metabolism, excretion and pharmacokinetics of [ 14 c]crizotinib...
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http://informahealthcare.com/xenISSN: 0049-8254 (print), 1366-5928 (electronic)
Xenobiotica, Early Online: 1–15! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.941964
RESEARCH ARTICLE
Metabolism, excretion and pharmacokinetics of [14C]crizotinib followingoral administration to healthy subjects
Theodore R. Johnson1, Weiwei Tan2, Lance Goulet1*, Evan B. Smith1*, Shinji Yamazaki1, Gregory S. Walker3,Melissa T. O’Gorman4, Gabriella Bedarida5, Helen Y. Zou6, James G. Christensen6*, Leslie N. Nguyen1*,Zhongzhou Shen1*, Deepak Dalvie1, Akintunde Bello7, and Bill J. Smith1
1Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., San Diego, CA, USA, 2Department of Clinical Pharmacology,
Pfizer Inc., San Diego, CA, USA, 3Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT, USA, 4Department of
Clinical Pharmacology, Pfizer Inc., Groton, CT, USA, 5Clinical Research Unit, Pfizer Inc., New Haven, CT, USA, 6Oncology Research, Pfizer Inc.,
San Diego, CA, USA, and 7Department of Clinical Pharmacology, Pfizer Inc., New York, NY, USA
Abstract
1. Crizotinib (XALKORI�), an oral inhibitor of anaplastic lymphoma kinase (ALK) andmesenchymal-epithelial transition factor kinase (c-Met), is currently approved for thetreatment of patients with non-small cell lung cancer that is ALK-positive.
2. The metabolism, excretion and pharmacokinetics of crizotinib were investigated followingadministration of a single oral dose of 250 mg/100 mCi [14C]crizotinib to six healthy malesubjects.
3. Mean recovery of [14C]crizotinib-related radioactivity in excreta samples was 85% of the dose(63% in feces and 22% in urine).
4. Crizotinib and its metabolite, crizotinib lactam, were the major components circulating inplasma, accounting for 33% and 10%, respectively, of the 0–96 h plasma radioactivity.Unchanged crizotinib was the major excreted component in feces (�53% of the dose).In urine, crizotinib and O-desalkyl crizotinib lactam accounted for �2% and 5% of the dose,respectively. Collectively, these data indicate that the primary clearance pathway forcrizotinib in humans is oxidative metabolism/hepatic elimination.
5. Based on plasma exposure in healthy subjects following a single dose of crizotinib andin vitro potency against ALK and c-Met, the crizotinib lactam diastereomers are notanticipated to contribute significantly to in vivo activity; however, additional assessment incancer patients is warranted.
Keywords
Crizotinib, excretion, human, metabolism,oncology, pharmacokinetics
History
Received 9 May 2014Revised 1 July 2014Accepted 2 July 2014Published online 18 July 2014
Introduction
Lung cancer is the most common and lethal cancer worldwide
(Janku et al., 2010), and the majority of lung cancers (85%)
are non-small cell lung cancer (NSCLC) (Tyczynski et al.,
2003). Recently, advances in the understanding and treatment
of NSCLC have been made based on the identification of
molecular alterations specific to tumor cells (Herbst et al.,
2008). Rearrangements of anaplastic lymphoma kinase
(ALK), a receptor tyrosine kinase (RTK), in NSCLC were
reported in 2007, primarily as fusions to echinoderm micro-
tubule-like protein 4 (EML4) (Soda et al., 2007). The potent
oncogenic activity of the EML4-ALK fusion kinase was
confirmed when expressed in the lungs of transgenic mice
(Soda et al., 2008). These fusion proteins are found in �3–7%
of NSCLC patients overall, frequently in younger patients and
in never or light smokers (Gandhi & Janne, 2012).
Collectively, these findings suggested that a pharmacotherapy
specifically targeting ALK may prove beneficial in treatment
of patients with advanced NSCLC.
Crizotinib (XALKORI�, PF-02341066) is a potent small-
molecule inhibitor of ALK and its oncogenic variants
(i.e. ALK fusion events and selected ALK mutations)
(Figure 1) (Cui et al., 2011). It is also a potent inhibitor of
the RTK mesenchymal-epithelial transition (c-Met), also
known as hepatocyte growth factor receptor, as well as
ROS1 (c-ros) and Recepteur d’Origine Nantais RTKs
(Cui et al., 2008; Zou et al., 2007). In mice bearing NSCLC
*Present addresses: Lance Goulet, PharmAkea Therapeutics, San Diego,CA, USA. Evan B. Smith, Neurocrine Pharmaceuticals, San Diego, CA,USA. James G. Christensen, Mirati Therapeutics, San Diego, CA, USA.Leslie N. Nguyen, Johnson & Johnson Pharmaceutical Research &Development, San Diego, CA, USA. Zhongzhou Shen, DartNeuroScience, San Diego, CA, USA.
Address for correspondence: Theodore R. Johnson, Ph.D.,Pharmacokinetics, Dynamics and Metabolism, La Jolla Laboratories,Pfizer Worldwide Research and Development, 10646 Science CenterDrive, San Diego, CA 92121, USA. Tel: +858-622-7988.Fax: +484-323-8332. E-mail: [email protected]
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tumor xenografts that expressed ALK fusion protein,
crizotinib demonstrated marked, dose-dependent antitumor
efficacy, which correlated to pharmacodynamic inhibition of
phosphorylation of ALK fusion proteins (including
EML4-ALK) in tumors in vivo (Yamazaki et al., 2012; Zou
et al., 2011). Crizotinib is approved globally for the treatment
of patients with locally advanced or metastatic NSCLC that is
ALK-positive. The recommended dose schedule of crizotinib
is 250 mg taken orally twice daily. Results from a phase III
trial demonstrated that crizotinib was superior to standard
chemotherapy (pemetrexed or docetaxel) in patients with
previously treated, advanced ALK-positive NSCLC (Shaw
et al., 2013). In 347 patients who had previously been treated
with platinum-based chemotherapy, crizotinib prolonged
progression-free survival to a median of 7.7 months compared
with 3.0 months in the chemotherapy group. Likewise,
objective response rate was significantly higher in those
treated with crizotinib (65% versus 20%).
The disposition of crizotinib has been evaluated in rats and
dogs to support safety assessment and the pharmacokinetics
(PK) of crizotinib have been characterized in healthy subjects
and cancer patients in a number of clinical studies. The major
metabolic pathways in rats and dogs were oxidation of the
piperidine ring, with direct sulfation and O-dealkylation
also observed in rat (Smith et al., 2011; Zhong et al., 2010).
In nonclinical mass balance studies, recovery of total
radioactivity was essentially complete (>89%), and fecal/
biliary excretion was the major route of elimination.
Crizotinib is primarily metabolized by CYP3A and is also a
time-dependent inhibitor and inducer of CYP3A in vitro
(Johnson et al., 2011; Mao et al., 2013). In vivo, crizotinib is a
moderate CYP3A inhibitor, with a 3.7-fold increase in the
area under the plasma concentration–time curve (AUC) of
oral midazolam observed following 28 days of crizotinib
dosing at 250 mg BID (Tan et al., 2010). In cancer patients,
peak plasma concentrations of crizotinib were reached 4 h
after a single 250-mg oral dose and declined with an apparent
terminal half-life of 42 h (Li et al., 2011; Tan et al., 2010).
Oral bioavailability of crizotinib in healthy volunteers was
43% following a single 250-mg oral dose (Xu et al., 2011a).
Crizotinib exhibits time-dependent PK, as reflected by a
40% decrease in oral clearance with repeat-dose adminis-
tration, likely due to auto-inhibition/inactivation of CYP3A
(Li et al., 2011).
Given the complex pharmacokinetic profile of crizotinib in
humans, a more thorough understanding of its disposition was
warranted. Herein, we report the findings from a study of the
metabolism, excretion and PK of [14C]crizotinib in
healthy human subjects following a single 250-mg oral dose
as well as exploratory assessments of the PK and pharmaco-
logical activity of the primary oxidative metabolites of
crizotinib.
Materials and methods
Chemicals and reference compounds
Crizotinib (PF-02341066) was synthesized by Medicinal
Chemistry and Pharmaceutical Sciences, Pfizer Worldwide
Research and Development (San Diego, CA and Sandwich,
UK, respectively) (Cui et al., 2011). Synthetic standards of
crizotinib metabolites, crizotinib lactam (PF-06260182), the
constituent diastereomers of crizotinib lactam (PF-06270079
and PF-06270080), O-desalkyl crizotinib (PF-03255243) and
O-desalkyl crizotinib lactam (PF-06268935), were synthe-
sized by Medicinal Chemistry, Pfizer Worldwide Research
and Development (San Diego, CA). The absolute configur-
ation of the crizotinib lactam constituent diastereomers,
PF-06270079 and PF-06270080, were not determined.
[14C]Crizotinib (specific activity 0.407mCi/mg; radiochem-
ical purity >99%) and [2H5]crizotinib (PF-03623192) were
synthesized by Radiochemistry, Pfizer Worldwide Research
and Development (Groton, CT). Perma Fluor, Carbo-Sorb and
Ultima Gold liquid scintillation fluid was obtained from
PerkinElmer (Waltham, MA). Reagents and solvents of
analytical or high pressure liquid chromatography (HPLC)
grade were purchased from commercial manufacturers.
Study subjects
Subjects were healthy males between the ages of 18 and 55
years with body mass index of 17.5 to 30.5 kg/m2 and a total
body weight of >50 kg (110 lbs). All subjects signed an
informed consent document before screening. Exclusion
criteria included, but was not limited to: evidence or history
of clinical significant diseases, any condition possibly affect-
ing drug absorption, a positive drug screen, history of excess
alcohol consumption, use of tobacco- or nicotine-containing
products, QTc >450 ms at screening, use of drugs and dietary
supplement within 7 days or 5 half-lives prior to the start of
study drug treatment, radionucleotide study or radiotherapy
within 12 months prior to screening or such that total
radioactivity would have exceeded acceptable dosimetry
(i.e. occupational exposure of 5 rem per year), blood donation
of �500 mL within 56 days prior to dosing and a positive
serology for hepatitis B and C.
Study design
This was an open label, single dose, single center study
(Study A8081009) to evaluate the mass balance and PK of
crizotinib in six healthy male subjects, conducted at the Pfizer
Clinical Research Unit (New Haven, CT). Based on previous
clinical experiences where 250 mg twice daily dosing was
determined to be the maximum tolerated dose in cancer
patients (Kwak et al., 2010), a single 250-mg dose was
selected as the dose for this study. Dosimetry estimations
allow the use of the 100 mCi radiolabel dose based on a
[14C]crizotinib study in rats.
ON
NN
NH
ClF
ClNH2
* *
Figure 1. Chemical structure of crizotinib and position of [14C] label(asterisk).
2 T. R. Johnson et al. Xenobiotica, Early Online: 1–15
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Study conduct and sample collection
The study was conducted in compliance with the ethical
principles originating in or derived from the Declaration of
Helsinki and in compliance with all International Conference
on Harmonization Good Practice Guidelines. The study
protocol and informed consent documentation were approved
by an institutional review board, and all local regulatory
requirements were followed, in particular, those affording
greater protection to the safety of study participants.
Following an 8-h fast, each subject received a single oral
250-mg crizotinib dose, containing �100 mCi of
[14C]crizotinib, suspended in 100 mL of 0.1% (w/v) methyl-
cellulose, followed by an additional 140 mL of water (total
aqueous volume administered, 240 mL). Blood was collected
by an in-dwelling catheter or venipuncture into K2EDTA
vacutainer tubes pre-dose and at 1, 2, 3, 4, 6, 8, 10, 12, 16, 24,
36, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288 and
312 h post-dose in all subjects and 336, 360, 384 and 408 h in
subjects where recovery of radioactivity in excreta was
incomplete (vide infra). Plasma was prepared from blood
samples by centrifuging for 10 min at 1700 g and at �4 �C.
Urine was collected pre-dose, from 0 to 4, 4 to 8, 8 to 16
and 16 to 24 h post-dose, and at intervals of 24 h up to 480 h
post-dose. The collected urine from each void was stored
at 4 �C throughout the collection interval after the addition
of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesul-
fonate (2% weight/volume). Feces were collected pre-dose
and as passed, at intervals of 24 h up to 480 h post-dose. Daily
sample collections continued beyond 312 h until one of the
following conditions were met: the amount of radioactivity
recovered in excreta was �90% of administered radioactivity,
or51% was recovered in excreta from two consecutive days.
Total weight of urine and fecal output was recorded for each
collection interval. All samples were stored at ��20 �C or
below until the time of analysis.
Determination of total radioactivity
Radioactivity in blood, plasma, urine and feces was measured
using PerkinElmer Tri-Carb Model 2900TR liquid scintilla-
tion counters (Waltham, MA). All sample combustions were
performed in a PerkinElmer Model 307 Sample Oxidizer
(Waltham, MA) and the resulting 14CO2 was trapped in a
mixture of Perma Fluor and Carbo-Sorb scintillation fluids.
Oxidation efficiency was evaluated on each day of sample
combustion by analyzing a commercial radiolabeled standard
both directly in scintillation cocktail and by oxidation. Liquid
scintillation counting (LSC) data (cpm) were automatically
corrected for counting efficiency using the external standard-
ization technique and an instrument-stored quench curve
generated from a series of sealed quenched standards to
obtain dpm. Samples were counted for at least 5 min or
100 000 counts for urine and feces and for at least 30 min or
100 000 counts for plasma.
Blood samples were mixed and single-weighed aliquots
(�0.4 g) were combusted and analyzed by LSC. Plasma
samples were mixed, and single-weighed aliquots (�1 g) were
analyzed directly by LSC. Specific gravity values of 1.05 and
1.02 g/mL for blood and plasma, respectively, were used to
calculate radioactivity concentration as nanogram-equivalents
per milliliter (Trudnowski & Rico, 1974). Total radioactivity
concentrations in red blood cells (RBCs) were calculated for
each time point (where possible) as [Cb � Cp�(1 � H)]/H,
where Cb, Cp and H were the whole blood radioactivity
concentration, plasma radioactivity concentration and the
hematocrit, respectively (average of Day 1 and Day 14 H
values). Negative RBC concentration (CRBC) values were
reported as zero. Urine samples were mixed and triplicate-
weighed aliquots (�0.2 g) were analyzed directly by LSC.
Fecal samples were homogenized with water (1:2, w/w, feces/
water) and triplicate-weighed aliquots (�0.2 g) were com-
busted and analyzed by LSC. The limits of quantitation for
blood and plasma were 59.6 and 19.8 ng-equivalents/mL,
respectively.
Quantification of crizotinib in plasma and urine
Concentrations of crizotinib in plasma and urine samples
were determined with validated liquid chromatography–
tandem mass spectrometry (LC–MS/MS) analytical methods
in compliance with Pfizer standard operating procedures
at Covance Bioanalytical Services, LLC (Indianapolis, IN).
Briefly, isotopically labeled internal standard ([2H5]crizotinib,
PF-03623192) was added to 200 mL of plasma or 100 mL of
urine. For plasma samples, crizotinib was extracted by protein
precipitation with acetonitrile. Samples were centrifuged, and
a 500 mL aliquot was evaporated to dryness. Plasma extracts
were reconstituted in 200 mL water:formic acid (10:1, v/v),
mixed and centrifuged. For urine samples, urine (100 mL) was
treated with internal standard and diluted with 100 mL
water:formic acid (10:1, v/v). The diluted urine sample was
mixed and centrifuged. Aliquots (10 mL) of plasma extracts or
diluted urine were injected onto a Phenomenex Onyx
Monolith HPLC column (50� 4.6 mm; Torrance, CA) main-
tained at 40 �C. Analytes were eluted with mobile phases
containing mixtures of water, formic acid and methanol using
a step gradient and flow rate of 1.5 mL/min. Detection was
performed by positive ion MS/MS on a SciEx API 4000 mass
spectrometer (Applied Biosystems, Foster City, CA) with
multiple reaction monitoring (m/z 450 to 260 for crizotinib
and m/z 455 to 265 for internal standard). The LLOQ for
crizotinib measurement was 0.200 ng/mL. Calibration stand-
ard responses were linear over the range of 0.200 ng/mL to
200 ng/mL, using a weighted (l/concentration2) linear least
squares regression. For plasma samples, the between-day
assay accuracy, expressed as percent relative error for quality
control (QC) concentrations, ranged from 1.3% to 3.0% for
the low (0.600 ng/mL), medium (70.0 ng/mL) and high
(150.0 ng/mL) QC samples. Assay precision, expressed as
the between-day percent coefficients of variation (% CV) of
the mean estimated concentrations of QC samples was
�4.1%. For urine QC samples, the between-day assay
accuracy ranged from �4.7% to �2.4%, and assay precision
was �5.2%.
Pharmacokinetic analysis
PK parameters for total radioactivity (plasma, whole blood
and RBC), crizotinib (plasma), and crizotinib lactam
diastereomers (plasma) were calculated for each subject
using noncompartmental analysis of concentration–time data
DOI: 10.3109/00498254.2014.941964 Crizotinib disposition in humans 3
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using Pfizer internal software eNCA (version 2.2.2). Actual
sample collection times were used for the PK analysis. The
maximum plasma concentration (Cmax) and the time at which
Cmax was achieved (tmax) were estimated directly from the
concentration data. The area under the plasma concentration–
time curve (AUClast) from time 0 to the last time (tlast) with a
quantifiable concentration (Clast) was estimated using linear-
log trapezoidal approximation. AUC extrapolated to infinity
(AUC1) was calculated using the equation AUClast +
(Clast/�), where � was the elimination rate constant calculated
by a linear regression of the log-linear concentration–time
curve. Apparent terminal elimination half-life (t1/2) was
calculated as ln2/�. A well-characterized terminal phase was
defined as one with at least three data points, a goodness-of-
fit statistic for the log-linear regression (r2) �0.9, a span ratio
�2 (duration over which t1/2 was assessed, divided by the t1/2
estimate) and percent of AUC1 due to forward extrapolation
(AUCextrap%) �20%.
The amount of crizotinib excreted in urine during each
collection interval was calculated by multiplying the concen-
tration of crizotinib by the volume of urine collected over that
interval. The amount of unchanged crizotinib excreted in the
urine expressed as percent of dose was calculated as the
cumulative amount excreted over all the collection periods
divided by the dose. Renal clearance was calculated as the
cumulative amount of crizotinib excreted divided by AUC1.
Recovery of total radioactivity in urine and feces as a
percentage of dose was calculated using a similar method.
Preparation of samples for radioactivity profiling
Plasma
Time-normalized pooled plasma samples representing 0–96 h
post-dose were prepared for each subject (Hamilton et al.,
1981). A global pool was then prepared by mixing equal
volumes of each individual pool. The time interval selected
for sample pooling was limited due to low levels of circulating
radioactivity in all subjects. Two samples (�2.5 g each) of
pooled plasma were extracted three times with four volumes
of 80:20 acetonitrile:methanol. Each extraction was carried
out by vortex mixing for �1 min, sonication for �5 min and
centrifugation for 10 min at 800 g. The supernatants from each
extraction were combined, and 100mL of dimethyl sulfoxide
was added. Solvent was removed with heat under vacuum.
The concentrated extracts were reconstituted in 250 mL of
70:30 water:acetonitrile containing 0.1% formic acid and
centrifuged at 17 000 g for 5 min.
Urine
Urine samples were prepared for each subject such that the
amount of radioactivity contained in the pool collectively
represented 90% or greater of the total radioactivity recovered
in urine for a given subject (�0–120 h post-dose). The amount
of sample added for each time point was normalized by
weight of sample within each given subject. Samples (1 mL)
of pooled urine from each subject were combined with 1 mL
acetonitrile, vortex-mixed for 15 min and centrifuged for
10 min at 3000 g. Supernatants were evaporated to dryness
with heat under vacuum and then reconstituted in 200 mL of
80:20 water:acetonitrile containing 0.1% formic acid. The
reconstituted extracts were sonicated for 5 min, vortex-mixed
for 10 min and centrifuged for 10 min at 2000 g.
Feces
Weight-normalized pooled fecal homogenate samples from
each individual were generated in the same manner as used
for urine (representing �0–120 h post-dose). Samples
(�0.5 g) of pooled fecal homogenate from each subject
were extracted by solid phase using Sep Pak C18 6 cc
cartridges with a 1 g bed volume (Waters, Milford, MA).
Cartridges were conditioned with 15 mL acetonitrile/water
(98:2, v/v) containing 0.2% formic acid, and then equilibrated
with acetonitrile/water (2:98, v/v) containing 0.2% formic
acid. Fecal homogenates were loaded and cartridges were
washed with 10 mL acetonitrile/water (2:98, v/v) containing
0.2% formic acid. Analytes were eluted with 10 mL aceto-
nitrile/water (1:1, v/v) containing 0.2% formic acid into tubes
containing 50 mL dimethyl sulfoxide. The solutions resulting
from the elution step were concentrated with heat under
vacuum and reconstituted in 200 mL of acetonitrile/water
(2:8, v/v) containing 0.1% formic acid. The reconstituted
extracts were sonicated for 5 min, vortex-mixed for 10 min
and centrifuged for 10 min at 2000 g.
Supernatants of the reconstituted plasma, urine or fecal
extracts were transferred to pre-tared HPLC vials and the total
weights of the samples were recorded. Aliquots were weighed
into scintillation vials, and radioactivity was determined
(LSC) to estimate recovery. Aliquots (200mL for plasma and
100 mL for urine and feces) of the concentrated extracts were
injected into an HPLC system for analysis.
Metabolite profiling
Metabolic profiling was performed on an Agilent 1100 series
HPLC equipped with binary pumps, an autosampler and a
diode array detector (Agilent Technologies, Santa Clara, CA).
Two HPLC methods were used for generation of metabolic
profiles. The first method was used for initial metabolite
profile generation and the second method was used to
separate early eluting (55 min), unresolved components in
the initial profiles.
HPLC method 1
A Kromasil C4 column (4.6� 150 mm, 5 m) (AkzoNobel,
Brewster, NY) was used for metabolite separation. HPLC
analyses were performed at 1 mL/min flow rate and ambient
temperature. The HPLC mobile phase consisted of water with
0.1% formic acid (A) and acetonitrile with 0.1% formic acid
(B). The following linear gradient was used: 0 min, 2% B;
5 min, 2% B; 10 min, 10% B; 50 min, 30% B; 56 min, 95% B;
58 min, 95% B; and 60 min, 2% B. The column was re-
equilibrated at 2% B for 10 min prior to the next injection.
HPLC method 2
A Synergi Polar-RP column (2.0� 250 mm, 4 m)
(Phenomenex, Torrance, CA)was utilized for metabolite
separation at flow rate of 0.2 mL/min flow and ambient
temperature. The HPLC mobile phase consisted of water with
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10 mM ammonium formate (A) and acetonitrile (B). The
following linear gradient was used: 0 min, 2% B; 7 min, 2% B;
15 min, 10% B; 35 min, 20% B; 45 min, 40% B; 62 min, 95%
B; 64 min, 95% B; and 65 min, 2% B. The column was re-
equilibrated at 2% B for 25 min prior to the next injection.
For the plasma profiles, fraction collection of the HPLC
column effluent with subsequent off-line determination of
radioactivity by LSC was used. Fractions were collected at
0.5 min intervals using a Gilson FC 204 Fraction Collector
(Middleton, WI). Aliquots of each fraction were removed for
analysis by LC/MS, and the radioactivity in the remaining
fractions was determined by LSC using a 30 min count time.
Radiochromatographic metabolite profiles were constructed
by plotting the dpm values from the LSC of the HPLC
fractions versus time post-injection using Microsoft Excel
(Microsoft, Redmond, WA). The relative abundance of drug-
related materials observed in the chromatogram (expressed as
a percent of total radioactivity in the chromatogram) were
determined by dividing the sum of the radioactive content of
fractions deemed to contribute to a particular peak by the sum
of the radioactive content of all fractions used to construct the
radio chromatogram. To assign the relative abundance of
drug-related materials in plasma resolved using HPLC
method 2, the percent of total radioactivity corresponding to
each resolved peak was normalized by multiplying by the
relative abundance of radioactivity corresponding to the early
eluting fractions (55 min) from the profile generated using
HPLC method 1 (i.e. 24%).
For urine and feces samples, the radioactivity in the HPLC
column effluent was measured using an IN/US System Model
3 b-ram radio-HPLC detector equipped with a 600 mL flow cell
operated in stop-flow modes using an AIM research Company
model C StopFlow System controller. The fraction time was
8.64 s, the scintillation cocktail was StopFlow AD (AIM
Research Company, Hockessin, DE), and the HPLC effluent:
scintillation cocktail ratio was 1:1.5. The count time was 120 s
for urine and 20 s for feces. Urinary and fecal metabolite
abundance (expressed as percent of administered dose) was
calculated by multiplying the fractional contribution of
radioactive response for the peak in the radio-HPLC chro-
matogram by the percent of administered dose recovered in the
matrix.
Fractionation of samples for metabolite identification
Plasma
Fractions generated from radio-HPLC profiling containing
radioactivity, as described previously, were analyzed directly
by LC/MS to identify metabolites.
Urine
Pooled urine from two subjects (2.5 mL from each) was
extracted as described previously. The evaporated extract was
reconstituted in 2 mL of acetonitrile/water (2:8) containing
0.1% formic acid. Repeated injections (10� 100 mL) of the
urine extract were performed using HPLC method 1 with the
effluent from each run collected into a single set of
scintillation vials such that the same time interval for all
runs was collected in the same vial. Fractions were collected
at 0.33 min intervals from 0 to 18 min. Aliquots of each
fraction were removed for analysis by LC/MS and the
radioactivity in the remaining fractions was determined by
LSC using a 5 min count time. Fractions eluting before 5 min
that contained significant radioactivity were combined,
evaporated to dryness and reconstituted in 600 mL of aceto-
nitrile/water (2:8) containing 0.1% formic acid. Repeated
injections (3� 200 mL) were performed using HPLC method
2 with the effluent from each run collected into a single set of
scintillation vials. Fractions were collected at 0.25 min
intervals from 0 to 44 min. Aliquots of each fraction were
removed for analysis by LC/MS and the radioactivity in the
remaining fractions was determined by LSC using a 15 min
count time. Fractions containing radioactivity from the two
chromatographic methods were analyzed by LC/MS to
identify metabolites.
Feces
Samples of pooled feces were extracted as described previ-
ously. The evaporated extract was reconstituted in 0.8 mL of
acetonitrile/water (2:8) containing 0.1% formic acid.
Repeated injections (4� 200 mL) of the fecal extract were
performed using HPLC method 1 with the effluent from each
run collected into a single set of scintillation vials. Fractions
were collected at 0.5 min intervals from 0 to 50 min. Aliquots
of each fraction were removed for analysis by LC/MS and the
radioactivity in the remaining fractions was determined by
LSC using a 5 min count time. Fractions eluting before 5 min
that contained significant radioactivity were combined,
evaporated to dryness and reconstituted in 600 mL of aceto-
nitrile/water (2:8) containing 0.1% formic acid. These fecal
extracts were fractioned by HPLC method 2 as described for
the urine samples. Fractions containing radioactivity from the
two chromatographic methods were analyzed by LC/MS to
identify metabolites.
Metabolite identification by LC/MS
Metabolite identification by mass spectrometry was per-
formed using a hybrid LC/MS system consisting of two
Shimadzu pumps, a CTC Analytics autosampler, an Agilent
1100 series diode array detector and an API 4000 QTRAP
mass spectrometer (Applied Biosystems, Foster City, CA).
All components of the system were controlled using Analyst
1.4.1 (Build 1200) software (Applied Biosystems). For all
analyses, the mass spectrometer was operated in
TurboIonSpray mode with conditions adjusted as necessary.
Scan modes utilized included enhanced MS, multiple reaction
monitoring, neutral loss, precursor ion and MS2. Fractions
were analyzed by direct infusion and/or LC/MS. Infusions
were conducted with a Harvard Apparatus Pump 11 syringe
pump. For LC/MS analyses a Synergi Polar-RP column
(2.0� 250 mm, 4 m) was utilized for metabolite separation at
flow rate of 0.2 mL/min flow and ambient temperature. The
HPLC mobile phase and gradient was the same as described
for HPLC method 2.
Quantification of the diastereomers of crizotiniblactam (M10) in plasma
As there was insufficient plasma volume remaining from the
study, the quantification of diastereomers of crizotinib lactam
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was conducted by using plasma samples from a sub-set of
subjects (n¼ 4) receiving a single oral 250-mg dose of
crizotinib from a relative bioavailability study (A8081008;
ClinicalTrials.gov identifier NCT00939731). Blood samples
were collected by an indwelling catheter or venipuncture into
K2EDTA vacutainer tubes pre-dose and at 0.5, 1, 2, 3, 4, 6, 8,
10, 12, 16, 24, 36, 48, 60, 72, 84 and 96 h post-dose. Plasma
was prepared from blood samples as described previously, and
samples were stored at or below�20 �C until the time of
analysis.
Concentrations of the constituent diastereomers of crizo-
tinib lactam (M10, PF-06270079 and PF-06270080) in
plasma samples were determined with a non-validated
LC–MS/MS analytical method. Briefly, internal standard
([2H5]crizotinib, PF-03623192) was added to 100mL of
plasma followed by addition of 100mL of sodium bicarbonate
buffer, pH 7.4. Two hundred mL of each sample was added
into an Isolute solid–liquid extraction (SLE)+ 200 mg plate,
and a light vacuum was applied to the SLE block. Samples
were eluted with 1.2 mL of methyl tert-butyl ether and
evaporated under nitrogen. Samples were reconstituted with
100mL of water/acetonitrile (1:1, v/v). Aliquots (5 mL) of
plasma extracts were injected onto ChiralPAK, RH-AS
column (5mm, 150� 4.6 mm; Chiral Technologies, Inc.,
West Chester, PA) maintained at ambient temperature.
Analytes were eluted using an isocratic mobile phase of
water/acetonitrile (48:52, v/v) containing 10 mM ammonium
acetate and a flow rate of 0.4 mL/min. Detection was
performed by positive ion MS/MS on a SciEx API 4000
mass spectrometer (Applied Biosystems, Foster City, CA)
with multiple reaction monitoring (m/z 464 to 274 for
PF-06270079 and PF-06270080 and m/z 455 to 265 for
internal standard). The LLOQ for PF-06270079 and
PF-06270080 measurement was 0.1 ng/mL. Calibration stand-
ard responses were linear over the range of 0.1–125 ng/mL,
using a weighted (l/concentration2) linear least squares
regression. Calibration standards and QC samples (prepared
in triplicate at 0.75, 15 and 100 ng/mL) were within ± 20% of
the nominal value.
Cellular pharmacodynamic assays
Crizotinib and its metabolites, crizotinib lactam
(PF-06260182), the constituent diastereomers of crizotinib
lactam (PF-06270079 and PF-06270080), O-desalkyl crizoti-
nib (PF-03255243) and O-desalkyl crizotinib lactam
(PF-06268935), were evaluated for their ability to inhibit
the phosphorylation of ALK fusion variants and c-Met in cells
lines expressing these targets. Inhibition of ALK was
evaluated in NCI-H3122 and NCI-H2228 cells, two human
lung adenocarcinoma cell lines harboring chromosomal
inversion events resulting in the expression of EML4-ALK
fusion protein variant 1 (V1) and variant 3 (V3), respectively
(Koivunen et al., 2008). Inhibition of c-Met phosphorylation
was evaluated in human A549 lung adenocarcinoma cells.
Briefly, cells were seeded in 96-well plates in medium
supplemented with 10% fetal bovine serum and transferred to
serum-free medium (with 0.04% bovine serum albumin) after
being cultured overnight. After incubation of cells with an
inhibitor for 1 h, cells were washed once with Hank’s
balanced salt solution supplemented with 1 mM Na3VO4
and protein lysates were generated from cells. In experiments
investigating ligand-dependent RTK phosphorylation, corres-
ponding growth factors were added for up to 20 min.
Subsequently, phosphorylation of selected protein kinases
was assessed by sandwich enzyme-linked immunosorbent
assays using specific capture antibodies used to coat 96-well
plates and a detection antibody specific for phosphorylated
tyrosine residues as described previously (Cui et al., 2011;
Zou et al., 2007). EC50 values were calculated by concentra-
tion–response curve fitting using a Microsoft Excel-based
four-parameter method. Statistical significance of differences
in mean EC50 values was determined by an unpaired two-
sample t-test using Microsoft Excel.
Pharmacological activity index
The pharmacological activity index (PAI) (Leclercq et al.,
2009) was used as a framework within which the potential
contribution of metabolites to pharmacological activity could
be evaluated. The PAI index was calculated as:
PAI ¼ AUCm
AUCp
� fum
fup
� EC50, p
EC50, m
where, AUCm and AUCp are the area under the plasma
concentration–time curves for metabolite and parent, respect-
ively; fum and fup are the unbound fraction values in plasma
for metabolite and parent, respectively; and EC50,p and EC50,m
are the half maximal inhibitory concentrations for parent and
metabolite, respectively, in the cellular pharmacodynamic
assays.
Safety assessments
Safety evaluations included clinical monitoring, collection of
subject-reported adverse events, including serious adverse
events, vital signs, 12-lead electrocardiograms and clinical
laboratory tests.
Results
Subjects
Six male subjects (three African-American, two White and
one Hispanic) enrolled and completed the study. The mean
(SD) age of the subjects was 43.2 (1.8) years, and the mean
body weight was 85.2 (9.5) kg. In the relative bioavailability
study, samples from four male subjects were evaluated for
metabolite PK (three African-American, one other). The
mean age of these subjects was 29.5 (11.1) years, and the
mean body weight was 68.8 (9.0) kg.
Excretion and mass balance
The mean cumulative recovery of radioactivity in urine and
feces from 0 to 480 h post-dose in six healthy male subjects
administered a single 250-mg (100mCi) oral dose of
[14C]crizotinib is presented in Figure 2 and Table 1. The
overall mean recovery of drug-derived radioactivity was
85.2% of the dose with recovery in individual subjects ranging
from 68.6% to 91.3%. Radioactivity was recovered predom-
inantly in feces, with a mean (±SD) cumulative amount of the
dose recovered of 63.1 ± 5.9%. The mean cumulative amount
6 T. R. Johnson et al. Xenobiotica, Early Online: 1–15
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of the dose recovered in urine was 22.2 ± 4.6%. A majority of
the administered radioactivity was recovered in the first 120 h
post-dose (77.9 ± 9.4%).
Pharmacokinetics
The mean concentration–time profiles and pharmacokinetic
parameters of unchanged crizotinib (plasma) and total
radioactivity (plasma, whole blood and RBCs) in healthy
male subjects administered a single 250-mg (100 mCi) oral
dose of [14C]crizotinib are presented in Figure 3(A, B) and
Table 2, respectively. The peak concentrations of crizotinib
occurred between 2.0 and 6.0 h after oral dosing and the mean
Cmax was 109 ng/mL. The mean apparent t1/2 value for
crizotinib in plasma was 94 h and was longer than determin-
ations in other studies (29–51 h), likely due to the extended
sampling period of up to 408 h post-dose (Li et al., 2011;
Tan et al., 2010; Xu et al., 2011a). The variability, evaluated
by the CV, was 46 and 38% for crizotinib Cmax and AUC1,
respectively. Total radioactivity measured in plasma peaked
between 3.0 and 6.0 h after dosing, similar to the tmax for
crizotinib. Plasma exposure of crizotinib was considerably
lower than that of the total radioactivity. Unchanged drug
represented �12% of drug-related material in plasma based
on AUClast values, indicating the presence of circulating
metabolic products in plasma. The apparent t1/2 of total
radioactivity in plasma was prolonged relative to unchanged
drug (Figure 3A). Total radioactivity in whole blood paral-
leled the profile of total radioactivity in plasma up to 48 h, the
last quantifiable concentration of radioactivity in blood, with
peak radioactivity occurring at similar times (3.0–6.0 h after
dosing; Figure 3B). Mean RBC concentrations of total
Figure 3. Mean (±SD) concentration versus time profiles for total radioactivity and crizotinib in plasma (A) and total radioactivity (to 48 h) in plasma,blood and RBCs (B) after a single 250-mg oral dose of [14C]crizotinib to male volunteers (n¼ 6).
Table 2. Pharmacokinetic parameters of crizotinib in plasma and totalradioactivity in plasma, whole blood and RBC after a single 250-mg oraldose of [14C]crizotinib to six healthy male subjects.
Crizotinib Total radioactivityb
Parametera Plasma Plasma Whole Blood RBC
Cmax (ng/mL) 109 (46) 436 (19) 312 (20) 175 (26)tmax (h) 3 (2–6) 5 (3–6) 4 (3–6) 5 (3–8)AUClast
(ng�h/mL)2686 (40)c 22 830 (11)d 7032 (18)e 3641 (23)e
AUC1(ng�h/mL)
2777 (38) 29 000, 29 600f nc nc
t1/2 (h) 94.0 (15) 134, 178f nc nc
nc, not calculated.aGeometric mean (%CV) for Cmax and AUC; arithmetic mean (%CV) for
t1/2; median (range) for tmax; n¼ 6.bTotal radioactivity expressed in units of nanogram-equivalents per
milliliter (Cmax) or nanogram-equivalents�hour per milliliter (AUC).cTime of last quantifiable concentration was 312 h in three subjects,
336 h in one subject, 360 h in one subject and 408 h in one subject.dTime of last quantifiable concentration was 312 h in three subjects,
336 h in one subject and 360 h in two subjects.eTime of last quantifiable concentration was 36 h in one subject and 48 h
in five subjects.fIndividual values for the two evaluable subjects. For the remaining four
subjects, AUC1 and t1/2 could not be estimated.
Figure 2. Mean (±SD) cumulative excretion of total radioactivity inurine, feces and overall after a single 250-mg oral dose of [14C]crizotinibto male volunteers (n¼ 6).
Table 1. Cumulative excretion of total radioactivity in urine and fecesafter a single 250-mg oral dose of [14C]crizotinib to six healthy malesubjects.
Percent of radioactive dose recovered by subjecta
Matrix 1001 1002 1003 1004 1005 1006 Mean (SD)
Urine 22.6 28.8 15.1 23.0 24.3 19.2 22.2 (4.6)Feces 63.2 58.9 53.5 68.2 66.0 68.7 63.1 (5.9)Total 85.8 87.7 68.6 91.3 90.2 87.8 85.2 (8.4)
aTime period of excretion was �480 h.
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radioactivity were 29–45% of plasma radioactivity concen-
trations at corresponding time points, indicating that crizoti-
nib or its metabolites do not preferentially distribute to RBCs.
A mean of 2.3% of the dose was excreted in urine as
unchanged crizotinib, based on a specific LC-MS/MS
assay for crizotinib, with a mean renal clearance estimate of
2.5 L/h.
Metabolite profiling
The metabolic profile in a pooled (0–96 h) plasma sample is
presented in Figure 4(A) and the distribution of metabolites is
summarized in Table 3. The recovery of radioactivity
following acetonitrile/methanol extraction of this pooled
plasma sample was �60%. Unchanged crizotinib was the
major circulating component, accounting for 33% of radio-
activity in the profile. M10 represented 10% of circulating
radioactivity and early eluting components (55 min)
accounted for 24% of circulating radioactivity. A second
pooled plasma sample was analyzed with HPLC conditions
optimized to retain and resolve these early eluting compo-
nents and the resulting metabolic profile is shown in
Figure 4(B). The early eluting peaks were composed of
multiple components (M1, M2, M3 and M8), each accounting
for510% of the recovered radioactivity individually.
Representative radio-HPLC profiles of pooled feces
samples and pooled urine samples are shown in Figure 5(A
and B), respectively. Unchanged crizotinib was the major
excreted component in feces, accounting for an average of
53% of total administered dose (Figure 5A). In urine, an
average of 2.4% of the dose was excreted as unchanged
crizotinib, based on radio-HPLC profiling. M8 was the major
excreted component in urine, accounting for an average of
4.5% of dose (Figure 5B). No other metabolites accounted for
�1% of the total administered dose in excreta.
Metabolite identification
Nine metabolites were identified in this study. Metabolite
structures were elucidated by LC–MS analysis (full scan
and MS2), and in the case of M2 (PF-06268935), M4
(PF-03255243) and M10 (PF-06260182), by comparison to
Figure 4. HPLC radiochromatograms of pooled human plasma (0–96 h)after a single 250-mg oral dose of [14C]crizotinib to male volunteers(n¼ 6). Primary full profile (A) and secondary profile resolving earlyeluting (55 min) components in the primary profile (B).
Figure 5. Representative HPLC radiochromatograms of individualsubject-pooled (0–120 h) human feces (A) and urine (B) after a single250-mg oral dose of [14C]crizotinib to male volunteers.
Table 3. Relative percent distribution of radioactivemetabolites in 0–96 h-pooled plasma after a single250-mg oral dose of [14C]crizotinib to six healthy malesubjects.
ComponentPercent of radioactivity
(0–96 h)a
M1 3.0M2 1.1M3 2.5M8 0.8M10 10.2Crizotinib 33.1Total 50.7
aPercent of total radioactivity in 0–96 h plasma AUC.
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retention time and MS fragmentation of authentic metabolite
standards. The MS fragmentation patterns of crizotinib and
metabolites are presented in Table 4. The proposed metabolic
pathways of crizotinib in humans is presented in Figure 6 and
identification of metabolites is described in order of primary
metabolites, followed by secondary products, as outlined in
this scheme.
The fragmentation pattern of crizotinib was studied to
facilitate interpretation of mass spectra of its metabolites. The
protonated molecular ion for crizotinib was at m/z 450.
The collision-induced dissociation MS2 spectrum showed
fragment ions at m/z 367, 260, 177 and 84. The fragment ion
at m/z 367 resulted from a loss of the piperidine ring from the
molecule, whereas fragment ions at m/z 260 and 177 were
Table 4. Mass spectral data and proposed structures of crizotinib metabolites.
Metabolite ID/MS data Proposed structure Metabolite ID/MS data Proposed structure
Crizotinib [M + H]+: 450Product ions (m/z): 367, 260, 177, 84
OF
N N
NCl
Cl
NH
84367
260
+2H
+2H+2H
+H
+H 177
NH2
M5 [M + H]+: 452Product ions (m/z): 276
N N
NHO
HO
OHO
HOHOOC
OH
NH
276+H
NH2
M1 [M + H]+: 436Product ions (m/z): 353, 260, 177
O
N N
NHO
HO353 HO
HOHOOC
NH
177
260
+2H
+H
+H
NH2
M6 [M + H]+: 290Product ions (m/z): 177
N N
NHO
OOHNH
177
+2H
NH2
M2 (PF-06268935) [M + H]+: 274Product ions (m/z): 177, 98
ON N
NHO
NH
98
177
+2H
+H
NH2
M8 [M + H]+: 354Product ions (m/z): 274, 177, 98
N N
NHO
OO
OSHO
NH98
274
177
+2H
+H
+H
NH2
M3 [M + H]+: 340Product ions (m/z): 260, 177
O
OSHO
N N
NHO
NH
260
177
+2H
+H
NH2
M9 [M + H]+: 393Product ions (m/z): 296, 279, 235, 209, 98
N N
NHO
O
O
SHO
NH
98
279
209235
296
+2H
+2H
+3H+2H
+H
NH2
NH2
M4 (PF-03255243) [M + H]+: 260Product ions (m/z): 177, 84
N N
NHO
NH
84
177
+2H
+H
NH2
M10 (PF-06260182) [M + H]+: 464Product ions (m/z): 367, 274, 177, 98
NH2
ON
NN
NH
ClF
Cl
O367
274
98
177
+2H
+2H
+H
+2H
+H
PF-number, synthetic standard available.
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products of cleavage of the dichloro-fluorobenzyl group and
subsequent loss of the piperidine ring, respectively. The
fragment ion at m/z 84 was indicative of the piperidine
moiety.
Metabolite M10
M10 (PF-06260182) was detected in plasma and feces
(MS only). The protonated molecular ion of M10 was at
m/z 464 and indicated the addition of 14 amu to the
molecular ion of crizotinib. The mass spectrum showed
fragment ions at m/z 367, 274, 177 and 98. The ions at
m/z 367 and 177 were characteristic of ions present in
the mass spectrum of crizotinib, as described previously.
The ions at m/z 274 and 98 showed additions of 14 amu
to characteristic fragment ions of m/z 260 and 84 in the
spectrum of crizotinib, suggesting oxidation of the
piperidine ring.
Metabolite M4.
M4 (PF-03255243) was detected in feces (early eluting
component; radiochromatographic data not shown). The
protonated molecular ion of M4 was at m/z 260, indicative
of an O-desalkyl metabolite of crizotinib. Fragments at
177 and 84 were consistent with the fragmentation spectrum
of crizotinib, as described previously.
Metabolite M1
M1 was detected in plasma, urine and feces (early eluting
component in excreta; radiochromatographic data not shown).
The protonated molecular ion of M1 was at m/z 436, 176 amu
greater than M4 (O-desalkyl crizotinib), suggesting that M1
was a glucuronide conjugate of M4. The mass spectrum of the
metabolite showed fragment ions at m/z 353, 260 and 177.
The ions at m/z 260 and 177 were similar to those observed in
the mass spectrum of crizotinib. The ion at m/z 353 suggested a
loss of the piperidine ring from the molecule and indicated
that the glucuronide was not conjugated to the piperidine ring
and was possibly attached to the hydroxyaminopyridine
moiety.
Metabolite M3
M3 was detected in plasma, urine and feces (early eluting
component in excreta; radiochromatographic data not shown).
The protonated molecular ion of M3 was at m/z 340, 80 amu
greater than M4 (O-desalkyl crizotinib). This molecular ion
was indicative of a sulfate conjugate of M4. The mass
ON
NN
NH
ClF
Cl NH2 NH2
Crizotinib
HON
NN
NH
NH2
O
HON
NN
NH
NH2
O
O N
NN
NH
ClF
Cl
O
SHOO
O
HON
NN
NH
NH2
O
HO
HOOCHO
HO
M8
M2
M10
M1
HON
NN
NH
NH2
O
M9
HON
NN
NH
NH2
M4
HON
NN
NH
NH2
SHOO
O
M3
HON
NN
NH
NH2
O
M6
OH
HON
NN
NH
NH2
M5
O
HO
HOOCHO
HOOH
SHOOC
NH2
Figure 6. Proposed biotransformation pathways of crizotinib based on metabolites identified in plasma, urine and fecal samples from male volunteersadministered a single 250-mg oral dose of [14C]crizotinib.
10 T. R. Johnson et al. Xenobiotica, Early Online: 1–15
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spectrum showed fragment ions at m/z 260 and 177, which
were consistent with those observed in the mass spectrum of
crizotinib, as described previously.
Metabolite M5
M5 was detected in urine (early eluting component; radio-
chromatographic data not shown). The protonated molecular
ion of M5 was at m/z 452. The molecular ion was 192 amu
greater than M4 (O-desalkyl crizotinib), indicative of a
hydroxylation and glucuronidation of M4. A fragment ion at
m/z 276 was detected, indicative of the characteristic neutral
loss of 176 amu from a glucuronide conjugate. Moreover, this
fragment (m/z 276) was 16 amu greater than the molecular ion
of M4 (m/z 260), indicating a hydroxylation of O-desalkyl
crizotinib.
Metabolite M2
M2 (PF-06268935) was detected in plasma, urine and feces
(early eluting component in excreta; radiochromatographic
data not shown). The protonated molecular ion of M2 was at
m/z 274. This molecular ion was 14 amu greater than the ion
of M4 (O-desalkyl crizotinib), indicative of a lactam metab-
olite of M4. The absence of a fragment ion at m/z 84 and the
presence of an ion at m/z 98 suggested that the piperidine was
the site of oxidation.
Metabolite M6
M6 was detected in feces (early eluting component; radio-
chromatographic data not shown). The protonated molecular
ion of M6 was at m/z 290. The molecular ion was 16 amu
greater than M2 (O-desalkyl crizotinib lactam), indicative of a
hydroxylation of M2. The fragment at m/z 177 was consistent
with the fragmentation of crizotinib and indicated that the
oxidation occurred on the piperidine ring.
Metabolite M8
M8 was detected in plasma and urine. The protonated
molecular ion of M8 was at m/z 354. The molecular ion
was 80 amu greater than M2 (O-desalkyl crizotinib lactam),
indicative of a sulfate conjugate of M2. The mass spectrum of
the metabolite showed fragment ions at m/z 274, 177 and 98.
The fragment ion at m/z 274 results from the loss of the
sulfate moiety. The fragment ion at m/z 177 was consistent
with the fragmentation pattern of crizotinib, whereas, the ion
at m/z 98 represented the oxopiperidine moiety.
Metabolite M9
M9 was a trace metabolite detected in urine (MS only). The
protonated molecular ion of M9 was m/z 393. This molecular
ion was 119 amu greater than M2 (O-desalkyl crizotinib
lactam), indicative of a cysteine conjugate of M2. The
fragment at m/z 98 represented the oxopiperidine moiety,
and the fragment at m/z 296 resulted from the loss of
this moiety. This fragmentation pattern indicated that the
cysteine was not conjugated to the oxopiperidine ring and was
possibly attached to the hydroxyaminopyridine moiety.
Additional fragment ions at m/z 279, 235 and 209 were
attributed to loss of the oxopiperidine moiety and cleavages of
the cysteine. M9 is proposed as a downstream metabolite of a
glutathione adduct of M2. A proposed mechanism of
nucleophilic attack by glutathione via a quinone imine
intermediate leading to the formation of M9 is presented in
Figure 7.
Plasma pharmacokinetics of the diastereomers ofcrizotinib lactam (M10)
The mean plasma concentration–time profiles and pharma-
cokinetic parameters of crizotinib and the diastereomers of
crizotinib lactam (M10), PF-06270079 and PF-06270080,
from an exploratory assessment of four healthy male subjects
administered a single 250-mg oral dose of crizotinib as part of
a relative bioavailability study are presented in Figure 8 and
Table 5, respectively. Plasma exposure (as assessed by Cmax
and AUC1) of PF-06270079 was greater than PF-06270080,
with a mean AUC1 ratio of 1.66 (range: 1.59–1.76). The
mean metabolite:parent AUC1 ratios (%) for PF-006270079
and PF-06270080 were 14% and 8%, respectively. Values of
tmax for the lactam metabolites (range: 3–6 h) were similar to
those observed for crizotinib. The mean apparent t1/2 values
for PF-006270079 and PF-06270080 were similar (20.3 and
20.2 h, respectively).
Activities of crizotinib and its primary metabolitesagainst ALK and c-Met kinases
The inhibitory activity of crizotinib and its primary metab-
olites M2 (O-desalkyl crizotinib lactam, PF-06268935), M4
(O-desalkyl crizotinib, PF-03255243) and M10 (crizotinib
lactam, constituent diastereomers PF-06270079 and
PF-06270080) against phosphorylation of ALK fusion vari-
ants and c-Met was evaluated in cell lines expressing these
targets, as summarized in Table 6. Crizotinib was a potent
inhibitor of these target kinases in vitro, with mean EC50
values of 63 nM, 74 nM and 5 nM in cells lines expressing
EML4-ALK V1, EML4-ALK V3 and c-Met, respectively.
M10 (crizotinib lactam) was �2.5- to 7.7-fold less potent than
crizotinib against ALK and 2.5- to 4-fold less potent against
c-Met. One of the constituent diastereomers of M10
(PF-06270080) was more potent compared with the other
diastereomer (PF-06270079). The two O-desalkyl crizotinib
metabolites, M2 and M4, did not inhibit EML4-ALK or
c-Met activity in cell-based assays at concentrations up to
10 mM.
Safety summary
There were no serious adverse events, severe adverse events
or deaths during the clinical studies. In addition, there were no
permanent or temporary discontinuations due to adverse
events. No laboratory test results, vital signs or electrocar-
diogram abnormalities met the criteria for being clinically
relevant, and therefore, none were recorded as adverse events.
In the human absorption, distribution, metabolism and
excretion (ADME) study, all subjects experienced treatment-
related gastrointestinal adverse events; however, all adverse
events observed during the study were considered mild in
severity.
DOI: 10.3109/00498254.2014.941964 Crizotinib disposition in humans 11
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ON
NN
NH
HN
H
H
ON
NN
NH
NH
ON
NN
NH
NH2
H
GS
quinone-imine
HON
NN
NH
NH2
SHOOC
NH2
O
M9
ON
NN
NH
ClF
ClNH2
O O
OO
ON
NN
NH
NH2
O
SGH
HON
NN
NH
NH2
O
M9
SCOOH
NH2
M10 M2
ON
NN
NH
ClF
ClNH2
Crizotinib
GSH
(or)
−H2
GSH
[O] [O]
[O]
GSH
GSH
Figure 7. Proposed glutathione conjugation mechanism via a quinone imine intermediate leading to the formation of the cysteine conjugatemetabolite (M9).
Figure 8. Mean ( ± SD) concentration versus time profiles for crizotiniband the diastereomers of the lactam metabolite (M10), PF-06270079 andPF-06270080, in plasma after a single 250-mg oral dose of crizotinib tomale volunteers (n¼ 4).
Table 5. Pharmacokinetic parameters of crizotinib and the diastereomersof the lactam metabolite (M10), PF-06270079 and PF-06270080, inplasma after a single 250-mg oral dose of crizotinib to four healthy malesubjects.
Parameter Crizotinib PF-06270079 PF-06270080
Cmax (ng/mL) 124 (55) 32.5 (51) 20.7 (55)tmax (h) 5 (3–6) 5 (3–6) 5 (4–6)AUClast (ng�h/mL) 2948 (53) 443 (66) 272 (69)AUC1 (ng�h/mL) 3000 (53) 451 (66) 277 (69)t1/2 (h) 31.7 (15) 20.3 (11) 20.2 (35)MRAUC (%)a na 14 (18) 8 (22)
Arithmetic mean (percent CV); median (range) for tmax; n¼ 4.na¼ not applicable.aMetabolite-to-parent ratio based on AUC1, expressed as a percentage.
AUC1 values were corrected for the molecular weight of crizotnib andlactam metabolite diastereomers (i.e. ng�h/mL converted to nM�h).
12 T. R. Johnson et al. Xenobiotica, Early Online: 1–15
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Discussion
The utility of conducting human ADME studies with
radiolabeled drugs has been extensively reviewed (Beumer
et al., 2006; Penner et al., 2009; Roffey et al., 2007). The
major objectives of these studies include determination of:
(1) mass balance and routes of elimination, (2) circulatory and
excretory metabolites, (3) exposure of parent drug and
metabolites in circulation, (4) the potential contribution of
metabolites to the pharmacological/toxicological effects,
(5) clearance mechanisms and potential contributors to
interpatient variability and drug–drug interactions (DDIs)
and (6) the validity of the animal species used in toxicological
evaluation. All of these objectives were achieved for the
described investigation with crizotinib.
Following a single 250-mg/100 mCi dose of [14C]crizotinib
to healthy human subjects, the overall mean recovery of the
radioactive dose in the 20-day collection period was 85%,
with 78% recovering in the first 5 days. In a retrospective
analysis of human ADME studies with 27 radiolabeled
compounds, the overall mean (±SD) total recovery value
was 87 ± 10% (Roffey et al., 2007). Therefore, the extent of
recovery in the present study was well within this historical
range, suggesting that mass balance was achieved.
[14C]Crizotinib-related radioactivity was primarily recovered
in the feces after oral administration (63% of dose), consistent
with findings in nonclinical mass balance studies (Smith
et al., 2011).
Crizotinib represented �12% (AUClast) to 25% (Cmax) of
drug-related material in plasma. The radioactive profile of a
0–96 h Hamilton pool of plasma from all six subjects revealed
that unchanged crizotinib was the major circulating compo-
nent, representing 33% of radioactivity and crizotinib lactam
(M10) accounted for 10% of circulating radioactivity. No
other single circulating component accounted for >10% of
radioactivity in plasma over the time interval profiled. Other
minor metabolites included O-desalkyl metabolites and
related conjugates. The difference in the percentage of
drug-related material attributed to crizotinib using the two
orthogonal methods (i.e. 12% versus 33%) could potentially
be due to: (1) different time-intervals assessed in the two
methods (i.e. �312 h for AUClast versus 96 h for metabolite
profiling) and/or (2) non-extractable radioactivity in plasma
impacting the value obtained via metabolite profiling. In total,
�51% of the radioactivity in human plasma was accounted for
by crizotinib and identified metabolites. The remaining
radioactivity was attributed to multiple minor metabolites.
The low levels of radioactivity in plasma in the 0–96 h
Hamilton pool (�132 dpm/g) and the wide range of
metabolite polarity made identifying components with
510% abundance extremely challenging. This illustrates one
limitation of the Hamilton pooling technique when working
with low levels of radioactivity, because the sample is diluted
making low abundance metabolites even more difficult to
quantify and/or identify without resorting to high sensitivity
methods such as accelerator mass spectrometry.
Unchanged crizotinib was the major component in feces
(53% of dose). This significant recovery of unchanged drug in
the feces likely represents unabsorbed parent drug. Although
biliary excretion of intact crizotinib in humans cannot be
conclusively ruled out, unchanged crizotinib was not observed
in bile collected from rats following oral administration of
[14C]crizotinib (Smith et al., 2011). Therefore, biliary excre-
tion of unchanged crizotinib is not anticipated to be a
significant clearance pathway in humans, as rats are typically
considered to excrete xenobiotics into bile more readily than
humans (Kwon, 2001). Moreover, intestinal deconjugation of
direct glucuronide metabolites of crizotinib and/or reduction
of crizotinib N-oxide is not anticipated to play a significant
role in crizotinib clearance, as these metabolites were not
identified in the present study or in rats or dogs (Smith et al.,
2011). In urine, an average of �2% of the dose was excreted
as unchanged crizotinib based on radiochromatographic or
LC–MS/MS quantification methods. The major excreted
component in urine (4.5% of dose) was the sulfate conjugate
of O-desalkyl crizotinib lactam (M8). The remaining radio-
activity in urine was attributed to multiple minor metabolites,
each accounting for51% of the administered dose.
Upon oxidation of crizotinib to crizotinib lactam (M10), a
new chiral center is introduced, resulting in the formation of
two possible diastereomers of M10, PF-06270079 and
PF-06270080. In plasma samples from a subset of subjects
(n¼ 4) administered a single oral 250-mg dose of crizotinib in
a relative bioavailability study, PF-06270079 exposure
Table 6. In vitro pharmacodynamic inhibition of target tyrosine kinases and fusion variants by crizotinib and the diastereomers of the lactam metabolite(M10), PF-06270079 and PF-06270080 and corresponding PAIs.
Cellular kinase assay Compound EC50 (nM)a nb EC50 M:P ratioc,d p Valued PAI (%)e
EML4-ALK V1 phosphorylation Crizotinib 63 ± 31 20PF-06270079 284 ± 182 7 3.7 50.0001 2PF-06270080 194 ± 118 6 2.5 50.0001 2
EML4-ALK V3 phosphorylation Crizotinib 74 ± 23 6PF-06270079 554 ± 102 3 7.5 50.0001 1PF-06270080 355 ± 66 3 5.0 0.0026 1
c-Met phosphorylation Crizotinib 5.0 ± 3.8 164PF-06270079 18.5 ± 7.3 3 4.0 0.0065 2PF-06270080 11.8 ± 2.7 3 2.5 0.0308 2
aMean ± SD.bNumber of replicate kinase inhibition experiments.cMetabolite-to-parent ratio for EC50.dRatios and p values were derived by comparing EC50 values of crizotinib versus metabolites within the same experiment.ePAI expressed as a percentage. For calculation of unbound exposures, fraction unbound in human plasma was 0.093 for crizotinib (Yamazaki et al.,
2011). For PF-06270079 and PF-06270080, fraction unbound values in human plasma were 0.055 and 0.059, respectively (unpublished data).
DOI: 10.3109/00498254.2014.941964 Crizotinib disposition in humans 13
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exceeded that of PF-06270080 (mean AUC ratio of 1.66;
range: 1.59–1.76). The AUC values of PF-06270079 and PF-
06270080 represented 14% and 8%, respectively, of the AUC
of crizotinib. Therefore, the overall ratio of M10 to crizotinib
based on this exploratory analysis (22%) was in general
agreement with the ratio of derived from the radioactive
plasma profile (30%). In vitro, PF-06270079 and
PF-06270080 were �2.5- to 7.7-fold less potent than
crizotinib in ALK and c-Met cellular pharmacodynamic
assays. O-Desalkyl crizotinib (M4, PF 03255243) and
O-desalkyl crizotinib lactam (M2, PF 06268935) were
inactive against both ALK and c-Met kinases
(EC50> 10 mM). This finding is consistent with the known
SAR for crizotinib, where the 3-benzyloxy group is known to
be a critical structural feature for binding to the hydrophobic
pocket of the c-Met kinase (Cui et al., 2011). It has been
proposed that a PAI, vide supra, of >25% can be used as
a criterion to assess the significance of contribution of a
metabolite to the pharmacology of the parent drug (Leclercq
et al., 2009). The PAI values for both PF-06270079 and
PF-06270080 are estimated to be �2% (Table 6). Based on
this preliminary assessment, neither metabolite is anticipated
to contribute significantly to the pharmacological activity of
crizotinib. However, the limitations of this exploratory
assessment include data obtained from a low number of
healthy subjects following a single 250-mg crizotinib dose.
Given that crizotinib exhibits time-dependent PK, likely due
to auto-inhibition/inactivation of CYP3A, and is also primar-
ily metabolized by CYP3A, subsequent studies have included
the measurement of plasma M10 concentrations following
multiple doses to better evaluate its potential contribution to
in vivo activity at steady state.
The long-lived radioactivity in human plasma, relative to
parent drug and the incomplete extraction recovery of total
radioactivity from plasma (�60%) may suggest the potential
for a proportion of crizotinib-derived radioactivity to bind to
plasma proteins and/or tissue macromolecules. In addition,
the observation of a trace cysteine conjugate metabolite (M9),
a possible downstream metabolite of a glutathione adduct of
M2, in urine suggests the potential for a low level of
bioactivation of crizotinib in vivo (Figure 7). To date, the
nature of this apparent binding has not been fully character-
ized. In this study, the mean recovery of total radioactivity
was 85% of the dose, indicating that any apparent binding to
macromolecules would comprise only a small fraction of the
total crizotinib dose (Roffey et al., 2007).
Based on the excretion profile, where a majority of the
radioactive dose was recovered in feces with limited urinary
recovery of unchanged crizotinib, and the identity of metab-
olites, the primary elimination pathway for crizotinib in
humans appears to be via hepatic clearance, specifically,
oxidative metabolism. In vitro studies demonstrated that
crizotinib was predominantly metabolized by CYP3A, which
also mediated the formation of the major oxidative lactam
(M10) and O-desalkyl (M4) metabolites to a significant extent
(Figure 6) (Johnson et al., 2011). Based on these in vitro data,
the fraction of systemically available crizotinib metabolized
by CYP3A (fm,CYP3A) was estimated to be �0.8. In humans, a
major role for CYP3A in the clearance of crizotinib was
confirmed in clinical studies with the potent CYP3A inhibitor
ketoconazole and inducer rifampin (Xu et al., 2011b,c). Using
the fm,CYP3A value derived in vitro, the observed magnitude of
the interaction between crizotinib and ketoconazole (AUC
ratio¼ 3.2) or rifampin (AUC ratio¼ 0.18) was reasonably
predicted using mechanistic static models (Ito et al., 1998;
Johnson et al., 2011; Shou et al., 2008). Thus, the under-
standing of the major clearance mechanism and metabolic
pathways derived from the present study were consistent with
findings from clinical DDI studies.
Circulating metabolites observed in humans were also
observed in at least one of the nonclinical safety species, rats
and dogs, confirming that the choice of these species for
nonclinical safety assessment was acceptable (Smith et al.,
2011). Qualitatively, the metabolism of crizotinib in rats was
representative of that observed in humans. Quantitatively,
plasma exposure of the M10 diastereomers in the rat was
similar to or exceeded human exposure (unpublished data).
Conclusion
The disposition of crizotinib has been characterized in healthy
subjects to augment understanding of its complex PK. After a
single oral dose of [14C]crizotinib, mass balance was
achieved, with a majority of drug-related radioactivity
recovered in the feces. The primary clearance pathway for
crizotinib is hepatic elimination/oxidative metabolism, and
the major metabolic pathways in humans were oxidation of
the piperidine ring to crizotinib lactam (M10) and
O-dealkylation, with subsequent conjugation of O-desalkyl
metabolites. Unchanged crizotinib and M10 were the princi-
pal drug-related components in circulation. Exploratory
assessments suggest that M10 is unlikely to be a major
contributor to crizotinib’s pharmacological activity.
Definitive assessments of the clinical relevance of M10 are
ongoing.
Acknowledgements
We thank personnel from Covance Laboratories Inc.
(Madison, WI and Indianapolis, IN) for radioanalysis support
of the human ADME study and for quantitation of crizotinib
in human plasma and urine samples; Yao (Grace) Ni for
facilitating clinical study conduct; Chunze Li for preliminary
pharmacokinetic analyses; Nicoletta Brega for clinical sup-
port; and Klaas Schildknegt for preparation of radiolabeled
crizotinib. The support of these colleagues is gratefully
acknowledged.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the writing and content of this article.
These studies were sponsored by Pfizer Inc. All authors are
employees of Pfizer Inc. or were at the time of conducting
these studies.
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