Metabolism, excretion and pharmacokinetics of [ 14 C]crizotinib following oral administration to healthy subjects

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  • http://informahealthcare.com/xenISSN: 0049-8254 (print), 1366-5928 (electronic)

    Xenobiotica, Early Online: 115! 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. OGorman4, 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 250mg/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 096 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 37%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 dOrigine 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: theodore.johnson@pfizer.com

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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 concentrationtime 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) weresynthesized 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 aninformed 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 dietarysupplement 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 positiveserology 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: 115

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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 additionof 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 orbelow 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. Plasmasamples were mixed, and single-weighed aliquots (1 g) wereanalyzed 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 spec...

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