In Vitro Hepatic Metabolism of Cediranib, a Potent ...dmd.aspetjournals.org/content/dmd/early/2010/07/15/dmd.110.033159... · DMD #33159 3 Abstract The in vitro metabolism of cediranib,

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In Vitro Hepatic Metabolism of Cediranib, a Potent Vascular Endothelial

Growth Factor Tyrosine Kinase Inhibitor: Interspecies Comparison and Human

Enzymology

Timothy Schulz-Utermoehl1, Michael Spear1, Christopher R.J. Pollard, Christine Pattison,

Helen Rollison, Sunil Sarda, Michelle Ward, Nick Bushby, Angela Jordan & Mike Harrison

Clinical Pharmacology & DMPK Department, Alderley Park, AstraZeneca UK Ltd,

Macclesfield, Cheshire SK10 4TG, United Kingdom.

DMD Fast Forward. Published on July 15, 2010 as doi:10.1124/dmd.110.033159

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on July 15, 2010 as DOI: 10.1124/dmd.110.033159

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Running title: In Vitro Hepatic Metabolism of Cediranib

Corresponding Author:

Dr Timothy Schulz-Utermoehl, Clinical Pharmacology and DMPK Department, Alderley

Park, AstraZeneca UK Ltd, Macclesfield, Cheshire SK10 4TG, United Kingdom.

Telephone: +44-1625-233632

Fax: +44-1625-230614

Email: [email protected]

Number of Text Pages : 35

Number of Tables : 3

Number of Figures : 6

Number of References : 29

Number of Words

Abstract : 248

Introduction : 232

Discussion : 1065

Non-standard abbreviations: 1-ABT, 1-aminobenzotriazole; CRC, colorectal cancer; HLM,

human liver microsomes; HPLC-MS, high performance liquid chromatography-mass

spectrometry; KIT, stem cell factor receptor; TKI, tyrosine kinase inhibitor; UGT; uridine

glucuronosyl transferase; UDPGA, uridine diphosphate glucuronic acid; VEGF, vascular

endothelial growth factor


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Abstract

The in vitro metabolism of cediranib, a vascular endothelial growth factor (VEGF) tyrosine

kinase inhibitor (TKI) of all three VEGF receptors in late stage development for the treatment

of colorectal cancer and recurrent glioblastoma was investigated in hepatic proteins from

pre-clinical species and humans using radiolabelled material. In human hepatocyte cultures,

oxidative and conjugative metabolic pathways were identified, with pyrrolidine

N+-glucuronidation being the major route. The primary oxidative pathways were di-and

tri-oxidations and pyrrolidine N-oxidation. All metabolites with the exception of the

N+-glucuronide metabolite were observed in rat and cynomolgus monkey hepatocyte

preparations. Additional metabolism studies in liver microsomes from these or other

preclinical species (CD-1 mouse, Han Wistar rat, Dunkin Hartley guinea-pig, Göttingen

mini-pig, New Zealand White rabbit, Beagle dog, cynomolgus and Rhesus monkey) indicated

that the N+-glucuronide metabolite was not formed in these additional species. Incubations

with recombinant flavin-containing monooxygenase (FMO) and uridine

glucuronosyltransferase (UGT) enzymes and inhibition studies using the non-selective

cytochrome P450 (CYP450) chemical inhibitor 1-aminobenzotriazole (1-ABT) in human

hepatocytes indicated that FMO1 and FMO3 contributed to cediranib N-oxidation, whilst

UGT1A4 had a major role in cediranib N+-glucuronidation. CYP450 enzymes contributed

only a minor role to the metabolism of cediranib. In conclusion, species differences in the

formation of the N+-glucuronide metabolite of cediranib were observed. All other

metabolites of cediranib found in humans were also detected in rat and cynomolgus monkey.

Non-CYP450 enzymes are predominantly involved in the metabolism of cediranib and this

suggests that clinical drug interactions involving other co-administered drugs are unlikely.


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Introduction

Cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-

pyrrolidinyl)propoxy]quinazoline; RECENTIN™; AZD2171)) (Fig. 1) is a highly potent

vascular endothelial growth factor (VEGF) inhibitor, with activity against all three VEGF

receptor tyrosine kinases, and stem cell factor receptor (KIT). It is currently in Phase III

clinical trials for the treatment of colorectal cancer (CRC) and recurrent glioblastoma. VEGF

is involved in the regulation of key processes throughout the angiogenic cascade, and over-

production of VEGF in tumours facilitates tumour progression by stimulating angiogenesis

and increasing vascular permeability. Inhibition of VEGF receptor tyrosine kinases stabilizes

the progression of tumours by disrupting tumour-induced angiogenesis (Kim et al., 1993).

Pre-clinical studies have shown that cediranib prevents VEGF-induced angiogenesis in vivo

and inhibits the growth of established human tumour xenografts in athmic mice in a

dose-dependent manner (Wedge et al., 2005). In the clinic, cediranib has exhibited activity in

patients with advanced CRC and recurrent glioblastoma (Batchelor et al., 2007; Drevs et al.,

2007; Chen et al., 2009).

To gain an understanding of the species differences in the disposition of cediranib and

pre-clinical coverage of human metabolites, the hepatic metabolism of [14C]-cediranib was

investigated in liver preparations from preclinical species and human. The in vitro metabolic

profiles and identity of cediranib metabolites were assessed and a proposed metabolic

pathway suggested. The identity of the hepatic human Phase I and II enzymes involved in

the oxidation and conjugation of cediranib were confirmed and are reported here.


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Materials and Methods

Chemicals & Materials. Dexamethasone, insulin, penicillin-streptomycin, Williams’ E

media, β-NADPH, alamethicin, D-(+)-glucuronic acid, acetaminophen, 1-aminobenzotriazole

(1-ABT), DMSO-d6, [14C]-7-ethoxycoumarin (specific activity, 56 mCi/mmol) and

[14C]-acetaminophen (specific activity, 5.4 mCi/mmol) were purchased from Sigma-Aldrich

(Poole, UK). 10 % foetal bovine serum, gentamycin, Leibovitz L15 media and HEPES buffer

were purchased from Invitrogen (Paisley, UK). Matrigel was obtained from BD Biosciences

(San Jose, CA). Ultima Flo-M scintillation cocktail was purchased from Packard Instruments,

(Pangbourne, UK). [14C]-cediranib maleate (4-[(4-fluoro-[2-14C]-2-methyl-1H-indol-5-

yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline maleate) (batch 1R4, specific

activity of 26.9 mCi/mmol; batch 1R5, specific activity of 32.9 mCi/mmol; batch SEL/1713

(1R6), specific activity of 59 mCi/mmol; batch 016, specific activity of 56 mCi/mmol) was

synthesised by the Isotope Chemistry Group at AstraZeneca UK Ltd (batches 1R4, 1R5 and

016; Macclesfield, UK) or by Selcia Limited (batch SEL/1713 (1R6); Ongar, UK). The

radiochemical purity of 1R4, 1R5, 1R6 and 016 was >98, >98, 99 and >98%, respectively.

An authentic metabolite standard, cediranib pyrrolidine N-oxide, was synthesised by

AstraZeneca UK Ltd (Macclesfield, UK). The N+-glucuronide of cediranib was

biosynthesised by Novacta Biosystems (Welwyn Garden City, UK). Fisher Scientific

supplied acetonitrile and methanol. All other chemicals or solvents were purchased from

commercial suppliers and were of analytical grade or the best equivalent.

Hepatocytes. Viable fresh hepatocytes were isolated from male Han-Wistar rats

(AstraZeneca Alderley Park; Macclesfield, UK) using the standard collagenase perfusion

technique. Freshly isolated human hepatocytes from 2 male (donor 1 - unknown race aged

39; donor 3 - Asian, aged 38) and 3 female donors (donor 2 - unknown race, aged 68; donor 4


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- Caucasian, aged 60 years and donor 5 - unknown race, aged 45 years) were purchased from

the United Kingdom Human Tissue Bank (UKHTB; Leicester, UK). All human liver-derived

samples were obtained with permission from the relevant local ethical committees. Freshly

isolated cynomolgus monkey hepatocytes were purchased from Covance (Harrogate, UK).

Cell yield and viability were assessed using Trypan Blue.

Liver Microsomes. Pooled human liver microsomes (28 male donors and 5 female donors;

lot 63103) were obtained from BD Biosciences (San Jose, CA). Pooled liver microsomes

from cynomolgus monkey (8 male donors; lot 3), Dunkin-Hartley guinea-pig (8 male donors;

lot 12635), Göttingen mini-pig (3 male donors; lot 71911), Rhesus monkey (4 male donors;

lot 1), New Zealand White rabbit (3 male donors; lot 98358), CD-1 mouse (150 male donors;

lot 80662), beagle dog (20 male donors; lot 11969) and Han-Wistar rat (30 male donors;

lot 37975) were purchased from BD Biosciences (San Jose, CA). These samples were stored

at -80°C in a controlled tissue collection until use.

Recombinant Enzymes. Samples of microsomal fractions prepared from insect cells

transfected with plasmid vectors expressing human UGT1A1 (lot 13), UGT1A3 (lot 13),

UGT1A4 (lots 9, 10, 11, 34582 and 59840), UGT1A6 (lot 10), UGT1A7 (lot 41931),

UGT1A8 (lot 36867), UGT1A9 (lot 8), UGT1A10 (lot 51067), UGT2B4 (lot 4), UGT2B7

(lot 55297), UGT2B15 (lot 8) and UGT2B17 (lot 4) were purchased from BD Biosciences

(San Jose, CA). Microsomes prepared from insect cells that expressed human FMO1

(lot 52151), FMO3 (lot 64186) and FMO5 (lot 56381) and microsomes from cells that

contained the empty transfection vector were also obtained from BD Biosciences.


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Characterisation of [14C]-cediranib. Stock solutions of [14C]-cediranib were diluted in

either 0.2% (v/v) formic acid in water: 0.2% formic acid in acetonitrile 95:5 (v/v) or

water: acetonitrile 80:20 (v/v) to concentrations in the range of 74 to 100 µM (dependent on

specific activity of the batch used for each experiment), before the radiochemical purity was

assessed by HPLC with radiochemical detection (HPLC-RAD).

Hepatocyte Suspension Incubations. Human hepatocytes (donors 1, 2 and 3; approximately

1 X 106 cells/mL) were pre-incubated at 37°C in Leibovitz L15 media in the absence and

presence of 1-aminobenzotriazole (1-ABT; 1 mM final) for 15 min prior to incubation with

[14C]-cediranib. [14C]-cediranib (prepared as a 3 mM solution in methanol) was diluted 1 in

300 with the microsomal suspension to achieve a final concentration of 10 µM (arbitrary

concentration). Incubations with [14C]-cediranib were maintained at 37°C for up to 4 hours

in a shaking water bath and were terminated with an equal volume of ethanol. Appropriate

control incubations were also performed. The positive control compound, [14C]-7-

ethoxycoumarin (20 µM), was incubated in parallel to [14C]-cediranib incubations. All

samples were vortex mixed, centrifuged at 14171 g for up to 10 min and the resulting

supernatants stored at –20°C until analysis by HPLC with radiometric (RAD) and mass

spectrometry detection (MSn). Assays were performed in duplicate.

Hepatocyte Culture Incubations. Incubations of hepatocytes (donors 4 and 5) with

[14C]-cediranib were based on the method described previously (LeCluyse et al., 2000) with

minor modifications. Hepatocytes (132,000 viable cells/cm2) were plated onto 12 well plates

(growth areas of 3.8 cm2 per well) pre-coated with collagen I (BD Biosciences, San Jose, CA)

and sandwiched with a top layer of Matrigel (250 µg/mL). Following a 12 to 24 hour

stabilisation period, hepatocytes were incubated with [14C]-cediranib (10 µM final (arbitrary


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concentration); initially dissolved in methanol as a 3 mM stock solution) in Williams’ E

media containing dexamethasone (30 nM), gentamycin (50 µg/mL), HEPES buffer (10 mM),

insulin (170 nM) and penicillin-streptomycin (102 U/mL; 100 µg/mL). The total incubation

volume was 1 mL. Hepatocytes were incubated for up to 72 hours (24 hours for donor 4) at

37°C in an atmosphere of 5 % CO2 in air. Reactions were terminated with one half volume

of ethanol. Appropriate control incubations were also performed. The positive control

compound, [14C]-7-ethoxycoumarin (20 µM), was incubated in parallel to [14C]-cediranib

incubations. Samples were vortexed and centrifuged 14171 g for 10 min to remove

precipitated protein/cell pellets, and the resulting supernatants were stored at –20°C until

analysis by HPLC with radiometric (RAD) and mass spectrometry detection (MSn). Assays

were performed in duplicate.

Hepatic Subcellular Fractions Incubations. [14C]-cediranib (10 µM final (arbitrary

concentration); initially dissolved in methanol as a 2 mM stock solution) was incubated with

human liver microsomes, cytosol and S9. The incubations were carried out at a protein

concentration of 1 mg/mL in phosphate buffer (100 mM; pH 7.4), with 1 mM NADPH or

NAD. Incubations were maintained at 37°C for up to 120 min in a shaking water bath and

terminated with an equal volume of ethanol. All samples were centrifuged and stored in the

dark at -20°C until analysis by HPLC with radiometric detection.

Phase II Hepatic Microsomal and Recombinant UGT Enzyme Incubations. Incubations

(1 mL) of liver microsomes from human, cynomolgus monkey, Dunkin-Hartley guinea-pig,

Göttingen mini-pig, rhesus monkey, New Zealand White rabbit, CD-1 mouse, beagle dog and

Han-Wistar rat (all at 1 mg protein/mL) with [14C]-cediranib (10 µM final (arbitrary

concentration); initially dissolved in methanol as a 3 mM stock solution) were conducted in


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phosphate buffer (100 mM; pH 7.4) containing 3.3 mM magnesium chloride (MgCl2) and

50 µg alamethicin/mg protein. The solvent attributable to the alamethicin was evaporated to

dryness under a stream of nitrogen at 35°C in the incubation tubes prior to the addition of the

remaining pre-incubation components. After a 5 min pre-incubation period, the reactions

were initiated by the addition of UDPGA (4 mM final). Following a 60-min incubation at

37°C, aliquots were removed into an equal volume of ethanol. Appropriate control

incubations were also performed. The positive control compound, [14C]-acetaminophen

(10 µM), was incubated in parallel to [14C]-cediranib incubations. Samples were centrifuged

(14171 g) for 10 min to remove precipitated protein and the resulting supernatant stored at

-80°C until subjected to analysis by HPLC-RAD and HPLC-MSn. Assays were performed in

duplicate.

For incubations with individual recombinant UGT enzymes, the incubations mixtures

contained the same components, except that a final protein concentration of

0.25 mg UGT protein/mL was used. In addition, following a 60-min incubation at 37°C,

aliquots were removed into an equal volume of 0.05% formic acid in ethanol.

Recombinant FMO Enzyme Incubations. Microsomes prepared from insect cell lines that

heterologously expressed human FMO1, FMO3 and FMO5 (all at 0.2 mg/mL) were

incubated with [14C]-cediranib (5 µM final (arbitrary concentration); initially dissolved in

methanol as a 1 mM stock solution) in phosphate buffer (100 mM; pH 7.4), with 1 mM

NADPH. Incubations were maintained at 37°C for 120 min and terminated with an equal

volume of ethanol. Additionally, control incubations were performed with microsomes from

cells that contained the empty transfection vector. All samples were centrifuged and stored in

the dark at -20°C until analysis by HPLC with radiometric detection.


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Enzyme Kinetics of [14C]-cediranib N-oxidation in recombinant FMO1 and FMO3

Enzymes. The N-oxidation of [14C]-cediranib was conducted under linear conditions with

respect to incubation time and protein concentration. A range of concentrations (0.2 to

50 µM) of [14C]-cediranib was incubated with either 0.02 mg/mL FMO1 or 0.2 mg/mL

FMO3 protein in phosphate buffer (100 mM; pH 7.4) for 10 (FMO1) or 20 min (FMO3) at

37°C in the presence of NADPH (1 mM). Incubations were terminated by the addition of an

equal volume of ethanol followed by centrifugation at 14171 g for 10 min. The resultant

supernatants were stored at –20°C until analysis by HPLC with radiochemical detection.

Metabolite Profiling by HPLC-RAD. [14C]-cediranib and its metabolites were separated on

an ACE AQ column (4.6 X 150 mm, 5 µm; Advanced Chromatography Technologies (ACT),

Aberdeen, UK) using Perkin Elmer 200 Series pumps (Beaconsfield, UK) at a flow rate of 1

mL/min. The aqueous mobile phase (solvent A) consisted of 0.2% (v/v) formic acid in

deionised water while the organic mobile phase (solvent B) consisted of 0.2% (v/v) formic

acid in acetonitrile. The initial mobile phase consisted of 5% solvent B, which was increased

to 20% over a period of 15 min, then further increased to 25% in 20 min, maintained for

25 min, and finally to 50% over 5 min. This value was held for 10 min before returning to

5% solvent B for column equilibration for 10 min before the next injection. Radiochemical

detection was performed using a Packard Radiomatic Flo-One Beta 500 TR series detector

(Packard Instruments, Pangbourne, UK) with a 500 µL liquid flow cell and Ultima Flo-M

scintillation cocktail (Packard Instruments, Pangbourne, UK) running at 3 mL/min. The data

were collected and analysed using either FLO-ONE version 3.65 (Packard Instruments,

Pangbourne, UK) or LAURA version 3.4.1.10e (Lablogic Systems Ltd, Sheffield, UK).

Recovery of sample radioactivity following centrifugation of the quenched incubates was


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determined prior to injection onto the HPLC system. To ensure the system suitability, the

recovery of radioactivity from the column was evaluated by comparing the radioactive

content (determined by liquid scintillation counting) of the post-column eluent to the pre-

column eluent. No loss of radioactivity on the column was found for any of the incubated

samples.

For the UGT and FMO phenotyping experiments and Phase II microsomal incubate samples,

a shorter HPLC gradient was used. [14C]-cediranib and its metabolites were separated on a

Polaris C18-A column (4.6 X 150 mm, 5 µm, Varian, Yarnton, UK) using either Jasco

PU-1580 Intelligent (Jasco (UK), Great Dunmow, UK) or Agilent 1200 Series (Agilent

Technologies UK Ltd, Stockport, UK) HPLC pumps. The initial mobile phase consisted of

5% solvent B, which was maintained for 2 min, increased to 25% over a period of 4.5 min,

further increased to 29% in 15.5 min, to 33% in 4 min, and finally to 95% in 1.5 min. This

value was held for 4.5 min before returning to 5% solvent B for column equilibration for

4.9 min before the next injection.

[14C]-acetaminophen and its metabolites were separated on an Eclipse XDB C18 column

(4.6 X 150 mm, 5 µm; Agilent Technologies UK Ltd, Stockport, UK) using Agilent 1200

Series HPLC pumps (Stockport, UK) with a flow rate of 1 mL/min. The aqueous mobile

phase (solvent A) consisted of ammonium acetate in deionised water (10 mM; pH 5.5) while

the organic mobile phase (solvent B) consisted of methanol. The initial mobile phase

consisted of 5% solvent B, which was maintained for 15 min and increased to 95% over a

period of 1 min. This value was returned to 5% solvent B within a 1 min for column

equilibration for 3 min before the next injection.


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[14C]-7-ethoxycoumarin and its metabolites were separated on a Zorbax SB-C8 column

(4.6 X 150 mm, 5 µm; Agilent Technologies UK Ltd, Stockport, UK) using Jasco PU-2080

Intelligent HPLC pumps (Jasco (UK), Great Dunmow, UK) with a flow rate of 1 mL/min.

The aqueous mobile phase (solvent A) consisted of ammonium acetate in deionised water

(10 mM; pH 5.5) while the organic mobile phase (solvent B) consisted of methanol. The

initial mobile phase consisted of 10% solvent B, which was increased to 17% over a period of

7 min, further increased to 29% in 2min, to 55% in 10 min and finally to 70% in 6 min. This

value was returned to 10% solvent B within a 5 min for column equilibration for 5 min before

the next injection.

Metabolite identification by HPLC-MSn. [14C]-Cediranib and its metabolites were

separated on a Polaris C18 column (4.6x150 mm, 5 μm, Varian, Yarnton, UK) using Agilent

HP1200SL binary pumps (Agilent Technologies UK Ltd, Stockport, UK) with a flow rate of

1 mL/min. The aqueous mobile phase (solvent A) consisted of 0.2% formic acid in deionised

water while the organic mobile phase (solvent B) consisted of 0.2% formic acid in

acetonitrile. Mass spectrometry data were collected either using Xcalibur version 2.0

(Thermo Fisher Scientific, Waltham, MA) on a Thermo Finnigan LTQ ion trap instrument

(Thermo Fisher Scientific, Waltham, MA) fitted with an electrospray ionisation (ESI) source

operating in positive mode under optimised conditions (voltage 3.5 kV; sheath gas nitrogen,

collision energy 20 eV, capillary temperature 350 °C), or using the MassLynx v4.0 software

(Waters) on a Waters Micromass Quattro MicroTM API instrument (Waters, Elstree, UK)

fitted with an electrospray ionisation (ESI) source operating in positive ion mode under

optimised conditions (cone voltage 20 V; collision energy 20 eV).

Metabolite Identification/Characterisation by 1H NMR spectroscopy. 1H NMR spectra

were acquired on a Bruker DRX500 NMR spectrometer operating at 500 MHz resonance


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frequency). 1H NMR data were collected and analysed using Topspin software (version 2.0).

Cediranib pyrrolidine N-oxide was reconstituted in DMSO-d6.

Data Analysis. The assessment of the metabolism of [14C]-cediranib was made by both

qualitative and quantitative analysis of the chromatographic patterns. Radiolabelled

components were assumed to have the same specific activity as parent molecule.

MSn data were obtained for the metabolites identified in the analysis by HPLC-MSn with

radiometric detection. Components were identified as being derived from [14C]-cediranib if

they demonstrated elements of the characteristic isotopic and fragmentation patterns observed

with the parent molecule.

Enzyme kinetic parameters (Km and Vmax) were determined by non-linear regression using

Grafit version 5.0.10 (Erithacus Software Limited; Horley, UK).


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Results

Metabolism profile of [14C]-cediranib in hepatocytes. The metabolic profiles of

[14C]-cediranib in human hepatocyte suspension and culture are highlighted in Figures 2 and

3, respectively. In human hepatocyte suspension incubations, only one major metabolite,

M3, was detected, which accounted for up to 10.8% of total chromatogram radioactivity

(Figure 2A; Table 1). Minor components included metabolites M1, M2 and M4. The

formation of M3 as well as M1 and M4 were not inhibited in the presence of the

non-selective CYP450 chemical inhibitor, 1-ABT (Figure 2B).

Consistent with the findings in human hepatocyte suspensions, [14C]-cediranib was

metabolised to these 4 components as well as to two further minor metabolites M5 and M7,

in human hepatocyte cultures (Figure 2C; Table 1). With the exception of metabolite M3, all

other metabolites detected in human hepatocytes were also observed in either rat and/or

cynomolgus monkey hepatocyte cultures (Figure 3). Unlike human, M4 was the major

metabolite in both rat and cynomolgus hepatocyte incubations and accounted for 30 and 9%,

respectively, of total chromatogram radioactivity. Metabolites M8, M10 and M11 were

detected in rat and cynomolgus monkey hepatocyte samples but not in human hepatocytes,

whilst metabolites M6 and M10 were detected only in cynomolgus monkey hepatocytes

(Table 1). The mass spectral data for cediranib and its metabolites are summarised in

Table 2.

The recovery of sample radioactivity from the HPLC column for human hepatocyte

suspension (donor 3) and culture (donor 5) incubations was 102 and 99.3%, respectively. For

rat and cynomolgus monkey hepatocyte cultures, these values were 98.5 and 94.3%,

respectively.


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Metabolite identification in hepatocytes. Based on mass spectrometric data, metabolite M3

showed a [M+H]+ ion of m/z 629 corresponding to a gain of 176 atomic mass units (amu)

from [14C]-cediranib (m/z 453) (Table 2), consistent with the addition of a glucuronic acid

moiety to the molecule. The exact site of conjugation in metabolite M3 was confirmed by

LC-MSn and [1H]-NMR, which indicated conjugation on the pyrrolidine nitrogen (Lenz et al.,

(2010)). The proton NMR chemical shifts and coupling constants of a bio-synthesised

standard of metabolite M3 are included in Table 3.

For metabolite M4, an [M+H]+ ion at m/z 469 was characteristic of mono-oxygenation. With

a major fragment at m/z 128, corresponding to a gain of 16 amu on the propyl pyrrolidinyl

moiety (m/z 112), this suggested that oxidation occurred on this group (Table 2). The

synthetic standard for cediranib pyrrolidine N-oxide showed a similar retention time and mass

fragmentation profile, thus confirming that the structural identity of metabolite M4 and

cediranib N-oxide were identical. The proton NMR chemical shifts and coupling constants of

the synthetic standard for cediranib pyrrolidine N-oxide (M4) are included in Table 3.

The mass spectrum of metabolite M7 showed an [M+H]+ ion at m/z 483, 30 amu higher than

that of protonated [14C]-cediranib. With two major fragments at m/z 356 and 372,

corresponding to increments of +30 amu on the fluoro-indole quinazoline moiety (m/z 326

and 342 respectively), this suggested that oxidations had occurred on this group, resulting in

the formation of the carboxylic acid M7 (Table 2).


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Metabolites M1, M2 and M5 had mass spectral profile characteristics of di- and

tri-oxygenations, although precise positional assignment of these oxidations was not possible

based on the MSn fragmentation data.

The mass spectrum of metabolite M8 showed an [M+H]+ ion at m/z 248, 205 amu less than

that of protonated [14C]-cediranib. The MS/MS of protonated M8 produced one major

fragment at m/z 168, corresponding to the loss of a sulfate moiety. The results suggest that

metabolite M8 is a fluoro-indole monooxygenated sulfate.

Metabolite M11 displayed an [M+H]+ ion of m/z 414 corresponding to a loss of 39 amu from

[14C]-cediranib (m/z 453) (Table 2), and a predominant MS/MS fragment at m/z 342. This is

consistent with the loss of the pyrrolidine ring and oxidation to form the propanoic acid.

No molecular ion assignments were possible for metabolites M6, M9, M10 and M12.

Oxidative metabolic profiles of [14C]-cediranib in human hepatic microsomal fractions.

Minor metabolism (<12% depletion of [14C]-cediranib total chromatogram radioactivity) was

observed following incubation of [14C]-cediranib with human liver microsomes and S9 in the

presence of NADPH (data not shown). Up to five components were observed, however the

structure of only one could be elucidated. This identified component in S9 accounted for

6.7 % of the total radiochromatogram radioactivity and the mass fragmentation profile was

consistent with that for metabolite M4.

The oxidative metabolites M1, M2 and M5 that were detected in human hepatocytes were not

formed in the presence of NADPH in either human liver microsomes or S9. No metabolism


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was observed in the human cytosolic incubate samples. Similarly, no metabolites were

detected in the incubation of [14C]-cediranib with human liver microsomes or S9 in the

presence of the cofactor, NAD.

Involvement of FMO enzymes in the metabolism of [14C]-cediranib. A role for

flavin-containing monooxygenase (FMO) enzymes in the metabolism of [14C]-cediranib was

anticipated based on the role of these enzymes in N-oxidation reactions (Cashman, 1995) and

on the inability of the non-selective CYP inhibitor, 1-ABT, to inhibit the formation of the

N-oxide metabolite of [14C]-cediranib (metabolite M4) in human hepatocytes (Figure 2).

Incubation of [14C]-cediranib (5 μM) with microsomes prepared from cell lines expressing a

single FMO isoform indicated that in the presence of FMO1, extensive metabolism of

[14C]-cediranib was observed, with only metabolite M4 (accounting for 85.0% of the total

chromatogram radioactivity), being generated (data not shown). Similarly for FMO3, only

metabolite M4 was detected, which accounted for 60.6% of the total chromatogram

radioactivity. FMO5 and insect control enzymes displayed no catalytic activity for the

formation of metabolite M4.

Enzyme kinetic experiments for [14C]-cediranib N-oxide (metabolite M4) in recombinant

FMO1 showed that the rate of formation conformed to Michaelis-Menten kinetics

characterised by values for apparent Km of 11.9 µM and Vmax of 8.2 nmol equiv/min/mg

protein (Figure 4). The kinetic parameters for metabolite M4 in recombinant FMO3 could

not be calculated, as the rate of formation did not reach Vmax over the concentration range

tested (Figure 4). Solubility limitations prevented the use of higher [14C]-cediranib

concentrations.


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Phase II metabolic profiles of [14C]-cediranib in liver microsomes. Human liver

microsomal incubations containing [14C]-cediranib and UDPGA resulted in the formation of

a single metabolite (M3; 15.2% of total chromatogram radioactivity), as observed by HPLC

with radiochemical detection (results not shown). A small component representing

approximately 1% of the total chromatogram radioactivity with a retention time of 27.5 min

was also detected in the HLM sample, however as this component was also present in the

control incubates, it was considered not of metabolic origin. No metabolites were detected

following incubation of [14C]-cediranib in microsomes from cynomolgus monkey,

Dunkin-Hartley guinea-pig, Göttingen mini-pig, rhesus monkey, New Zealand White rabbit,

CD-1 mouse, beagle dog or Han Wister rat in the presence of UDPGA (data not shown).

Involvement of UGT enzymes in the metabolism of [14C]-cediranib. The identification of

the UGT enzymes responsible for the N+-glucuronidation of [14C]-cediranib was performed

using recombinant UGT enzymes. UGT enzyme identification studies in human liver

microsomes were not performed due to the limited availability of a complete set of UGT

selective chemical inhibitors (Miners et al., 2004; Uchaipichat et al., 2006).

Incubation of [14C]-cediranib (10 μM) with microsomes prepared from cell lines expressing a

single UGT isoform indicated that the metabolism of [14C]-cediranib was limited to UGT1A4

(Figure 5), with a reduction observed in the amount of chromatogram radioactivity

represented by [14C]-cediranib and the formation of one major component (M3; 16.2 % of

total chromatogram radioactivity). Incubations with UGTs 1A1, 1A3, 1A6, 1A7, 1A8, 1A9,

1A10, 2B4, 2B7, 2B15 and 2B17 showed no evidence of metabolism of [14C]-cediranib

(Figure 5).


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Discussion

The present study was undertaken to elucidate the biotransformation pathways and identify

the enzymes involved in the in vitro metabolism of cediranib. It has been demonstrated that

the in vitro metabolism of cediranib proceeds predominantly via non-CYP450 enzymes, with

the UGT and FMO enzymes playing a major role.

In vitro metabolism studies have shown that cediranib was metabolised to at least 11

components in hepatic proteins from human, rat and cynomolgus monkey. A summary of the

proposed metabolic pathway of [14C]-cediranib is highlighted in Figure 6.

Incubations with hepatic in vitro systems have highlighted that marked species differences in

the N+-glucuronidation of cediranib was observed. The formation of the pyrrolidine

N+-glucuronide metabolite (M3) was only produced in human and was not detected in the

other species tested. This finding was supported by previous studies where quaternary

N-glucuronidation of cyclic tertiary amines were shown to exhibit distinct species

differences, with a general trend that only human and higher primates have the capability of

catalysing such reactions (Chaudhuri et al., 1976; Fisher et al., 1980; Delbressine et al., 1992;

Chiu & Huskey, 1998). These studies have shown that several cyclic tertiary amine-

containing drugs, the anti-histamines cyproheptadine and tripelennamine, and the

anti-depressant mianserin, also undergo human-specific N+-glucuronidation. Of all the

species studied, the N+-glucuronide metabolites of these drugs were only detected in the urine

of higher primates and/or human. For cediranib, the N+-glucuronide metabolite (M3) has

been identified in the plasma and faeces of humans following a single oral dose of 45 mg

[14C]-cediranib; however, it has not been detected in vivo in plasma, urine or faeces following

a single oral dose of 0.5 mg/kg [14C]-cediranib to beagle dogs, in vivo in urine or faeces


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following a single oral dose of 1.0 mg/kg [14C]-cediranib to cynomolgus monkeys or in vivo

in urine, plasma or bile following a single oral dose of 30 mg/kg [14C]-cediranib to Sprague

Dawley rats (unpublished data). Additional data to be published at a later date will support

the conclusion that M3 is a major metabolite in human and should be considered in this

context.

The enzyme catalysing the N+-glucuronidation of cediranib was shown using recombinant

proteins to be human UGT1A4. This observation is consistent with previous studies

confirming that N+-glucuronidation of cyclic tertiary amines usually proceeds via UGT1A4

(Green et al., 1995; Green et al., 1998; Green & Tephly, 1996; 1998; Kubota et al., 2007).

Information on the interspecies variation in the expression of this enzyme is limited, although

it is well established that in the rat, a pseudo-gene is present in the UGT1 gene complex that

encodes for an incomplete UGT1A4 protein, leaving the rat without a functional UGT1A4

enzyme (Green & Tephly 1998). This could potentially provide an explanation for the lack

of N+-glucuronidation observed in this species. With the exception of rabbit, little data is

available on UGT1A4 expression levels in cynomolgus monkey and the other laboratory

species and thus the inability of the other species to catalyse the formation of a quaternary

N+-glucuronide of cediranib remains unexplained (Green & Tephly 1998).

Functional polymorphisms of hepatic UGT1A4 that are predicted to alter the hepatic

metabolism and detoxification of selective xenobiotics and steroids have been identified in

German Caucasian and Japanese populations (Ehmer et al., 2004; Mori et al., 2005; Saeki et

al., 2005). The frequencies of two genetic variations, P24T (UGT1A4.2) and L48V

(UGT1A4.3) were up to 8 and 17%, respectively (Benoit-Biancamano et al., 2009). Whether

these UGT1A4 polymorphisms could potentially influence the inter-individual variability in


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cediranib disposition in vivo and impact on an individual’s response to cediranib treatment

remains to be assessed, given that functional investigations of UGT1A4 polymorphisms to

date have been limited to in vitro systems. In addition, it has also been suggested that the

effects of these functional polymorphisms may be dependent on the xenobiotic administered

(Mori et al., 2005).

In this study, the human FMO enzymes were shown to be primarily involved in the

N-oxidation of cediranib to metabolite M4. Amongst the FMO enzymes tested, only human

FMO1 and FMO3 were able to catalyse this reaction in vitro, with FMO1 being most

efficient in catalysing this reaction. Although FMO1 is the most prominent isoform in adult

kidney, its expression is very low in adult liver, with FMO3 being the major isoform in this

tissue (Cashman & Zhang, 2006). Based on the kinetic parameters and absolute levels of

each enzyme, it is conceivable that in vivo, both enzymes may be involved to a similar extent

in the N-oxidation of cediranib. A role for the CYP450 enzymes in the formation of this

metabolite was excluded, due to a lack of significant inhibition of cediranib N-oxide

formation by 1-ABT.

The human enzyme(s) responsible for the metabolism of cediranib to metabolite M2 were

CYP450 enzymes. As for metabolite M1, the enzymes involved in its formation are

unknown and have yet to be determined. However, it is clear that CYP450 enzymes do not

play a role, given the lack of inhibition of metabolite formation in the presence of 1-ABT.

Based on the structure of cediranib, a possible role for aldehyde oxidase may exist (Beedham,

2001). Whilst no metabolism of cediranib was observed following incubation with human

cytosolic fractions, it should be noted that this may be due to the instability of aldehyde

oxidase upon preparation and storage of the human hepatic tissue (Lang & Kalgutkar, 2003).


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For the formation of metabolite M11, which was only formed in rat and cynomolgus monkey,

a potential mechanism would involve the hydroxylation of the methylene group adjacent to

the pyrrolidine nitrogen, followed by the intermediate formation of the aldehyde through

N-dealkylation, which would then be rapidly oxidised to the carboxylic acid M11.

In conclusion, the present study has shown that the major biotransformation pathways of

cediranib in humans involve UGT1A4-mediated N+-glucuronidation and

non-CYP450-mediated oxidations, with the N-oxidation of cediranib catalysed by the FMO

enzymes. With the exception of the N+-glucuronide, all other metabolites of cediranib found

in humans were also detected in rat and cynomolgus monkey. As the metabolism of

cediranib proceeds via conjugative and non-CYP450 oxidative pathways, it is predicted that

cediranib may be less prone to potential drug-drug interactions from other co-administered

xenobiotics. The FMO enzymes are not readily inhibited or induced by xenobiotics

(Cashman & Zhang, 2006, Mitchell, 2008) and the magnitude of UGT-based drug

interactions is significantly less when compared to CYP450-mediated interactions (Williams

et al., 2004; Kiang et al., 2005).


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Acknowledgements

We thank Drs Ian Wilson and Eva Lenz (Clinical Pharmacology & DMPK department) for

critically reviewing this manuscript. We also thank Ian McFarlane (Pharmaceutical

Development department), Eva Lenz and Selcia Limited (Ongar, UK) for the preparation of

the authentic cediranib N-oxide metabolite standard, for providing support in the preparation

of the NMR data, and for the preparation of radiolabelled cediranib (batch 1R6), respectively.


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Footnotes

Parts of this work were previously presented at the following conference: 16th North

American Regional Meeting, International Society for the Study of Xenobiotics (ISSX),

Baltimore, MD

Reprint requests to:

Dr Timothy Schulz-Utermoehl, Clinical Pharmacology and DMPK Department, Alderley

Park, AstraZeneca UK Ltd, Macclesfield, Cheshire SK10 4TG, United Kingdom.

Email: [email protected]

1Joint First Authors


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Legends for figures

Figure 1. The chemical structure of [14C]-cediranib.

Figure 2. Representative HPLC-RAD metabolite profiles following incubation of

[14C]-cediranib (10 μM) with human hepatocyte suspensions (donor 2; 1 X 106 cells/mL, at

37°C for 240 min) in the absence (A) and presence (B) of 1-aminobenzotriazole (1-ABT;

1 mM) and with human hepatocyte cultures (C) (donor 5, 132,000 viable cells/cm2, at 37°C

for 72 hours; 5% CO2). The proportion of radioactivity attributed to each metabolite is

shown in Tables 1 and 2.

Figure 3. Representative HPLC-RAD metabolite profiles following incubation (at 37°C for

72 hours; 5% CO2) of [14C]-cediranib (10 μM) with rat (A) and cynomolgus monkey (B)

hepatocyte cultures (132,000 viable cells/cm2). The proportion of radioactivity attributed to

each metabolite is shown in Table 1.

Figure 4. Formation kinetics of the N-oxide metabolite (M4) over the [14C]-cediranib

concentration range of 0.2 to 50 µM in recombinant FMO1 (solid circle) and FMO3 (open

circle). Vmax and Km values of 8.2 nmol equiv/min/mg protein and 11.9 µM for recombinant

FMO1 were obtained. The corresponding values for recombinant FMO3 could not be

calculated.

Figure 5. Formation of [14C]-cediranib N-glucuronide (M3) by recombinant human UGT

enzymes and human liver microsomes. Incubations were carried out at 37°C with 10 µM

[14C]-cediranib for 60 min using 0.25 (recombinant UGT enzymes) and 1 mg/mL (human


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liver microsomes) protein concentration. Each column represents the mean of duplicate

determinations.

Figure 6. Proposed metabolic pathway of [14C]-cediranib in hepatocyte suspensions, cultures

and hepatic subcellular fractions from human and pre-clinical species. HHp, human

hepatocytes; RHp, rat hepatocytes; cMHp, cynomolgus monkey hepatocytes; HLM, human

liver microsomes; HS9, human liver S9.


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Table 1. Metabolite profiles following incubations of [14C]-cediranib (10 µM) with human hepatocyte suspensions (1 X 106 cells/mL,

240 min, in the absence and presence of 1-aminobenzotriazole (ABT; 1 mM)) and with hepatocyte cultures (132,000 viable cells/cm2,

72 hours (24 hours for human donor 4)) of rat, cynomolgus monkey and human

Human (Donor 1)a

Without ABTWithout

ABTWith ABT

Without ABT

With ABT

M1 1.6 1.2 1.3 1.4 1.7 4.2 8.9 2.9 1.9

M5 -c - 2.5M6 1.3M7 √ 2.4 √

M2 √b

√ √ 1.1 1.2 4.4 6.8M8 12.1 7.3M3 9.6 10.8 11.7 9.0 9.1 11.3 24.9M4 √ 1.1 1.2 1.6 1.3 6.8 6.1 30.1 9M9 1.3M10 2.3M11 3.1 1.1M12 0.4 1.8Cediranib 75.6 72.6 71.5 72.7 73.8 67.5 45.9 32.8 57.2

Incubations with Hepatocyte Suspensions Incubations with Hepatocyte Cultures

Human (Donor 4)

% total chromatogram radioactivity

Compound Human (Donor 5)

Wistar Rat (Male)

Cynomolgus Monkey (Male)

Human (Donor 2) Human (Donor 3)

a data with ABT is not available. b component was present but did not account for greater than 1% of the chromatogram radioactivity. c Peaks for M1 and M5 could not be separated chromatographically; the value stated for M1 is the combined proportion of total chromatogram radioactivity for metabolites M1 and M5.

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Table 2. Summary of mass spectrometric data for [14C]-cediranib and its metabolites

Assignment [M+H]+

(m/z )a

Proposed Structure Diagnostic MS2 Fragment Ions (m/z)

Cediranib453 (P)

342, 326, 112

Cediranib

M1485

(P+32)467 (loss of H2O), 358, 342

Di-oxygenated

M5501

(P+48)483 (loss of H2O), 389, 374

Tri-oxygenated

M6 -b Unassigned No data available

M7483

(P+30)465 (loss of H2O), 439, 372, 356

Fluoro-indole, carboxylic acid

M2485

(P+32)342

Di-oxygenated

C14NH

N N

NO

O

O

CH3

F

CH3

342326

112

C14NH

N N

NO

O

O

CH3

F

CH3

2[O]342

C14NH

N N

NO

O

O

CH3

F

CH3

3[O]

389 (-H)374

N O

O

CH3

N

N

O

NH

C14

F

O

OH

372

356

439

N O

O

CH3

N

N

O

NH

C14 CH3

F

[O]

[O]

358342


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Table 2. – Continued

Assignment [M+H]+

(m/z )a

Proposed Structure Diagnostic MS2 Fragment Ions (m/z)

M8248 (P-

285+80)168

Fluoro-indole monooxygenated sulfate

M3629

(P+176)453 (aglycone), 342, 326

N -glucuronide

M4469

(P+16)382, 342, 128, 84

N -oxide

M9 - Unassigned No data available

M10 - Unassigned No data available

M11414 (P-

69+30)342

Propanoic acid

C14NH

N+

N

NO

O

O

CH3

F

CH3

O342

84 (- O) 128

382

C14NH

N

NO

O

O

CH3

F

CH3

O

OH

342

N+

O

O

CH3

N

N

O

NH

C14 CH3

FOOH

OH

OH

COOH

342

326

453

NH

C14 CH3

F

O

SO3

168

a 14C isotope is the predominant ion where present. b no molecular ions assigned.


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Table 3. 1H NMR characteristics of cediranib and its metabolites M3 and M4

Cediranib M4 M3

(multiplicity) 1-2 NR NR NR

3 7.43 (s) 7.42 (s) 7.44 (s)4 7.65 (s) 7.60 (s) 7.67 (s)

5-7 NR NR NR8 8.53 (s) 8.53 (s) 8.53 (s)

9-15 NR NR NR16 7.17 (d) 7.2 (d) 7.18 (d)17 6.99 (dd) 7.01 (dd) 7.00 (dd)

18-19 NR NR NR20 6.25 (s) 6.26 (s) 6.25 (s)21 NR NR NR22 11.35 11.36 11.3523 2.42 (s) 2.43 (s) 2.42 (s)24 NR NR NR25 4.33 (t) 4.39 (t) 4.28 (t)26 2.24 (m) 2.52 (m) 2.41 (m)27 3.35 (t) 3.94 3.60, 3.7028 NR NR NR29 3.37 (m, br) 3.67 (m, br) 3.83 (m, br)30 1.97 (m, br) 2.09 (m, br) 2.08 (m, br)31 1.97 (m, br) 2.23 (m, br) 2.21 (m, br)32 3.37 (m, br) 3.93 (m, br) 3.92 (m, br)33 4.01 (s) 3.97 (s) 4.0134 NR NR 4.6835 NR NR 3.6736 NR NR 3.3237 NR NR 3.2038 NR NR 3.45

MetabolitePosition

1H (ppm)

NH

N+

N

NO

O

O

FOOH

OH

OH

COOH

1

23

45

67

8

9

10

13

14

17

1618

22

21 23

201915

24

11

33

12

25

2

27

28

29

30

31 32

M3

35

36

3738

34

NH

N N

NO

O

O

F1

23

45

67

8

9

10

13

14

17

1618

22

21 23

201915

24

11

33

12

25

2

27

28

29

30

31 32

CediranibNH

N+

N

NO

O

O

F

O

1

23

45

67

8

9

10

13

14

17

1618

22

21 23

201915

24

11

33

12

25

2

27

28

29

30

31 32

M4

Notations: br, broad peak; d, doublet; dd, doublet of a doublet; m, multiplet; NR, no result; s, singlet

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on July 15, 2010 as DO

I: 10.1124/dmd.110.033159

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ber 16, 2018dm


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at ASPE


ber 16, 2018dm


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In Vitro Hepatic Metabolism of Cediranib, a Potent ...dmd.aspetjournals.org/content/dmd/early/2010/07/15/dmd.110.033159... · DMD #33159 3 Abstract The in vitro metabolism of cediranib,