
ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; hERG, human ether-a-go-go-related gene; HPLC, high-performance
liquid chromatography; HUVEC, human vascular endothelial cell; LC, liquid chromatography; LC-MS/MS, liquid chromatography–tandem mass
spectrometry; [M+H+], protonated molecular ion; MCPBA, meta-chloroperbenzoic acid; MS, mass spectrometry; ng-eq, nanogram equivalents;
nOe, nuclear Overhauser effect; t1/2, elimination half-life; Tmax, time to maximal plasma concentration; VEGF, vascular endothelial growth factor;
VEGFR, vascular endothelial growth factor receptor.
918

Axitinib Human Metabolism
919
[
14C]axitinib powder. The container was capped tightly and then shaken
vigorously by hand 10 to 12 times until the powder was suspended in the
cranberry juice. Subjects drank the resulting suspension directly from the
plastic container. The container was then rinsed four times using 20 ml of
additional cranberry juice each time. At each rinse, the container was shaken
vigorously. Subjects immediately drank the rinsed solutions directly from the
container. After drinking the final rinse solution, the subjects swallowed at least
200 ml of tepid water. The oral cavity of each subject was examined after
dosing to ensure that all study medication was swallowed. After each drug
administration, the empty dosing container was capped and retained for assay
of residual radioactivity and, if necessary, for residual drug. Radioactivity
determinations of the dose solution and residual remaining in the container
confirmed that all subjects received .98% of the expected radioactive dose.
Recovery calculations were based on the actual amount of radioactivity
administered. Blood samples (7 ml) were collected in K3-EDTA-containing
glass Vacutainer tubes prior to dosing and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8,
12, 36, 48, 72, 96, 120, 144, and 168 hours after dosing. Additional blood
volume (20 ml) was collected at 1, 4, 8, and 12 hours after dosing for
metabolite profiling and identification. Following collection of blood
samples, plasma was prepared by centrifugation for drug and metabolite
analyses and stored frozen. Urine was collected over 4-hour intervals through
12 hours, 12-hour intervals through 48 hours, and every 24 hours thereafter.
Feces were collected over 24-hour intervals. Per protocol, excreta samples were
collected and tested daily until ,1% of the administered dose was recovered, at
which point the subject could be discharged from the study. Samples were
stored frozen prior to analysis. Precautions were taken during sample col-
lection, processing, and analysis to protect all samples from exposure to visible
light.
Fig. 1. Structure of axitinib. Asterisk denotes position of [14C] incorporation.
metabolites circulating in plasma, elucidate metabolite structures, de-
termine the primary clearance mechanisms of the drug, understand the
rate and route of excretion of parent drug and its metabolites, and validate
the species used in safety studies. These studies generate important
knowledge regarding the disposition of the drug in humans. Furthermore,
the studies are typically conducted during drug development as part of
drug registration packages and are seldom repeated due to the cost,
complexity, and challenges of performing human studies with radioac-
tivity. During the conduct of human absorption, distribution, metabolism,
and excretion (ADME) studies, challenges may arise that could com-
promise the ability to achieve the study objectives defined above. The
human ADME study of axitinib, conducted during early clinical de-
velopment, is an example of a study that advanced our knowledge of the
disposition of the drug but had the limitation of low and variable recovery
of radioactivity. Herein we describe the knowledge gained during the
conduct of the radiolabeled mass balance and metabolite structure elu-
cidation of a single oral 5-mg dose of [14C]axitinib to healthy volunteers.
Measurement of Radioactivity
Plasma (0.25 ml) and urine (1 ml) aliquots were added to Ultima Gold
(Perkin Elmer, Waltham, MA) scintillation fluid to a final volume of 20 ml
for radioactivity determination by liquid scintillation counting, in triplicate
and duplicate, respectively. The concentration of the radioactivity in the
whole-blood and red blood cell samples containing potassium EDTA was
determined by oxidizing triplicate 0.25-ml aliquots of well mixed whole
blood as described below for fecal samples. To each fecal collection was
added an equal weight of water followed by homogenization. Three aliquots
of each fecal homogenate (0.25–0.47 g) were burned in a Packard 307A
oxidizer (Perkin Elmer), which captured the evolved CO2 from each aliquot
into Carbosorb (10 ml; Perkin Elmer). Permafluor (10 ml; Perkin Elmer) was
added by the oxidizer into each 20-ml scintillation vial. Whole blood (0.25
ml) was processed similarly using the sample oxidizer. All radioactivity
measurements were determined using a Tricarb 2100 counter for 20 minutes
or a period of time that yielded a counting precision of 95%. Only values .2
times the background were reported. The dpm were converted to nanogram
equivalents (ng-eq) per milliliter for each matrix using the known specific
activity. For urine and feces, the data were also converted to the percentage of
dose for each time interval.
Materials and Methods
Chemicals
Axitinib was synthesized by Pfizer Global Research and Development, La
Jolla Laboratories (San Diego, CA). [14C]Axitinib was prepared by Pfizer Global
Research and Development Radiosynthesis Group (Groton, CT) using the same
synthetic route as used for unlabeled axitinib (Borchardt et al., 2006). This route
yielded 125 mg of [14C]axitinib with a specific activity of 58.3 mCi/mmol,
and the position of the incorporation of 14C is shown in Fig. 1. The resulting
[
14C]axitinib was mixed with 811 mg of nonradiolabeled axitinib and subjected to
polymorph control (crystal form IV) (Campeta et al., 2010), yielding 800 mg of
14C]axitinib drug substance mixture with a specific activity of 7.1 mCi/mmol.
[
The purity of [14C]axitinib drug substance was 99% by high-performance liquid
chromatography (HPLC). Reference standards for M9 (axitinib sulfoxide/
N-oxide), M12 (axitinib sulfoxide), and M15 (axitinib sulfone) were prepared
biosynthetically using microorganisms and isolated by HPLC and the structures
confirmed by liquid chromatography–mass spectrometry (LC-MS) and NMR
(Supplemental Material). M7 (axitinib N-glucuronide) was isolated from urine
from this study and characterized by NMR.
Determination of Axitinib in Plasma and Urine
Axitinib was determined in human plasma and urine by liquid chromatography–
tandem mass spectrometry (LC-MS/MS) with plasma methods as described
previously (Rugo et al., 2005; Pithavala et al., 2012b). The analysis of axitinib
in urine was similar to that described for plasma with the exception of glass
tubes instead of the use of a 96-well plate for extraction. These methods
employed sample extraction by an ethyl acetate/hexanes liquid-liquid extraction
Study Design, Dosing, and Sample Collection
This was a single-dose, open-label study conducted at a single center in eight followed by evaporation, reconstitution, and final extract analyses by LC-MS/
MS. The methods were validated for the analysis of axitinib over a concen-
tration range of 0.100–25.0 ng/ml using a 200-ml extraction volume for plasma
samples and 1.00–500 ng/ml using a 100-ml extraction volume for urine sam-
healthy male nonsmoker subjects (six Caucasian, two African American) with
a median age of 34 years (range, 25–46 years) and median weight of 83 kg
(range, 79–96 kg) who met inclusion/exclusion criteria. The study was
conducted in accordance with the Declaration of Helsinki, approved by an ples. Accuracy, evaluated by percent bias of quality control samples, ranged
between 22.0% and 4.0% for plasma and 20.5% and 1.3% for urine across
consent. All subjects received a single 5-mg oral dose of axitinib containing concentrations and analytical runs. Precision, evaluated by CV percentage of
100 mCi [14C]axitinib supplied as a powder for reconstitution. On the day of
quality control samples, ranged from 3.8% to 7.9% for plasma and 1.9% to
dosing, 20 ml of cranberry juice was added to each plastic container containing 3.5% for urine across concentrations and analytical runs.
institutional review board, and all subjects provided voluntary informed

920
Smith et al.
Axitinib and metabolites were eluted using a linear gradient in mobile-phase
Calculation of Pharmacokinetic Parameters
composition summarized as follows: 100% solvent A for 10 minutes, a linear
gradient to 50% solvent A/50% solvent B at 60 minutes, a linear gradient to
100% solvent A at 61 minutes, and held at 100% solvent A to 70 minutes. The
HPLC effluent was split so that 20% of the flow was introduced into the mass
spectrometer via the ESI source while 80% was diverted to the b-RAM detector.
The analog outputs from the ARC/b-RAM and MS detectors were recorded in
real time by the ARC data system Version 2.4 (AIM Research Company). The
major operating parameters for the ion-trap ESI/MS method were as follows:
positive ionization mode, spray voltage (5.0 kV), capillary voltage (5.0 V), tube
lens offset (55 V), capillary temperature (200°C), sheath gas flow rate (80,
arbitrary), auxiliary gas flow rate (20, arbitrary); and negative ionization mode,
spray voltage (5.0 kV), capillary voltage (247.0 V), tube lens offset (260 V),
capillary temperature (200°C), sheath gas flow rate (80, arbitrary), auxiliary gas
flow rate (20, arbitrary). LC-MS spectra were acquired over a mass range of 200–
1400 m/z for all samples. Mass spectra were processed using either Xcalibur
Version 1.4 (ThermoFinnigan) or the ARC data system. Radiochromatographic
data were processed using the ARC data system only. Metabolite screening was
performed to manually search for molecular ions representing possible
metabolites. Ion-trap experiments were performed to generate multistage mass
spectra for the selected molecular ions representing possible metabolites of
axitinib. Helium at a constant pressure of 40 psi was used as the damping and
collision gas for all MSn experiments. The mobile phase, gradient, guard
cartridge, column, and source conditions used were identical to the ion-trap LC-
ESI/MS method. Precursor isolation window, activation amplitude, activation Q,
and activation time were set at 1.5 amu, 30%–50%, 0.25 millisecond, and 30
millisecond, respectively. The chemical structures of the axitinib metabolites were
proposed based on metabolite prediction and mass spectral interpretation. Definitive
structures (Wilmad-LabGlass, Vineland, NJ) were assigned for isolated metabolites
using NMR spectroscopy or by comparison with authentic synthetic standards.
Plasma pharmacokinetic parameters [AUC from time 0 to infinity (AUCinf),
Cmax, Tmax, and t1/2] were estimated by using WinNonlin software, Version 3.2
(Pharsight Corp., Mountain View, CA), and summarized with descriptive
statistics as appropriate using the Statistical Analysis Systems software,
Version 8.2 (SAS Institute, Cary, NC). All other calculations, including the
estimation of urinary, fecal, and total recovery, were also performed using the
Statistical Analysis Systems software. As outlined in the study protocol, no
formal statistical analyses were performed for this human mass balance study
conducted in eight healthy volunteers.
Sample Preparation for Plasma, Urine, and Fecal Extracts
for Metabolite Profiling
Plasma samples collected at 0, 1, 4, 8, and 12 hours postdosing from each
subject were pooled based on the algorithm described by Hamilton et al. (1981)
to generate a plasma pool (7.2 ml). Plasma proteins were precipitated by the
addition of four volumes of acetonitrile/methanol [1:1 (v/v)], vortexed, and
centrifuged. The supernatant was removed and retained. The pellet was re-
extracted as above, and the two supernatants were combined and transferred
into glass tubes for evaporation to dryness under N2 at 40°C. The residues were
reconstituted in 1 ml of 30:70 (v/v) methanol/20 mM ammonium acetate (pH 4),
and the solutions were transferred into autosampler vials for analysis. The
injection volume was 900 ml.
Urine samples collected over prespecified time intervals postdosing from
each subject were pooled on a percent weight basis to generate a urine pool
(2 ml). After centrifugation, each pooled urine sample was transferred into
autosampler vials for analysis. The injection volume was 900 ml.
The fecal homogenates selected for metabolite profiling varied by subject to
cover time intervals where the majority of the radioactivity was excreted. The
selected fecal homogenates from each subject were pooled on a percent weight
basis to generate a homogenate pool (2 ml). Each pooled sample was precipitated
by the addition of four volumes of acetonitrile, vortexed, and centrifuged. The
supernatant was removed and retained. The pellet was re-extracted as above, and
the supernatants were combined and transferred into glass tubes for evaporation to
dryness under N2 at 40°C. The residues were reconstituted in 1 ml of 30:70 (v/v)
methanol/20 mM ammonium acetate (pH 4), and the solutions were transferred
into autosampler vials for analysis. The injection volume was 800 ml.
To investigate causes of observed variable fecal recovery of radioactivity in
this study, the fecal homogenization procedure was also put to additional
scrutiny. In an effort to ascertain if an alternate homogenization procedure
might produce better recovery of radioactivity from the fecal samples, a series
of experiments were undertaken. Briefly, the following alternate procedures
were evaluated using blank fecal samples as well as actual fecal samples from
this study: 1) manual vigorous shaking of the fecal homogenate slurry for 1 to
2 minutes before sampling for radioactivity counting; 2) sonication of the fecal
homogenate slurry after manual vigorous shaking and before sampling for
radioactivity counting; 3) liquid extraction of the axitinib-related material from
the fecal homogenate slurry (using an acetonitrile-acidified water system that is
routinely used for solubilizing axitinib), involving three successive extractions
of the fecal homogenate, followed by the pooled supernatant and extraction
pellet being counted for radioactivity; and 4) evaluation of any radioactivity
adsorbed to the polyethylene bag used for the homogenization by oxidation of
two cut sections of the polyethylene bag that held the fecal homogenate.
Metabolite Biosynthesis and Isolation and NMR Spectral Characterization
Isolation of Carboxylic Acid Metabolite (M5) and N-Glucuronide of
Axitinib (M7). Metabolites M5 and M7 were isolated from human urine.
Briefly, pooled urine samples from each subject were combined to generate
a mixed urine pool and treated with acetonitrile. Following vortexing and
centrifugation of the mixture, the supernatant was separated, concentrated, and
further lyophilized. The residue was reconstituted in 1 ml of mobile phase
and injected onto a 900-ml sample loop and separated by HPLC using the same
analytical column and the gradient system used for profiling the metabolites.
The fractions containing the glucuronide conjugate were collected (detection of
the metabolite was done by UV signal) and evaporated under a steady stream of
1
N2. The structures of M5 and M7 were characterized by H NMR. Metabolite
M7 was also investigated using one-dimensional (1D) nuclear Overhauser
effect (nOe) spectroscopy. Approximately 3 mg of the isolated M7 was
dissolved into 120 ml of dimethylsulfoxide-d6 (DMSO-d6; Cambridge Isotope
Laboratories, Inc., Tewksbury, MA) and transferred to a 3-mm NMR tube
(Wilmad 335-PP; Wilmad-LabGlass). The sample of axitinib was prepared by
dissolving 51 mg into 0.5 ml of DMSO-d6 and transferring the material to a
5-mm NMR tube (Wilmad 535-PP; Wilmad-LabGlass). NMR measurements
were made on a Bruker Avance DRX spectrometer (Bruker Corporation,
1
Billerica, MA) operating at 500.13 MHz H frequency at 30°C (rt 0.01) using
a 5-mm TXI Zgradient CryoProbe (Bruker Corporation). The M7 1H NMR
spectrum was acquired with 4000 scans and 32,768 complex data points using
a spectral width of 10,000 Hz and an acquisition time of 1.64 seconds. An
Metabolic Profiling and Structure Elucidation
All metabolite profiling and structure elucidation were performed using HPLC additional relaxation delay of 1.0 second was added between pulses to allow for
coupled in-line with radioactivity monitor (b-RAM) and MS detection with T1 relaxation. NMR data for axitinib was acquired under the same conditions,
electrospray ionization (ESI) source in positive mode. The instrumental with the exception that only 16 scans were needed. For the M7 nOe data,
components were as follows: Agilent 1100 HPLC Pumps/Autosampler (Agilent 20,000 scans were acquired using a 1D nOe experiment and selectively
Technologies, Wilmington, DE), 4.0 Â 3.0 mm guard column C18 (Phenomenex, inverting the glucuronic acid anomeric proton resonance. Spectra were acquired
Torrance, CA), Cosmosil 5PYE column 150 Â 4.6 mm (Waters, Milford, MA),
and processed using Bruker’s XWTNNMR software, version 3.5-patch level 6.
LCQ-Deca XP Ion Trap MS (ThermoFinnigan, San Jose, CA), Model C ARC Chemical shifts were referenced to an internal standard of tetramethylsilane.
StopFlow System (AIM Research Company, Newark, DE), Model 3 b-RAM Preparation of Metabolites M9, M12, and M15. Authentic metabolite
RadioDetector (500-ml cell) (IN/US Systems, Tampa, FL), and StopFlow AD standards of M9, M12, and M15 were prepared either using microbes or
Scintillation Cocktail (AIM Research Company). The mobile phase consisted of chemically using meta-chloroperbenzoic acid (MCPBA) in support of definitive
20 mM ammonium acetate in water adjusted to pH 4.0 with acetic acid (solvent structure determination. For microbial synthesis, 40 fungi and 40 bacteria, the
A) and acetonitrile (solvent B). The flow rate was maintained at 1.0 ml/min. majority obtained from the American Type Culture Collection (Manassas, VA)

Axitinib Human Metabolism
921
and a few from in-house sources (Pfizer Inc., Groton, CT), were screened for
M12 formation. The bacteria and fungi were grown in 20 g glucose, 5 g soy flour,
5 g yeast extract, 5 g K2HPO4, 5 g NaCl, and 1 g MgSO4×7H2O, adjusted to
a final volume of 1 liter with water, adjusted to pH 7.2, and sterilized. All strains
were grown at 28°C and 250 rpm on a rotary shaker. After 2 and 3 days, 0.2 mg
of axitinib from a 10% ethanol solution was added. After 3 and 6 days, the
reactions were analyzed by LC-MS using a Cosmosil 5PYE column (150 Â 4.6
mm) with a mobile phase of acetonitrile/50 mM ammonium acetate (pH 4.5), at
a flow rate of 1.0 ml/min and UV detection at 254 nm. For positive-mode MS
detection, the mobile phase consisted of 20 mM ammonium acetate in water
adjusted to pH 4.0 with acetic acid and acetonitrile. Among all strains screened,
Cunninghamella echinulata and Streptomyces griseus showed the highest
oxygenation activity. M12 was produced in abundance of .80%, and this was
used to prepare a large batch of the metabolite.
derived pharmacokinetic parameters are presented in Table 1. Total
radioactivity and parent axitinib reached maximal concentration in
plasma at ;2–3 hours postdose. The mean axitinib plasma Cmax and
AUC was about 32% and 15% of total radioactivity, suggesting that
total circulating axitinib metabolites exceeded the parent drug. The
terminal elimination half-life after oral dosing was similar for axitinib
and total radioactivity, suggesting similar parent and total metabolite
elimination rates from the body. The total [14C]axitinib-derived
radioactivity whole-blood exposure, assessed by Cmax and AUC, was
about half of the plasma values, indicating that the combination of
parent and metabolite was preferentially distributed in plasma (data
not shown).
For preparative transformation, C. echinulata obtained from the American Type
Culture Collection was grown from an agar plate into a 100-ml preculture using the
screening medium and conditions. After 2 days, 10 ml of the preculture was
inoculated into 1 liter of culture on a 4-liter shake flask (Â 5 liters). The culture was
grown for 24 hours, and then axitinib was added (0.2 g/l substrate for each 1 liter of
culture). Oxidation was followed by reverse-phase HPLC, and the reaction was
stopped after 8 days. The mycelium was removed from the culture by filtration, and
the oxidation products were extracted three times with one volume of chloroform
used each time. After removal of CHCl3 in vacuo, crude product (;500 mg) was
obtained. The crude product was purified by silica gel flash chromatography, using
CH2C12/acetone/MeOH (10:1:1) as eluent to afford 30 mg of the sulfoxide M12.
For preparative transformation of axitinib to M12 by S. griseus, the microbe was
grown from an agar plate into a 100-ml preculture using the screening medium and
conditions. After 2 days, 10 ml of the preculture was inoculated into 1 liter of
culture in a 4-liter shake flask (Â 10 liters). The culture was grown for 24 hours, and
axitinib was added (0.1 g/l substrate for each 1 liter of culture). Oxidation was
followed by reverse-phase HPLC and the reaction stopped after 8 days. The
mycelium was removed from the culture by filtration, and the oxidation products
were extracted three times with one volume of chloroform each. After removal of
CHCl3 in vacuo, the crude product (1.5 g) was obtained. The crude product was
purified by silica gel flash chromatography using CH2Cl2/acetone/MeOH (10:1:1)
as eluent to afford 80 mg of sulfoxide and yielded 8–9 mg pure sulfoxide.
Preparative Transformation of Axitinib to M15 by MCPBA. MCPBA
was added to 200 mg of axitinib suspended in 10 ml of methylene chloride at
0°C. The reaction mixture was stirred at room temperature for 7 hours. After
removal of solvent, the crude products were purified by silica gel chromatography
using methylene chloride/acetone/methanol (10:1:1) as eluent to afford M15 (63
mg, 30%) and M9 (11 mg, 5%). The structures of M9, M12, and M15 were
elucidated using 1H and 13C NMR methods by dissolving the compounds into
DMSO-d6. The chromatographic profiles and 1H and 13C NMRs of M9, M12,
and M15 are presented in Supplemental Figs. S1–S7.
Evaluation of M7 and M12 for Inhibition of Kinases and hERG. Axitinib
metabolites M7 (glucuronide) and M12 (sulfoxide) were evaluated in cellular
assays for their ability to inhibit kinase activities according to the methods
described in Hu-Lowe et al. (2008). M12 was tested for inhibition of vascular
endothelial growth factor (VEGF)-stimulated human vascular endothelial cell
(HUVEC) survival, platelet-derived growth factor receptor-b [enzyme-linked
immunosorbent assay (ELISA)], basic fibroblast growth factor–stimulated
HUVEC survival, and KIT (ELISA), while M7 (glucuronide) was evaluated
for inhibition of VEGFR2 (ELISA) in transfected porcine aorta endothelial cells.
M7 and M12 were also evaluated for the inhibition of human ether-a-go-go-
related gene (hERG) to assess their risk to prolong cardiac QTc interval. M7 was
evaluated for inhibition of IKr using whole-cell voltage-clamp electrophysiology,
and M12 was evaluated for inhibition of ligand-binding fluorescence polarization
according to methods described by Deacon et al. (2007).
Excretion of Axitinib Radioactive Equivalents in Urine and Feces
The urine and feces were collected for 144 hours and up to 288 hours
postdose from eight healthy male subjects following an oral 5-mg dose
of radiolabeled axitinib. The excretion of total radioactivity in urine and
feces in each subject is shown in Fig. 2. The recovery of total
radioactivity was lower than expected, especially in subjects 1001 and
1003, and variable between subjects. The total recovery was 37.9% and
16.0% in subjects 1001 and 1003, respectively. In the remaining six
subjects, the overall recovery of radioactivity ranged from 51.3% to
77.9% of the administered dose. The median recovery (all subjects) was
59.7%. In all but one subject (1001) with low recovery, the excretion of
radioactivity in feces was greater than in urine. The median recovery
across all subjects in feces and urine was 37.0% and 22.7%, respectively.
Urinary recovery was consistent across the eight subjects; however, fecal
recovery was quite variable (2.5%–60%). An investigation into potential
causes of the poor recovery was conducted. This included confirmation
of the administered radioactivity in the dose and additional homogeni-
zation and sampling of fecal homogenates. In subjects 1002 and 1003,
a change in bowel habits was noted, with no fecal sample obtained for
several consecutive collection intervals (Fig. 3). Fecal samples were
homogenized and resampled for radioactivity determination in three
randomly selected subjects; however, no difference was noted from the
original counts (data not shown).
Axitinib Plasma Metabolites
Plasma samples collected at 1, 4, 8, and 12 hours were combined
according to the pooling method of Hamilton et al. (1981) to create
a 0–12-hour plasma pool for each subject. A representative radio-
chromatogram is presented in Fig. 4. These samples were profiled by
Results
Pharmacokinetics of Axitinib Radioactive Equivalents and Axitinib
in Plasma
The mean plasma concentration–time profile for total radioactivity
and axitinib parent drug following the administration of 5 mg of
[14C]axitinib to healthy human subjects is presented in Fig. 2, and the
Fig. 2. Plasma concentration of [14C]axitinib total radioactivity and axitinib with
time following a 5-mg oral dose to healthy subjects. Each point represents the mean
and S.D. (N = 8). The inset graph shows the same results from 0–12 hours.

922
Smith et al.
TABLE 1
Plasma pharmacokinetic parameters of axitinib and total radioactivity after a 5-mg oral dose of [14C]axitinib to
healthy subjects
Axitinib
AUCinf
Total Radioactivity
Tmax
Cmax
t1/2
Tmax
Cmax
AUCinf
t1/2
h
ng/ml
ng×h/ml
h
h
ng/ml
ng-eq×h/ml
h
Geometric mean
Upper 90% CI
Lower 90% CI
2.22
2.72
1.81
29.2
43.4
19.7
136
187
98.8
10.6
20.7
5.38
2.46
3.02
2.00
91.2
132
63.0
916
1247
643
12.9
17.6
9.41
CI, confidence interval.
HPLC, and the individual percentage of each axitinib-derived Dalvie et al., 2010), suggested that M5 was a carboxylic acid derivative
component is presented in Table 2. The mean percentages of axitinib (Table 3). The structure of the metabolite was further confirmed by 1H
1
and related metabolites present in circulation were as follows: M7, NMR after isolating the metabolite from human urine. The H NMR
50.4%; M12, 16.2%; and axitinib, 22.5%; with a coefficient of spectrum of M5 revealed a loss of the pyridine ring and the vinyl
1
variation of ,27% for each component.
protons observed in axitinib (Table 4; the H NMR spectra of axitinib
and M5 are shown in Supplemental Figs. S8 and S9, respectively). This
was replaced by the singlet due to methylene protons at 3.8 ppm,
sandwiched between the carboxy group and the indazole ring. The
metabolite was therefore proposed as 2-(6-(2-(methylcarbamoyl)phenyl-
thio)-1H-indazol-3-yl)acetic acid (Fig. 5).
Axitinib Urinary and Fecal Metabolites
Also shown in Table 2 are the percentages of the dose excreted in the
urine and feces for each metabolite, and representative radiochromato-
grams are shown in Fig. 4. The mean percent metabolite excreted in
urine was largest for M5 (5.7%), followed by M12 (3.5%), M7 (2.6%),
M9 (1.7%), and M8a (1.3%), while unchanged axitinib was not
detectable. In addition, a more sensitive LC-MS assay was used to
detect and quantify lower concentrations of unchanged axitinib in urine.
In five out of eight subjects, there was no detectable axitinib (,1 ng/ml)
in any urine sample. In the remaining three subjects, low concentrations
of axitinib were noted (#3.1 ng/ml) in urine samples collected from
0–12 hours. This independently confirmed the results of radiochemical
profiling that there was no appreciable renal clearance of unchanged
axitinib. In fecal extracts the primary component was unchanged
axitinib (12.0%), followed by roughly equal amounts of M14/15
(5.7%), M12a (5.1%), and an unidentified metabolite (5.0%).
Metabolite M7. Metabolite M7 showed a protonated molecular ion
at m/z 563 in the full scan and was consistent with addition of 176 amu
to axitinib and suggested that M7 was a glucuronide conjugate of
axitinib. The mass spectrum of m/z 563 gave fragment ions at m/z 387
(the molecular ion of axitinib) and 356 (Table 3). The ion at m/z 387
indicated a loss of 176 amu from m/z 563 and corresponded to the
molecular ion of axitinib. The fragment ion at m/z 356 suggested a loss
of the methylamine moiety from m/z 387 as observed in the mass
spectrum of axitinib as described above. The exact site of glucur-
onidation could not be discerned from the mass spectral details. The site
of glucuronidation was evaluated further by isolating the metabolite
from human urine and subjecting it to NMR analysis. 1H NMR of M7
showed all the assigned resonances of axitinib in addition to the
resonance signals from protons in the glucuronic acid moiety (Table 4;
1
Identification of Axitinib Metabolites
the H NMR is shown in Supplemental Fig. S10, A and B). The key
Axitinib gave a molecular ion at m/z 387 ([M+H+]) in a positive ion change in the H NMR spectrum of M7 was the shift of the resonance
mode. The mass spectrum of m/z 387 gave characteristic major fragment signal at 7.65 ppm for the proton at the 7-position of the indazole ring to
ions at m/z 356, 222, and 166 (Table 3). The fragment ion at m/z 356 7.95 ppm (Table 4) and suggested attachment of the glucuronic acid
was a result of the loss of the methylamine moiety from axitinib and moiety to the indazole nitrogen. The point of attachment of the
subsequent formation of acylium ion, whereas the fragment ions at m/z glucuronic acid moiety to the indazole nitrogen was confirmed with
222 and 166 were obtained from the cleavage of the sulfide linkage. The additional 1D nOe measurements. A selective nOe to the nearby proton
structures (confirmed or proposed) of metabolites of axitinib are shown at the 7-position was observed (Fig. 6) upon selective inversion of
in Fig. 5, and Table 3 depicts the molecular and major fragment ions of resonance of the anomeric proton of glucuronic acid.
1
all the detected metabolites.
Metabolite M8a. Metabolite M8a gave a signal at m/z 579
Metabolite M5. Metabolite M5 showed a protonated molecular ion suggesting an addition of 192 amu to the molecular ion of axitinib. A
at m/z 342 in the full scan. The mass spectrum of m/z 342 gave major mass spectrum of M8a at m/z 579 showed major fragment ions at m/z
fragment ions at m/z 311, 296, 265, and 166 (Table 3). The presence of 403, 385, 373, and 356 (Table 3). The molecular ion at m/z 403 (loss of
a radiolabel peak in the chromatogram suggested that the metabolite was 176 amu) in the mass spectrum suggested that the metabolite was
drug-related. The molecular ion at m/z 342 suggested a loss of 45 amu a glucuronide conjugate of an oxidative metabolite of axitinib. The
from m/z 387 ([M+H+] of axitinib). An even molecular ion of the modification was possibly on the N-methyl amide moiety since the mass
metabolite suggested a loss of a nitrogen atom from the molecule. The spectrum also showed a fragment ion at m/z 356, which was consistent
fragment ions at m/z 311 (Table 3) were consistent with a loss of with the loss of the methylamine group in axitinib. The fragment ion at
a methylamine moiety from m/z 342, while the fragment ion at m/z 166 m/z 385 and 373 suggested a loss of water and a hydroxymethyl moiety
suggested a cleavage of the sulfide moiety. Both the loss of the from m/z 403, respectively, further confirming that the methyl group on
methylamino group and the fragment ion of m/z 166 were observed in the methylamine moiety was the site of hydroxylation. Although the
the mass spectral fragmentation of axitinib. Other fragment ions at m/z exact site of glucuronidation could not be discerned from the fragment
296 and 265 showed a loss of 46 amu from m/z 342 and 311, ions, the possible site of glucuronidation was either the nitrogen of the
respectively (Table 3). The loss of 46 amu, which is characteristic of the indazole moiety as observed in M7 or the hydroxyl group of the hy-
presence of a carboxylic acid group in the molecule (Ling et al., 2006; droxymethyl amide moiety.

Axitinib Human Metabolism
923
Fig. 3. Cumulative urinary and fecal excretion
and total recovery of
[
14C]axitinib-associated
radioactivity in individual healthy subjects fol-
lowing a 5-mg oral dose. The asterisks represent
collection intervals where no fecal sample was
obtained.
Metabolite M9. Metabolite M9 showed a protonated molecular ion amu, which was consistent with the loss observed in the fragmentation
at m/z 419 in the full scan, which was 32 amu greater than axitinib. of axitinib, while the fragment ions at m/z 324 and 296 resulted from
This indicated that M9 was a dioxygenated axitinib metabolite. Its a loss of 64 amu from m/z 388 and a subsequent loss of 28 amu from
mass spectrum at m/z 419 gave major fragment ions at m/z 388, 324, m/z 324, similar to the losses observed in the spectrum of M15 (as
and 296 (Table 3). The fragment ion at m/z 388 indicated a loss of 31 described below). Although the loss of 64 amu generally indicates

924
Smith et al.
Fig. 4. A typical reverse-phase HPLC radiochromatogram
of plasma (0–12 hours), urine (0–36 hours), and fecal
extracts (varied time interval) following a 5-mg oral dose of
[
14C]axitinib (100 mCi).
extrusion of sulfur dioxide and therefore the presence of a sulfone, the prepared using MCPBA, suggested that the oxidation in M9 occurred on
different retention times of M9 to M15 suggested that M9 was a different the sulfur as well as the pyridine nitrogen as shown in Fig. 5.
dioxygenated metabolite of axitinib. The regiochemistry of oxygenation
Metabolite M12. Metabolite M12 showed a protonated molec-
could not be discerned from the mass spectral data. Comparison of ular ion at m/z 403, suggesting an addition of 16 amu to 387 ([M+H+]
retention time of M9 with N-oxide derivative of axitinib sulfoxide, of axitinib). An addition of 16 amu indicated that M12 was a
TABLE 2
Individual and mean percentages of radioactivity present in plasma, urine, and feces in healthy male subjects, listed by subject number, following
oral administration of 5 mg [14C]axitinib (100 mCi)
Percentage of Metabolite in Radiochromatogram or Dose for Each Individual Subject and as Mean and S.D.
Metabolite
1001
1002
1003
1004
1005
1006
1009
1013
Mean
S.D.
Plasma (0–12 h)
M7
M12
Axitinib
59.2
9.3
18.9
50.3
15.9
22.0
40.5
20.5
30.4
45.9
20.2
23.7
42.2
15.9
32.4
50.7
17.7
18.9
50.9
21.5
17.2
63.5
8.7
16.8
50.4
16.2
22.5
7.9
4.9
6.0
Urine (0–36 h)
M5
M7
M8a
M9
4.1
5.5
2.3
2.1
6.3
5.5
2.0
1.3
1.8
3.8
3.3
1.1
0.6
1.1
0.9
7.6
2.1
1.4
1.8
1.9
5.1
1.5
0.5
1.4
3.2
7.3
3.4
1.0
1.7
6.6
6.6
2.7
1.7
1.9
3.3
6.2
2.2
1.8
1.8
2.2
5.7
2.6
1.3
1.7
3.5
1.5
1.4
0.6
0.3
2.0
M12
Feces (varied time interval)
UNK
M12a
M14/15
Axitinib
0.7
0.4
3.1
6.2
6.2
10.0
6.5
0.2
0.4
0.2
1.5
8.6
10.4
5.2
6.0
4.8
7.0
6.8
4.0
8.3
7.8
4.8
11.7
12.2
3.3
5.6
3.5
5.0
5.1
5.7
3.2
3.7
3.5
10.0
5.7
36.5
10.5
13.8
12.0
10.6
UNK, unidentified metabolite.

Axitinib Human Metabolism
925
TABLE 3
Mass spectral fragmentation and structures of axitinib and proposed metabolites
Structure
[M+H+]
387
Fragment Ions
Axitinib
356
222
296
166
265
M5
342
563
311
166
M7
387
356
M8a
M9
579
419
403
388
385
324
373
296
356
M12
403
403
372
372
355
354
344
344
327
M12a
M14
403
372
355
344
M15
419
388
324
296

926
Smith et al.
Fig. 5. Proposed biotransformation scheme of [14C]axitinib in humans.
mono-oxygenated metabolite. The mass spectrum at m/z 403 gave that the site of oxidation was the alkene moiety in the molecule, and
fragment ions at m/z 372, 355, 344, and 327 (Table 3). The fragment the possibility of the metabolite being the epoxide of axitinib could not
ion at m/z 372 indicated a loss of 31 amu, which was consistent with be ruled out. The proposed mechanism leading to the loss of a water
the loss observed in the fragmentation of axitinib. The fragment ion at molecule from an epoxide is presented in Fig. 7.
m/z 355 resulted from a loss of 48 amu and was characteristic of a loss
The mass spectrum of M14 at m/z 403 gave major fragment ions at
of a sulfoxide moiety from the molecule (Bu et al., 2007; Shimizu m/z 372, 355, and 344 (Table 3). As in the case of M12a, the fragment
et al., 2009). The ions at m/z 344 and 327 indicated a loss of 28 amu ions at m/z 372 and 344 indicated that pyridine was the likely site of
from m/z 372 and 355, respectively, and suggested a loss of either modification. The fragment ion at m/z 355 indicated a loss of 17 amu
a carbonyl group from the acylium intermediate that was formed from from m/z 372, which was characteristic of an N-oxide metabolite,
cleavage of the N-methylamide or loss of nitrogen from the indazole especially the pyridine N-oxide. This suggested that M14 was most
ring. The metabolite was confirmed by comparing it with the synthetic likely a pyridine N-oxide metabolite of axitinib. However, since
standard, which was prepared using microbial reactions.
neither M12a nor M14 was confirmed by NMR or their respective
Metabolite M12a/M14. Metabolites M12a and M14 gave a pro- synthetic standards, these metabolites have been represented as
tonated molecular ion at m/z 403 in the full scan, which suggested that Markush structures in Fig. 5 and Table 3.
both the metabolites were formed via mono-oxygenation of axitinib.
Metabolite M15. Metabolite M15 showed a protonated molecular
The mass spectrum of M12a gave major fragment ions at m/z 372, ion at m/z 419, suggesting an addition of 32 amu to 387 ([M+H+] of
354, and 344 (Table 3). The presence of the ion at m/z 372 indicated axitinib). This indicated that M15 was a dioxygenated metabolite. Its
a loss of 31 amu from m/z 403, suggesting a loss of the methylamine mass spectrum at m/z 419 gave major fragment ions at m/z 388, 324,
moiety, and the ion at m/z 344 was 28 amu lower than m/z 372, and 327 (Table 3). The fragment ion at m/z 388 indicated a loss of 31
corresponding to loss of N2 from the indazole or a carbonyl moiety. amu, which was consistent with the loss observed in the fragmentation
These losses were similar to those observed in the fragmentation of axitinib. The fragment ion at m/z 324 resulted from a loss of 64 amu
pattern of axitinib. The fragment ion at m/z 354, on the other hand, from m/z 388. Published reports indicate that compounds with a sul-
indicated a loss of a water molecule from m/z 372, which suggested fone moiety such as the sulfonamide and/or sulfones show a charac-
that hydroxylation was at a site that was susceptible to dehydration teristic loss of 64 amu due to extrusion of sulfur dioxide from the
and eliminated pyridine or indazole or the phenyl group in the axitinib molecule (Wang et al., 2003; Bu et al., 2007). The fragment ion at m/z
molecule as probable sites of oxygenation. It was therefore speculated 296 indicated a loss of 28 amu from m/z 324 and suggested a loss of

Axitinib Human Metabolism
927
TABLE 4
1H NMR chemical shift assignments for axitinib, M5, and M7 after an NMR analysis following isolation from human urine
All NMR spectra are presented in Supplemental Figs. S8, S9, and S10, A and B for axitinib, M5, and M7, respectively.
Chemical Shift for
Axitinib
M5
Protons
M7
ppm
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2.8 (d)
8.4 (q)
2.8 (d)
8.4 (q)
7.4 (d)
7.25 (d)
7.30 (m)
6.9 (d)
7.6 (s)
7.00 (d)
7.8 (d)
3.8 (s)
N.D.
2.8 (d)
8.4 (q)
7.51 (d)
7.28 (m)
7.32 (m)
7.06 (d)
7.65 (s)
7.2 (d)
7.81 (d)
7.58 (d)
7.99 (d)
7.67 (d)
7.27 (m)
8.21 (m)
8.62 (d)
7.51 (d)
7.28 (m)
7.32 (m)
7.06 (d)
7.95 (s)
7.2 (d)
7.81 (d)
7.58 (d)
7.99 (d)
7.67 (d)
7.27 (m)
8.21 (m)
8.62 (d)
4.3–5.8
N.D.
N.D.
N.D.
N.D.
Glucuronic Acid
COOH
N.D.
d, doublet; q, quartet; m, multiplet; N.D., not detected; s, singlet.
either a carbonyl group from the acylium intermediate that was formed that M7 and M12 were far less potent against the target and some related
from cleavage of the N-methylamine or loss of nitrogen from the kinases, and hence were considered pharmacologically inactive me-
indazole ring similar to that observed in the spectrum of the sulfoxide tabolites. M7 and M12 had hERG-inhibitory potencies of .30 mM and
metabolite (M12). This suggested that M15 was a sulfone metabolite .79 mM in cellular voltage-clamp and binding assays, respectively.
of axitinib. The modification of the sulfide to the sulfone was further
confirmed by comparing the spectral properties and the retention time
Discussion
with the synthetic standard that was prepared using MCPBA.
The objectives of the study were to investigate the disposition of an
oral 5-mg dose of [14C]axitinib in healthy human subjects to identify
Assessment of Kinase Activity of M7 and M12
Because M7 and M12 were the primary circulating metabolites in and quantify the exposure to metabolites circulating in plasma,
human plasma, they were evaluated for biologic activity in relevant elucidate metabolite structures, determine the primary clearance
assays. The activity of axitinib in the assays used to evaluate the mechanisms of axitinib, understand the rates and routes of excretion
metabolites has been reported previously (Hu-Lowe et al., 2008). M7 of axitinib and its metabolites from the body, and validate the species
(axitinib N-glucuronide) was not suspected to be active because it lacks used in safety studies. The majority of these study objectives were
the indazole NH that forms a critical hydrogen bond in the ATP-binding met, although not without some limitations.
site of the VEGFR kinase domain (McTigue et al., 2012). The IC50 for
Metabolism was the primary route of axitinib clearance because
inhibition of VEGFR2 autophosphorylation in porcine aorta endothelial only 12% of the unchanged drug was observed in the feces and none
(PAE) cells by M7 was 1990 nM (N = 7), which was 8300-fold higher was detected in the urine. Essentially, five primary metabolites
than axitinib. M12 (axitinib sulfoxide) was evaluated for inhibition of detected in human excreta comprised the key pathways for axitinib
VEGF-stimulated HUVEC survival and a few other related kinase clearance: sulfoxide (M12), N-glucuronide (M7), hydroxymethyl
activities. Unlike axitinib, M12 showed minimal activity against VEGF- (M8a precursor), oxidation products (M12a/M14), and N-oxide
stimulated HUVEC survival, platelet-derived growth factor receptor-b, (M9). In human plasma, the only metabolites detected were axitinib
and KIT in cell assays, with IC50 values of greater than 100, 810, and sulfoxide and axitinib N-glucuronide, and together they comprised
310 nM, which were greater than 400-, 470-, and 290-fold higher than about 66.6% of the drug-related plasma AUC. Many of the me-
the IC50 values for axitinib. The IC50 for inhibition of basic fibroblast tabolites in excreta were not detected in plasma. This suggested that
growth factor–stimulated HUVEC survival by M12 was .1000 nM, an the urinary metabolites M5, M8a, and M9 were cleared by the kidney
assay where axitinib also showed weak activity. These data indicated much more rapidly than their formation clearance, preventing

928
Smith et al.
Fig. 6. Comparison plot of 1D nOe difference
spectrum and normal 1D 1H NMR spectrum of
M7.
accumulation to detectable concentrations in blood. Similarly, the metabolic profile of a drug in humans has gained more importance since
fecal metabolites M12a, M14/15, and the unknown metabolite are the publication of position papers on drug metabolites in safety testing as
likely rapidly transported into the bile and are not substrates for well as regulatory guidance documents on nonclinical safety studies for
hepatic apical transporters that could facilitate their transfer to blood. the conduct of human clinical trials and marketing authorization for
Assessment of circulating metabolites in humans contributes to an pharmaceuticals (Davis-Bruno and Atrakchi, 2006; European Medicines
overall understanding of their potential impact on the safety and efficacy Agency, 2009; Nedderman et al., 2011). [14C]Axitinib disposition has been
of a drug. From a safety point of view, gathering knowledge of the in vivo studied in the mouse and dog (Smith et al., manuscript in preparation),
Fig. 7. Proposed mechanism for the formation of fragment ion m/z 354 from an epoxide metabolite.

Axitinib Human Metabolism
929
and the sulfoxide (M12), the metabolite of primary concern for such [14C]axitinib equivalents in the feces was the major route in three
assessments, was detected in sufficient concentrations in the plasma of subjects, whereas in other subjects fecal excretion was lower and/or the
both species when compared with the results from the present study in excretion pattern was unusual. The overall recovery of [14C]axitinib was
humans. Further, neither M7 nor M12 was pharmacologically active low across subjects, and this result did not meet expectations, initiating an
for VEGFR or other kinases, nor were they found to exert activity in investigation as to the potential causes. We confirmed that all subjects
hERG assays. Hence, both metabolites did not warrant further eval- received the entire dose by counting the radioactivity before and after
uation and quantification in nonclinical studies or clinical trials.
administration. Further, the plasma axitinib Cmax and AUC presented in
The enzymology of axitinib metabolism has been investigated in Table 1 was similar to what has been observed in other studies involving
human liver microsomes and recombinant enzymes (Zientek et al., healthy human subjects receiving the same 5-mg single oral dose of the
2010). CYP3A4/5 were the major enzymes involved in the oxidative drug (Pithavala et al., 2010, 2012a,b). The recovery of radiolabeled
metabolism of axitinib. This was consistent with the results from axitinib equivalents in urine was consistent between subjects, and the
several studies that investigated the clinical pharmacokinetics of urinary excretion rate was similar and aligned with the plasma phar-
axitinib. When ketoconazole 400 mg was given as an interacting drug macokinetics of total radioactivity and parent drug. These results
with 5 mg axitinib as the object, a 2-fold increase in Cmax and AUC indicated that the recovery of radioactivity from feces was variable and
was observed (Pithavala et al., 2012b). Axitinib has a moderate warranted further investigation. Several different procedures were
intravenous clearance and an oral bioavailability of 58% (Inlyta, evaluated to determine if homogenization of the fecal samples might
2012). Thus, a 2-fold interaction with ketoconazole is suggestive that have been incomplete and thus contributed to the recovery variability.
CYP3A is the major clearance mechanism.
Fecal samples from three subjects were rehomogenized and sampled in
The formation of M7 was primarily catalyzed by UGT1A1 (Zientek triplicate; however, the results did not differ from the original sampling
et al., 2010). Given the polymorphic nature of UGT1A1, several genetic (data not shown). We did note that the fecal output was low in some
polymorphisms were investigated using a meta-analysis of data from 11 subjects. In particular, one subject with the lowest fecal excretion of
axitinib healthy volunteer clinical studies in 315 subjects to determine if radioactivity only had reported fecal collections on 3 out of 10 days
there was association of specific genotypes with the observed variability during the study (Fig. 3). Another two subjects also had an unusual
in axitinib pharmacokinetics (Brennan et al., 2012). However, none of profile of generated fecal samples. Total fecal output collected during the
the evaluated genotypes were found to significantly contribute to the first 5 days of the study was lower in the subjects with the lowest
observed between-subject variability in axitinib pharmacokinetics. This recovery (data not shown). Finally, the radiolabel recovery in mouse and
suggests that the conversion of axitinib to the N-glucuronide is a minor dog mass balance studies with [14C]axitinib was .90%, which suggested
clearance mechanism even though it is a major metabolite in plasma. that the radiolabel was likely incorporated in a metabolically stable
The factors that cause the apparent disconnect between metabolite position in the molecule (Smith et al., manuscript in preparation), and
abundance in plasma and clearance are known and have been well thus radiolabel instability was an unlikely contributor to recovery
described by others (Lutz et al., 2010; Loi et al., 2013). Finally, taken variability. The exact reason(s) for incomplete total recovery is unknown,
together, these and other data suggest that liver is the primary organ but it was determined that it was not due to dosing errors or suboptimal
involved in the clearance of axitinib from the body.
fecal homogenization techniques and may have to do with irregular and
The formation of a carboxylic acid metabolite (M5) from axitinib was incomplete/sparse bowel movements. Beumer et al. (2006) conducted an
unusual in that it involves loss of a pyridine ring via a cleavage of extensive review of human mass balance studies for anticancer drugs and
a carbon-carbon bond. While the mechanism for its formation is found a range of recovery for anticancer antibiotics (28%–70%), topo-
unknown, one possible pathway might involve oxidation of axitinib to isomerase inhibitors (49%–102%), and tubulin inhibitors (27%–85%).
an epoxide metabolite, which could undergo rearrangement to a putative Roffey et al. (2007) reviewed human mass balance studies from 27
ketone intermediate (Fig. 8). Although not observed in any matrices, internal (recovery range, 61%–105%) and 171 literature compounds
this intermediate could undergo a carbon-carbon cleavage via a mech- (recovery range, 43%–105%) from a broad range of therapeutic
anism analogous to the Baeyer-Villiger rearrangement via a peroxy indications. The recovery of axitinib in this study, while not optimal,
intermediate, which involves insertion of an oxygen atom into the was within the range of those reported for these anticancer and other
carbon-carbon bond next to a carbonyl group (aldehyde or ketone) to therapeutic drug classes.
form an ester. Formation of this intermediate could occur through
In summary, the disposition of 5 mg [14C]axitinib in humans was
a cytochrome P450–catalyzed Fe(III)-OOH-mediated attack on the investigated. While the overall recovery of radioactivity was low, the
carbonyl carbon of the putative ketone metabolite to form an enzyme- majority of the study objectives were met. The plasma concentration–
bound peracid intermediate (Ortiz de Montellano and DeVoss, 2005). time profile of total radioactivity was similar to the parent drug. Only
Some evidence suggests that mammalian flavin mono-oxygenases may two metabolites were present in circulation, axitinib N-glucuronide
also catalyze these rearrangements in the metabolism of xenobiotics (M7) and axitinib sulfoxide (M12), both pharmacologically inactive.
(Chen et al., 1995; Lai et al., 2011). Hydrolysis of the corresponding The axitinib human ADME results were consistent with hepatic
ester would result in the carboxylic acid metabolite M5. Radical metabolism being the main clearance mechanism, with excretion of
decomposition of the peroxyhemiacetal intermediate can also lead to parent drug and metabolites in the feces as the main route of excretion
M5. A similar Baeyer-Villiger mechanism has been proposed pre- and excretion of axitinib metabolites in the urine as the secondary
viously by Mayol et al. (1994) in the formation of the triazolodione route. The metabolite structures for the majority of the metabolites
metabolite of nefazadone and Dalvie et al. (1996) in the formation of were elucidated with a proposed mechanism for the formation of an
a cleaved product of trovafloxacin.
unusual carboxylic acid metabolite.
Another important objective of this study was to determine the rates
and routes of excretion of [14C]axitinib in humans. In most subjects the
majority of the radioactive dose was recovered in the first 48 hours. The
median overall recovery in all subjects was 59.7% (16.0%–77.9%), with
22.7% (13.5%–28.2%) in urine and 37.0% (2.5%–60.2%) in feces.
Acknowledgments
The authors thank Dr. David Hoelscher at PPD Development (Austin, TX) for
the conduct of the clinical mass balance study, Andy Marquardt from PPD
Development for radioactivity counting, and Adrienne Manning from Charles River
When assessing the rates and routes of excretion, the excretion of Laboratories (Worcester, MA) for the bioanalysis of axitinib in plasma and urine.

930
Smith et al.
Fig. 8. Proposed mechanism for formation of the carboxylic acid metabolite (M5).
Bu HZ, Zhao P, Kang P, Pool WF, Wu EY, and Shetty BV (2007) Evaluation of capravirine as
a CYP3A probe substrate: in vitro and in vivo metabolism of capravirine in rats and dogs. Drug
Metab Dispos 35:1593–1602.
Campeta AM, Chekal BP, Abramov YA, Meenan PA, Henson MJ, Shi B, Singer RA,
and Horspool KR (2010) Development of a targeted polymorph screening approach for
a complex polymorphic and highly solvating API. J Pharm Sci 99:3874–3886.
Chen GP, Poulsen LL, and Ziegler DM (1995) Oxidation of aldehydes catalyzed by pig liver
flavin-containing monooxygenase. Drug Metab Dispos 23:1390–1393.
Dr. Shanghui Hu from the Pfizer Pharmaceutical Sciences group contributed to the
structure elucidation of axitinib N-glucuronide. Dr. Dana Hu-Lowe assessed
pharmacologic activity of axitinib metabolites. Maxwell Parker and Dr. Jill Steidl-
Nichols evaluated M7 and M12 for hERG interaction. Finally, Dr. Dennis Smith is
acknowledged for helpful discussions on the recovery of the radiolabel.
Dalvie D, Zhang C, Chen W, Smolarek T, Obach RS, and Loi CM (2010) Cross-species com-
parison of the metabolism and excretion of zoniporide: contribution of aldehyde oxidase to
interspecies differences. Drug Metab Dispos 38:641–654.
Dalvie DK, Khosla NB, Navetta KA, and Brighty KE (1996) Metabolism and excretion of
trovafloxacin, a new quinolone antibiotic, in Sprague-Dawley rats and beagle dogs. Effect of
bile duct cannulation on excretion pathways. Drug Metab Dispos 24:1231–1240.
Davis-Bruno KL and Atrakchi A (2006) A regulatory perspective on issues and approaches in
characterizing human metabolites. Chem Res Toxicol 19:1561–1563.
Deacon M, Singleton D, Szalkai N, Pasieczny R, Peacock C, Price D, Boyd J, Boyd H, Steidl-
Nichols JV, and Williams C (2007) Early evaluation of compound QT prolongation effects:
a predictive 384-well fluorescence polarization binding assay for measuring hERG blockade.
J Pharmacol Toxicol Methods 55:238–247.
Authorship Contributions
Participated in research design: Bu, Klamerus, Pithavala, Pool, Wu.
Conducted experiments: Bu, Deese, Kang.
Contributed new reagents or analytic tools: Bu, Deese.
Performed data analysis: Bu, Dalvie, Deese, Hee, Kang, Klamerus,
Pithavala, Pool.
Wrote or contributed to the writing of the manuscript: Bu, Dalvie, Deese,
Kang, Pithavala, Pool, Smith, Wu.
European Medicines Agency (2009) ICH guideline M3(R2) on non-clinical safety studies for the
conduct of human clinical trials and marketing authorisation for pharmaceuticals. EMA/CPMP/
ICH/286/1995. European Medicines Agency, London, UK. http://www.ema.europa.eu/docs/
en_GB/document_library/Scientific_guideline/2009/09/WC500002720.pdf.
Folkman J, Merler E, Abernathy C, and Williams G (1971) Isolation of a tumor factor responsible
for angiogenesis. J Exp Med 133:275–288.
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Address correspondence to: Bill J. Smith, Pfizer Worldwide Research and
Development, 10646 Science Center Drive, San Diego, CA 92121. E-mail: bill.j.
smith@pfizer.com