Characterization of in vitro and in vivo metabolism of AG-024322, a novel cyclin-dependent kinase (CDK)

doi:10.4236/health.2009.14041

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Vol.1, No.4, 249-262 (2009)

doi:10.4236/health.2009.14041

Health

Characterization of in vitro and in vivo metabolism of

AG-024322, a novel cyclin-dependent kinase (CDK)

inhibitor

Wei-Zhu Zhong*, Bojan Lalovic1, Jenny Zhan2

1Clinical Pharmacology, Pfizer Global Research and Development, New London, Connecticut, USA

2Metabolism and Pharmacokinetics, Novartis Institute of Biomedical Research, Cambridge, Massachusetts, USA

*Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, San Diego, California, USA;

wei-zhu.zhong@pfizer.com

Received 16 November 2009; revised 25 November 2009; accepted 28 November 2009.

ABSTRACT

Uncontrolled cell proliferation is the hall mark of

many cancers, and is typically manifested by a

deregulation of the cell-division cycle. CDKs

play critical roles in regulating cell cycle, apop-

tosis and cell differentiation. AG-024322 is a

multitargeted CDK inhibitor that has been

shown to induce cancer cell apoptosis and de-

monstrate significant antitumor activity in hu-

man tumor xenograft models. This compound is

under clinical development as an intravenous

anticancer agent. AG-024322 exhibited moder-

ate to high systemic clearance across preclini-

cal species. In vitro metabolism in human liver

microsomes and hepatocytes demonstrates that

glucuronidation and oxidation represent the

major metabolic pathways of AG-024322. The

experiments of chemical inhibition and micro-

somes containing individual CYP or UGT iso-

forms revealed that CYP3A and UGT1A1 appear

to predominantly mediate AG-024322 oxidation

and glucuronidation, respectively. Formation

kinetics of the two pathways in human liver mi-

crosomes suggested that the glucuronidation

activity of AG-024322 was approximately 3-fold

higher as compared to CYP-mediated oxidation,

contributing approximately 37% and 13% of the

total clearance, respectively, based on the pro-

jected human clearance. UGT1A1 is a poly-

morphic isoform involved in glucuronidation of

bilirubin. It is of concern if glucuronidation via

UGT1A1 plays a major role in the elimination of

AG-024322 in humans as competitive inhibition

of UGT1A1 has been associated with toxicity

(Gilbert and Crigler-Najjar syndromes). There-

fore, this information was used to influence the

clinical study design to only include subjects

having constitutive expression of UGT1A1 in

the first human study, thereby decreasing the

potential risk of toxicity to patients.

Keywords: Microsomes; Hepatocytes; Reaction

Phenotyping; Metabolite Formation Kinetics;

CYP3A4; UGT1A1; Mass Balance in Rat

1. INTRODUCTION

AG-024322, N-({5-[3-(4,6-difluoro-1H-benzimidazol-2-

yl)-1H-indazol-5-yl]-4-methylpyridin-3-yl}methyl)ethan-

amine, is a multitargeted CDK inhibitor under deve-

lopment as an anticancer agent. AG-024322 is a potent

ATP-competitive inhibitor of the cell cycle kinases

CDK1, CDK2, and CDK4, with kinetic inhibition con-

stants (Ki) in the 1-3 nM range [1]. CDKs play a key

role in regulating the cell cycle progression and to a

large degree control cellular transitions from growth

phases into phases associated with DNA replication and

mitosis by phosphorylating several cellular proteins

[2-4]. AG-024322 inhibits phosphorylation of CDK sub-

strates in cells, resulting in cell arrest and broad-spec-

trum anti-proliferative activity, and has been shown to

induce cancer cell apoptosis and significant antitumor

efficacy in human tumor xenograft models with tumor

growth inhibition (TGI) ranging from 32% to 86.4% [5].

Single-dose pharmacokinetics of AG-024322 was

evaluated in the rat, dog and monkey following a 30-

minute IV infusion, the intended administration route in

humans. AG-024322 demonstrated moderate clearance

of 26 and 27 ml/min/kg in monkeys and dogs, respec-

tively, and somewhat higher clearance in rats (67

ml/min/kg), with half-lives ranging from 5 to 7 hours.

Minimal contribution of renal clearance was observed in

all 3 species ( 1%). The volume of distribution greatly

exceeded the volume of total body water across species

(9 to 15 l/kg), and was consistent with rapidly and

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

250

widely tissue distribution observed in Long Evans rats

by quantitative whole body autoradiography (QWBA)

using [14C]AG-024322. The plasma protein binding of

AG-024322 was in the range of 85 to 95% across species.

In view of the predominant metabolic pathway of glu-

curonidation observed in human hepatocytes and the

generally low metabolic turnover in in vitro incubations,

the prediction of pharmacokinetics in human was per-

formed using interspecies allometric scaling of single-

dose animal pharmacokinetics data. This approach lead

to a reasonable prediction of human clearance (12.5 ml/

min/kg), which was similar to the clearance actually

observed in humans with IV doses from 20 to 340 mg.

Detailed nonclinical pharmacokinetics and the prediction

of human pharmacokinetics of AG-024322 will be re-

ported elsewhere.

To support the development of AG-024322, a series of

nonclinical in vitro and in vivo studies were conducted to

identify AG-024322 metabolites, and to characterize the

disposition of AG-024322. These studies involved with

either using animal and human liver microsomes and

hepatocytes incubated with C-14 labeled AG-024322

([14C]AG-024322) or using urine, plasma, bile, and fecal

samples from rats administered [14C]AG-024322. Fur-

ther studies to assess the two principal pathways of AG-

024322 biotransformation, CYP-mediated oxidative and

UGT-mediated glucuronidation, and to identify the ma-

jor enzymes involved in these metabolic pathways were

also carried out.

In the present paper, discussion was emphasized on the

in vitro and in vivo characterization of AG-024322 me-

tabolism and the relative importance of CYP3A4 and

UGT1A1 in the biotransformation of AG-024322 in HLM,

as well as the relative contributions of the two metabolic

pathways in the overall clearance of AG-024322.

2. MATERIALS AND METHODS

2.1. Materials

AG-024322 was synthesized at Pfizer Global Research

& Development (PGRD), La Jolla, CA. [14C]AG-

024322 (Specific activity 53.1 mCi/mmol) was synthe-

sized by the Radiosynthesis Group at PGRD (Groton,

CT), and the label was located in the C-1 position of the

benzimidazol ring (Scheme 1). Rat, dog, monkey and

human liver microsomes were obtained from Gentest

Corporation. (Woburn, MA). Human liver microsome

preparation (HL-Mix 14, 21.7 mg protein/ml, 0.3 nmol

CYP/mg protein) and microsomes from insect cells

stably expressing human cDNA CYPs and UGTs (su-

persomes) were also purchased from Gentest Corpora-

tion. Cryopreserved hepatocytes for dogs, monkeys and

humans, and the thawing media were obtained from In

Vitro Technologies (Baltimore, MD). Sprague-Dawley rat

hepatocytes and Hepatocyte Isolation Kit were pur-

Scheme 1. Chemical structure of AG-024322

(asterisk indicates the site of radiolabel).

chased from XenoTech LLC (Lenexa, KS). Ketocona-

zole, quindine, quercetin, furafyline, dibasic and mono-

basic potassium phosphate, NADPH, UDPGA, and

WEM were purchased from Sigma-Aldrich Co. (St.

Louis, MO). HEPES (4-(2-hydroxyethyl)-1-piperazi-

neethanesulfonic acid) buffer was purchased from Invi-

trogen (Carlsbad, CA). Commercially obtained chemi-

cals and solvents were of HPLC or the highest grade

available.

2.2. Liver Microsomal Incubations

Liver microsomes were preincubated with AG-024322

and [14C]AG-024322 in 100 mM potassium phosphate

buffer (pH 7.4) for 5 minutes in a 37ºC shaking water

bath. The reaction was initiated by the addition of

NADPH with a final incubation volume of 0.5 ml con-

taining 10 µM AG-024322, 0.87 µM [14C]AG-024322, 1

mM NADPH, 0.75 mg/ml microsomal protein and 100

mM potassium phosphate buffer (pH 7.4). The incuba-

tion was terminated at 0, 10, 20, 40 and 60 minutes by

the addition of 0.5 ml of acetonitrile and immediately

vortexed and centrifuged at 1900g for 25 minutes. The

supernatants were transferred to a fresh tube, evaporated

to dryness under nitrogen, and reconstituted with 200 µl

of Solvent A for LC/RAM/MS analysis as described

below.

2.3. Hepatocytes Incubation

Cryopreserved hepatocytes were thawed and used ac-

cording to the manufacturer’s instructions. The thawed

cryopreserved hepatocytes were re-suspended into CO2-

bubbled incubation media to determine the total cell

count using the Trypan Blue Exclusion method. The

incubation media consisted of WEM supplemented with

10 mM HEPES buffer. The final cell concentrations

were 1.5, 1.8, 1, and 1.23106 viable cells/ml for rat, dog,

monkey and human hepatocytes in WEM, respectively.

Hepatocytes were incubated in the media containing 10

µM AG-024322 and 0.87 µM [14C]AG-024322 in a

CO2:O2 (5:95) stationary incubator at 37ºC. The final

incubation volume was 1 ml. Reactions were terminated

at several time points from time zero to 5 hours by the

addition of 2 ml of acetonitrile. Samples were then soni-

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

251

cated, vortexed and centrifuged at 1900g for 25 min-

utes at 4ºC. The resulting supernatant was evaporated

under nitrogen and reconstituted in 100 µl of water for

LC/RAM/MS analysis.

2.4. CYP Reaction Phenotyping Screen

AG-024322 (1 and 10 µM), each in triplicate, were in-

cubated with a panel of microsomes from insect cells

expressing individual human CYPs (Supersomes) con-

taining 50 pmol of CYP1A2, 2C8, 2D6, 2E1, 3A4 and

3A5 protein or 100 pmol of 2C9*1 and 2C19. The final

incubation volume was 0.5 ml. Incubations containing

AG-024322 and 1 mM NADPH in 100 mM phosphate

buffer (pH 7.4) were preincubated for 5 minutes at 37ºC.

The reactions were started with the addition of 50 µl of

each CYP and were allowed to proceed up to 15 minutes.

Incubations were stopped with the addition of 0.5 ml of

acetonitrile and 20 µl of 1ng/µl AG-024397 (IS), and

immediately capped, vortexed and centrifuged at 1900g

for 25 minutes at 4ºC. The supernatant of each sample

was dried under N2 and reconstituted with 100 µL of

Solvent C for LC-MS analysis as described below.

2.5. Chemical Inhibition

CYP-specific chemical inhibitors, ketoconazole (CYP

3A4), quinidine (CYP2D6), quercetin (CYP2C8), and

furafyline (CYP1A2) were used to identify the isoforms

mediating the formation of N-desethyl AG-024322. In-

cubation mixtures were prepared with ketoconazole and

quinidine at 20, 100, 500 and 1 µM, quercetin at 0.3,

0.75 and 2 µM and furafyline at 1, 10 and 30 µM, and

were preincubated with AG-024322 (1 and 10 µM) and

15 mg HLM protein (0.3 mg/ml) for 5 minutes at 37ºC.

Incubations were started by the addition of NADPH to a

concentration of 1 mM in a final volume of 0.5 ml. In-

cubations with furafyline, a time-dependent inhibitor of

CYP1A2, required a preincubation step in the presence

of 1 mM NADPH for 20 minutes prior to the introduc-

tion of the substrate. The reactions were terminated after

8 minutes by the addition of 0.5 ml of acetonitrile and 20

µl of 1 ng/µl IS. The remaining part of the procedure

was performed as described above for CYP reaction

phenotyping screen.

2.6. UGT Reaction Phenotyping Screen

AG-024322 (1 and 10 µM), each in triplicate, was incu-

bated with 8 UGT isoforms (UGTlA1, 1A3, 1A4, 1A6,

1A8, 1A10, 2B7 and 2B15). The incubation procedure

was adapted from Fisher et al. [6]. Microsomes (0.5 mg/

ml) suspended in 100 mM phosphate buffer (pH 7.1)

were initially treated with alamethicin at 0.025 mg/mg

microsomal protein. The mixture also included 1 mM

MgCl2 and was kept on ice for 15 minutes to permealize

the microsomal membrane. AG-024322 was then added

and the mixture was preincubated at 37‘C for 5 minutes.

The reaction was started with the addition of 1 mM

UDPGA in a total incubation volume of 0.2 ml, and were

terminated after 15 minutes by the addition of 0.5 ml of

acetonitrile and 20 µl of 1 ng/µl IS. The remaining part

of the procedure was performed as described above for

CYP reaction phenotyping screen.

2.7. Metabolite Formation Kinetics in HLM

The formation kinetics of the N-desethyl metabolite and

the parent glucuronides were studies in the same liver

preparation from 14 different human livers (HL-Mix 14)

to afford a comparison of the oxidation and glucuronida-

tion pathways.

To determine the formation rates of N-desethyl me-

tabolite, AG-024322 with a concentration range of 0.5-

100 µM, each in triplicate, was pre-incubated with HLM

containing 0.75 mg/ml protein in 100 mM potassium

phosphate buffer (pH 7.4) for 5 minutes at 37ºC. The

reaction was initiated by the addition of 1 mM NADPH

in a final incubation volume of 0.5 ml. Reactions were

terminated at 15 minutes by the addition of 0.5 ml of

acetonitrile and 40 µl of 1 ng/µl IS. The remaining part

of the procedure was performed as described above for

CYP reaction phenotyping screen.

The experiment conditions to determine the formation

rates of parent glucuronides were optimized in respect to

time and protein concentration. Linear product formation

was observed for up to 1 hour and 2 mg/ml microsomal

protein. The experiments were following the similar

procedure as described above using UGT supersomes,

except that the incubation mixture contains 1 mg/ml

microsome protein, 1 mM MgCl2, 0.05 mg/mg micro-

some protein alamethicin, and 2.5 mM UDPGA in either

0.1 M phosphate buffer (pH 7.1) or WEM, incubated

with a range of AG-024322 concentrations (0.5 to 200

µM) in triplicate, and terminated at 35 minutes.

2.8. Formation Kinetics Calculation

The relative formation rate of each metabolite (peak area

ratio/mg protein/15 or 35 min) was calculated and plot-

ted against AG-024322 concentrations. The metabolite

formation kinetic parameter, Km, was estimated by fitting

the data to the sigmoid Emax model using SAAMII

(SAAM Institute, Seattle, WA). The maximum formation

rate (Vmax) was estimated from separate experiments of

HLM incubation with 10 µM [14C]AG-024322 at the

similar conditions as described above. The formation

rate (V) of each metabolite in the incubations was calcu-

lated based on the radioactivity. The Vmax value was then

calculated based on the Michaelist-Menten equation,

Vmax is the substrate concentration. The in vitro intrinsic

clearance (CLint) was determined by Vmax/Km ratio. The

predicted CLint was scaled up to in vivo by extrapolating

CLint assuming a human liver weight of 1500 g (70 kg body

weight) and that one gram liver tissue contains 45 mg HLM

protein. The total hepatic clearance (CLh) was calculated

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

252

from the well-stirred model: CLh = Q x CLint / Q + CLint,

where Q is the human hepatic blood flow (20 ml/min/kg).

The estimation of CLh was not incorporated with plasma

and microsome protein binding.

2.9. Mass Balance Study in Rats

In this study, [14C]AG-024322 in 5% dextrose in water

(pH 4) was administered as a single 30-minute IV infu-

sion to all animals at a target dose level of 10 mg/kg

(~50 µCi/250 g body weight). The intact animals in

Group 1 (3/gender) were individually housed in metabo-

lism cages for the separate collection of urine and feces

over intervals through 120 hours after dosing. The bile

duct-cannulated (BDC) rats in Group 3 (2/gender) were

individually housed in metabolism cages for the collec-

tion of bile over intervals through 48 hours. Blood sam-

ples were collected from intact animals in Group 2

(2/gender) before dosing and at 0.25, 0.5, 1, 3, 8, 24, 48,

72, 96, and 120 hours from the initiation of infusion.

Blood samples were collected from intact animals in

Group 4 by sacrificing 1 animal/gender at each time

point at 0.5, 2, 4, and 8 hours from the initiation of infu-

sion. Urine, feces, bile, cage wash, cage wipe, blood, and

plasma samples were processed and analyzed for radio-

activity. Selected plasma, urine, bile, and fecal samples

were used for metabolite identification and profiling.

2.10. Plasma, Urine, Fecal and Bile Sample

Preparation

Plasma samples collected from Group 4 animals were

pooled based on the pooling formula described by Ham-

ilton and coworkers [7] to generate plasma pools (2.0

mL). The pooled plasma samples were precipitated by

the addition of 4 volumes of acetonitrile: methanol (3:1,

v/v) with 0.2% formic acid, and then vortexed and cen-

trifuged. Urine samples were pooled on a percent weight

basis and centrifuged. The supernatants from the plasma

or urine pool extracts were concentrated to approxi-

mately 1 ml under N2 before LC/RAM/MS analysis.

Selected fecal homogenate from each animal were

pooled on a percent weight basis to generate fecal ho-

mogenate pools. Each pooled sample was precipitated by

the addition of 4 volumes of acetonitrile containing 0.2%

formic acid, vortexed and centrifuged. The pellet was re-

extracted using the same method, and supernatants were

combined for evaporation under N2 to dryness. The resi-

dues were reconstituted in 1 ml of 20:80 (v/v) metha-

nol:20 mM ammonium acetate with 0.1% formic acid for

LC/RAM/MS analysis. Selected bile samples were pooled

on a percent weight basis to generate bile pools. After

centrifugation, every 100 µl of bile sample was diluted

with 900 µl of 20:80 (v/v) methano l:20 mM ammonium

acetate containing 0.1% formic acid for LC/RAM/MS

analysis. All centrifugation was performed at 15850g

for 10 minutes using Brinkman eppendorf 5415c (West-

bury, NY). The injection volume for LC/RAM/MS was

300-900 µl.

2.11. Determination of Radioactivity

The radioactivity in urine, bile, fecal, blood, and plasma

samples was determined by liquid scintillation counting

(LSC). Triplicate weighed aliquots (approximately 0.02

to 0.1 g each) of urine, bile, and plasma samples were

mixed with scintillation fluid, and analyzed directly by

LSC. Triplicate weighed aliquots of fecal homogenate

(fecal:water, 20:80, w/w, approximately 0.1-0.5 g each)

or blood (approximately 0.02-0.03 g each) were placed

into cones and pad, dried, combusted, and then analyzed

by LSC.

2.12. Metabolite Characterization

Metabolite characterization was performed using an

Agilent 1100 HPLC system (Wilmington, DE) coupled

in-line with an ARC Model C StopFlow System (AIM

Research Company, Newark, DE) and an IN/US Model

3 -RAM radio-detector (Tampa, FL), and a ThermoF-

innigan LCQ-Deca XP ion trap mass spectrometer (San

Jose, CA). When MS detection was used, the HPLC ef-

fluent was split with approximately 20% of the flow

introduced into the MS via electrospray ionization (ESI)

source and 80% diverted to the -RAM detector. The

ion-trap ESI-MS was operated in positive ion model

with a spray voltage of 4.5 kV, sheath gas flow rate of 70

(arbitrary), auxiliary gas flow rate of 20 (arbitrary) and

capillary temperature at 200ºC. Laura 3 V3.0 (IN/US

system, Tampa, FL), ARC data system V2.4 (AIM Re-

search Company, Newark, DE) and Xcalibur V1.4

(ThermoFinnigan, San Jose, CA) software were em-

ployed to control the -RAM detector, the StopFlow

system and the LC-MS system for data acquisition and

processing. The MS spectra were acquired over a mass

range of m/z 100-900 for all samples. Ion-trap LC-ESI-

MS/MS experiments were performed to generate ion

mass spectra for the selected molecular ions representing

possible metabolites of AG-024322. AG-024322 me-

tabolites were identified by chromatographic resolution

and concurrent radiometric and mass-selective quantifi-

cation.

Separation of the parent drug and its metabolites was

accomplished using a Phenomenex Luna 5 µ C18 col-

umn (2504.6 mm, Torrance, CA) or an Agilent Zorbax

5µ C8 column (1504.6 mm, Palo Alto, CA). The mo-

bile phase consisting of a 20 mM ammonium acetate in

water (pH 4.0) as Solvent A and acetonitrile as Solvent B,

and were programmed for each matrix using a linear

gradient at a constant flow rate of 1.0 mL/min as follows:

1) for hepatocyte and microsome incubations: 0 min, 5%

B; 5 min, 5% B; 30 min, 35% B; 31 min, 95% B; 35 min,

95% B; 36 min, 5% B; and 43 min, 5% B; 2) for urine,

feces and bile samples: 0 min, 10% B; 10 min, 18% B;

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

253

20 min, 18% B; 42 min, 30% B; 50 min, 30% B; 60 min,

55% B; 61 min, 10% B; and 70 min, 10% B; and 3) for

plasma samples: 0 min, 10% B; 50 min, 30% B; 60 min,

55% B; 61 min, 10% B; and 70 min, 10% B.

Openly accessible at

The analysis of N-desethylation metabolite of AG-

024322 and the glucuronides of the parent drug was

using a Zorbax C8 column (2.15 mm, 5 µ) from

Agilent (Palo Alto, CA) to allow a chromatographic

resolution of the metabolites from the parent drug with

a run time of 12 minute. With an API 4000 MS (AB

Sciex Instruments, Toronto, Ontario), the monitored ion

transitions were 419374, 391374 and 595374 for

the parent compound, N-desethyl metabolite and parent

glucuronide, respectively. The mobile phase was pro-

grammed from the initial setting at 95% Solvent C (2

mM ammonium acetate with 0.1% formic acid in water)

and 5% Solvent D (acetonitrile with 0.1% formic acid)

to 35% C and 65% D at 10 minutes by linear gradient,

and restored to original composition at 10.1 min until

the end of run.. The flow rate was maintained constant

at 0.3 ml/min.

3. RESULTS

3.1. In Vitro Metabolism of AG-024322

Metabolic stability of [14C]AG-024322 in liver micro-

somes and hepatocytes are presented in Table 1 as per-

cent of parent drug remaining after incubation in the

preparations for rat, dog, monkey and human. The cor-

responding disappearance half-life was generated from

the available time course data. [14C]AG-024322 depleted

most extensively in rat liver microsome with 11% of

radiochromatogram of the parent drug remaining at

1-hour incubation time, followed by dog (55%). Com-

parable incubations in human and monkey liver micro-

somes yielded significantly less extensive parent drug

depletions with 95% and 92% remaining after 1-hour

incubation, respectively. [14C]AG-024322 also exhibited

metabolic stability in human hepatocytes relative to

other species. After 5 hours incubation, only 14% of the

compound was metabolized in human hepatocytes,

whereas 26% and 27% in dog and rat hepatocytes, re-

spectively. Monkey hepatocytes most extensively me-

tabolized AG-024322 up to 38%. Based on the chroma-

tographic retention time and molecule weight, several

metabolites were tentatively identified as parent glu-

curonides (M1, M2, and M3), N-desethylation (M6) and

metabolites that may arise from N-acetylation of M6 (M8)

or parent methylation (M10) in hepatocytes and micro-

somes of various species (Table 2). The radiochro- ma-

tograms of the 5-hour incubations of [14C]AG-024322 in

rat, dog, monkey and human hepatocytes are shown in

Figure 1. At 5 hours, glucuronidation of the parent drug

accounted for approximately 8% of the total radioactiv-

ity, while M6 and M8/M10 accounted for 4% and 2% of

Table 1. Metabolic stability of [14C]AG-024322 in liver mi-

crosomes and hepatocytes.

Microsomesa Hepatocytesb

Species Remaining

at 1-hour (%)

Half-life

(h)

Remaining

at 5-hour(%)

Half-life

(h)

Human95 12 86 50

Monkey92 37 62 8.5

Dog 55 1.0 74 14

Rat 11 0.3 73 15

a. Incubations contain 0.75 mg/ml microsomal protein; b. Incubations

contain 1.2, 1.0, 1.8 and 1.5 x106 cells/ml in WEM for human, monkey,

dog and rat, respectively.

Table 2. Metabolite profiles of AG-024322 in rat, dog, monkey and human liver microsomes and cryopreserved hepa-

tocytes incubated with [14C] AG-024322.

% Radiochromatogram

Species M1 M2 M3 M5 (Unk)M6 M7(Unk)AG-024322M7/M10 M9(Unk) M10/M7

Liver Microsomes (at 1 Hour of Incubation)

Rata - - - - 7.5 10.6 10.8 8.8 22.1 -

Dogb - - - - 5.2 12.3 55.3 14.0 - -

Monkey - - - - 8.1 - 91.9 - - -

Human - - - - 4.8 - 95.2 - -

Hepatocytes (at 5 hours of Incubation)

Rat - - 8.3 1.2 11.3- 73.1 6.1 - -

Dog - - - - 13.6- 74.2 2.4 3.9 5.8

Monkey 1.9 7.3 21.7 1.8 4.5 0.8 62 - - -

Human 1.5 1.5 5.2 - 4.1 - 85.7 2.1 - -

a. Novel unidentified metabolites accounted for 40.2% of radiochromatogram in rat liver microsome (not listed); b. Novel unidentified

metabolites accounted for 13.2% of radiochromatogram in dog liver microsome (not listed); -: Trace or no RAM detected metabolites;

M1, M2 and M3: Parent drug glucuronides (M.W. 594); M6: N-desethyl AG-024322 (M.W. 386); M7/M10: acetylated M6 or methy-

lated AG-024322 (M.W. 432); Unk: unknown/unidentified metabolite.

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

254

Time (Min)

Monkey

DPM

0:00 10:00 20:0030:00 40:00

100

200

300

400

500

600

700

800

AG-024322

Time (Min)

Monkey

DPM

0:00 10:00 20:0030:00 40:00

100

200

300

400

500

600

700

800

AG-024322

Human

Time (Min)

DPM

0:0010:00 20:00 30:00 40:00

100

200

300

400

500

600

700

800

900

1000

M1 M2 M3 M6

AG-024322

M8/M10

Human

Time (Min)

DPM

0:0010:00 20:00 30:00 40:00

100

200

300

400

500

600

700

800

900

1000

M1 M2 M3 M6

AG-024322

M8/M10

Dog

Time (Min)

DPM

0:00 10:00 20:00 30:00 40:00

100

200

300

400

500

600

700

800

DPM

M9( Unk)

M7(Unk)

AG-024322

M8/M10

Dog

Time (Min)

DPM

0:00 10:00 20:00 30:00 40:00

100

200

300

400

500

600

700

800

DPM

M9( Unk)

M7(Unk)

AG-024322

M8/M10

Rat

Time (Min)

DPM

M8/M10

M5 (Unk)M6

0:0010:0020:0030:00 40:00

100

200

300

400

500

600

700

AG -024322

M7 (Unk)

Rat

Time (Min)

DPM

M8/M10

M5 (Unk)M6

0:0010:0020:0030:00 40:00

100

200

300

400

500

600

700

AG -024322

M7 (Unk)

Figure 1. Representative HPLC radiochromatograms of a 5-hour [14C]AG-024322 (11 µM) incubation in rat, dog,

monkey and human cryopreserved hepatocytes (1106 cells)

total radioactivity, respectively, in human hepatocytes.

M6 is the only metabolite detected in human liver mi-

crosomes and accounted for 5% of the total radioactivity.

The preliminary proposed in vitro metabolic scheme is

show in Scheme 2.

3.2. CYP Reaction Phenotyping

The CYP enzymes involved in N-desethylation of AG-

024322 were examined by incubations with microsomes

from insect cells expressing cDNA (recombinant) human

CYPs and by CYP-selective chemical inhibition.

CYP3A appear to predominantly catalyzed the N-de-

sethylation of AG-024322 in vitro at 1 and 10 µM (Fig-

ure 2). Incubations of AG-024322 with recombinant

CYP1A2, 2C8 and 2D6, indicated their minor contribu-

tion after scale-up of the velocity using either CYP con-

tent or relative activity factors [8,9]. Follow-up incuba-

tions with CYP-specific inhibitors suggested that only

Scheme 2. Preliminary In Vitro AG-024322 metabolic scheme.

N-acetylation

M10

Gluc.

N-meth ylat ion

N-desethylation

AG-024322

UGT

M1, M2, M3

Oxidation

CYP

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

255

ketoconazole inhibited the N-desethylation of AG-024322

(Table 3). Despite the minor projected involvement of

CYP2D6, CYP2C8 and CYP1A2 (Figure 2), quinidine,

quercetin and furafyline had no inhibitory effect on the

formation rate of N-desethyl AG-024322, confirming a

minimal contribution of these enzymes. The involvement

of CYP2C9, CYP2C19 and CYP2E1 in N-desethylation of

AG-024322 was found to be negligible. Formation kinetics

of N-desethyl AG-024322 in human liver microsome were

further investigated over the concentration range of 0.5 to

100 µM AG-024322 and were best described by a single

enzyme system with an apparent Km of 10 µM using

SAAMII, a general nonlinear kinetic modeling program

(Figure 3). The estimated kinetic parameters, Km, Vmax, in

vitro CLint, and extrapolated in vivo intrinsic and hepatic

clearance are presented in Table 4.

3.3. UGT Reaction Phenotyping

The strategy used here for UGT phenotyping is based on-

well-established methods, including screening of re

Figure 2. Comparison of AG-024322 N-desethylation activity

at 1 and 10 µM in microsomes from insect cells individually

expressing human CYPs (CYP supersomes).

Table 3. Percent (%) inhibition or stimulation of AG-024322 metabolism in the presence of CYP-selective Inhibitors.

Inhibitor Concentration (µM)

% Desethylaton Activity

Remaining at 1 µM AG-0243222

% Desethylaton Activity

Remaining at 10 µM AG-0243222

0.02 77.5 ± 4.4 86.2 ± 3.9

0.1 31.4 ± 2.2 62.2 ± 5.2

0.5 16.9 ± 0.5 30.7 ± 1.7

Ketoconazole

(Inhibitor of CYP3A4)

1 14.2 ± 0.4 23.8 ± 2.1

0.02 131 ± 18 117 ± 7

0.1 157 ± 11 115 ± 4

0.5 158 ± 8 122 ± 11

Quinidine

(Inhibitor of CYP2D6)

1 165 ± 4 120 ± 7

0.3 145 ± 6 120 ± 9

0.75 160 ± 5 120 ± 8

Quercetin

(Inhibitor of CYP2C8) 2 156 ± 3 120 ± 9

1 - 126 ± 16

10 - 125 ± 7

Furafyline

(Inhibitor of CYP1A2) 30 - 131 ± 7

-: not determined

Table 4. Enzyme kinetic (michalis-menten) parameters estimates of the formation of AG-024322 oxidation and glu-

curonide metabolites using HL-14 human liver microsome preparation.

Formation Kinetics Buffer Km (µM)Vmax

a (pmol/mg/min) CLint

b (ml/min/kg) CLh

c (ml/min/kg)

N-desethyl AG-024322 KPI 10 20 1.86 1.70

Glucuronide M1 KPI 450 35 0.07 0.07

WEM 98 77 0.74 0.72

Glucuronide M2 KPI 9.1 5.3 0.55 0.54

WEM 19 27 1.34 1.26

Glucuronide M3 KPI 7.8 5.2 0.63 0.61

WEM 11 37 3.17 2.74

a. Formation velocity V was estimated from [14C]AG-024322 incubation. Vmax was calculated from V = Vmax x S/(Km+ S), where S is the sub-

strate concentration; b. CLint was scaled up to in vivo by extrapolating in vitro CLint assuming a human liver weight of 1500 g (70 kg body

weight) and that one gram liver tissue contains 45 mg HLM protein; c. CLh was calculated from the well-stirred model: CLh = Q x CLint / Q +

CLint , where Q is the human hepatic blood flow (20 ml/min/kg); KPI: phosphate buffer, pH 7.1, WEM: William E Medium.

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

256

200

400

600

800

1000

1200

1400

1600

1800

020406080100 120 140 160

AG-024322 (M)

Pear Area x 10

/0.375 mg/15 min

Observed

Predicted

Figure 3. Formation kinetics of N-desethyl AG-024322 in

human liver microsomes over the AG-024322 concentration

range of 0.5 to 100 µM.

binant UGTs for activity and comparative enzyme ki-

netic analysis. Incubations with microsomes from cells

individually expressing human UGT1A enzymes (UGT

supersomes) yield 3 distinct glucuronide products of the

parent drug (M1, M2 and M3), corresponding to the

peaks seen after incubation in human hepatocytes. A

majority involvement of UGT1A1 at 1 and 10 µM of

AG-024322 (Figure 4) in the formation of parent drug

glucuronides, M2 and M3, and minor involvement of

UGT1A8 in the formation of M1 and M3 were observed.

The involvement of other UGT1A and UGT2B enzymes

in glucuronidation of AG-024322 was negligible. The

formation of these three glucuronides was further inves-

tigated in HLM incubated at an AG-024322 concentra-

tion range of 0.5 to 200 µM, with the system optimized

for glucuronidation. The formation of AG-024322 glu-

curonides in HLM incubations was also tested in two

buffer systems, the phosphate buffer (pH 7.1) and WEM.

The data fits and the estimated kinetic parameters for the

Figure 4. Comparison of AG-024322 glucuronidation (UGT)

activity at 1 and 10 µM in microsomes from insect cells indivi-

dually expressing human UGT1A enzymes (UGT supersomes).

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.1

0.2

0.3

relative velocity (peak area/mg/30 min)

KPI buffer

WME

AG-024322 (µM)

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.1

0.2

0.3

relative velocity (peak area/mg/30 min)

KPI buffer

WME

AG-024322 (µM)

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.1

0.2

0.3

relative velocity (peak area/mg/30 min)

KPI buffer

WME

AG-024322 (µM)

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.5

1.0

1.5

2.0

2.5

050100 150200

0.0

0.1

0.2

0.3

relative velocity (peak area/mg/30 min)

KPI buffer

WME

AG-024322 (µM)

Figure 5. Comparison of formation kinetics of AG-

024322 glucuronides (M1, M2 and M3) in human liver

microsomes (HL-14) using 100 mM phosphate buffer

(KPI) or WEM (glucose 16 mM) in the liver microsome

incubation.

glucuronidation pathways are shown in Figure 5 and

Table 4, respectively. The extrapolated total in vivo he-

patic clearance via parent drug glucuronidation was 1.22

and 4.72 ml/min/kg using the phosphate buffer and

WEM, respectively.

3.4. [14C]AG-024322 Excretion in Rats

After a 30-min IV infusion of [14C]AG-024322 to intact

Sprague-Dawley rats at 10 mg/kg (~50 µCi/250 g ani-

mal), the total recovery of radioactivity dose averaged

84% by 120 hours, as shown in Table 5. The primary

route of excretion of radioactivity was via the feces

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

257

(76.5%), indicating extensive biliary excretion. A mean

total of 56% of dose was recovered in bile within 48

hours of administration to bile duct-cannulated rats, con-

sisting with the high recovery of radioactivity in feces

from the intact rats. Urinary excretion was minimal with

an average radioactivity recovery of 3.2%. These data

confirmed that the biliary excretion is the major route of

elimination in rats, which is consistent with the findings

from in vitro metabolism studies. The rate of elimination

of radioactivity was moderately rapid and appeared to be

essentially complete by 96 hours postdose (76.1% in

feces and 3.2% in urine). The overall excretion of radio-

activity was similar for males and females.

3.5. Metabolism of AG-024322 in Rats

Profiling and identification of metabolites of AG-024322

in rats were conducted for plasma, urine, fecal and bile

samples from the excretion mass balance study in rats as

described above. AG-024322 underwent extensive me-

tabolism in rats. The representative HPLC-radiochroma-

tograms from rat plasma, urine, feces, and bile are pre-

sented in Figure 6. Two major metabolites were identi-

fied including a direct glucuronide of the parent drug

and M13, a glucuronidation product of M15. M15 is a

desethyl aminated metabolite (an alcohol) of AG-024322.

Other metabolites identified included the N-desethylated

product of AG-024322 (M6), a combined mono-xygen-

ated and dehydrogenated product at the ethyl amine

group of AG-024322 (M16), M15, and the glucuronides

of M6 and M16 (M12 and M14, respectively). The pro-

posed metabolic scheme of AG-024322 in rats is shown

in Scheme 3. The profiles of these metabolites in plasma,

urine, feces, and bile from intact rats and bile-duct can-

nulated rats are presented in Table 6. In plasma, the

Table 5. Recovery (MeanSD) of [14C]AG-024322 in Rats Following 30-min IV Infusion of [14C]AG-024322 at 10

mg/kg (~50 Ci/Rat).

Total Radioactivity Recovered (%)

Sample

Time Interval Male (n=3) Female (n=2)a Overallb

Urine 0-120 h 4.06  0.24 2.4 3.4  0.94

Feces 0-120 h 74.2  6.7 78.8 76.1  6.3

Cage Wash 0-120 h 7.28  12.3 0.31 4.47  9.52

Cage Wipe 0-120 h 0.24  0.21 0.36 0.29  0.23

Intact Rats

Total 0-120 h 85.8  9.4 81.8 84.2  7.7

Bile Duct-Cannulated Rats Bile 0-48 h 55.5  11.2 (n=2)

a. one female rat excluded from calculation of the mean, as a result of difficulties during infusion; b. male and female combined.

1400.0

1200.0

1000.0

800.0

600.0

400.0

200.0

0.0

0.020. 0040.0060.00

Parent Glucuronide

AG-02432 2

M12

M13

M14

M6 M16

DPM

Time (min)

1.9

1.4

0.9

0.4

-0.1

Openly accessible at

Plasma AG -0 2432 2

Parent Glucuronide

0.0020. 0040.0060.00

Time (min)

ARC(CPM)

i n e

Urine

0.00

700.0

600.0

500.0

400.0

300.0

200.0

100.0

0.0

20.0040.00 60.00

Parent Glucuronide

AG -024322

M12

M13

M14

UN K

M15

M16

Feces

DPM

Time (min)

Feces

Plasma

650.

600.0

550.0

500.0

450.0

400.0

350.0

300.0

250.0

200.0

150.0

100.0

50.0

0.0

0.0020.0040.00 60.00

Parent Glucuronide

AG -02 4322

M12

M13

M14 M6

M16

Bile

DPM

Time (min)

Figure 6. Representative radiochromatograms of plasma, urine, fecal, and bile samples from rats following 30-minutes IV

infusion of [14C]AG-024322 at 10 mg/kg (50 µCi/250 g body weight).

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

258

parent drug was the predominant radioactive component,

accounting for 80-88% of the circulating radioactivity,

whereas the parent glucuronide represented 3-5% of

circulating radioactivity. In urine, the parent drug and

parent glucuronide represented the two major radioactive

components, accounting for 1-1.2% and 0.6-1.2% of the

dose, respectively. In feces, the major drug-related com-

ponents included the parent drug, the parent glucuronide

and M13, accounting for 9-19%, 12-29% and 9-21% of

the dose, respectively. In bile-duct cannulated male rats,

the major biliary drug-related components were the par-

ent drug, the parent glucuronide and M13, accounting

for 16%, 9% and 8% of the dose, respectively.

3.6. Structure Elucidation of In Vivo

Metabolites in Rats

The tentative structures of the metabolites observed in

rats were proposed based on the product ion mass spec-

trum generated for each identified metabolite peak by

ionspray LC-MS/MS. The combined Q1, product ion,

and multiple reaction monitoring scanning techniques

were used for the structure elucidation. The proposed

structure of each metabolite, along with the interpreta-

tion of its MS/MS spectrum is presented in Scheme 3.

The parent glucuronide had an radiochromatogram re-

tention time of 25.4 min and an [M+H]+ ion at m/z 595

(=419+176) consistent with a direct glucuronide of the

parent AG-024322. The product ion mass spectrum

showed major fragment ions at 550, 419, and 374. M6

had a retention time of 38.2 min and showed an [M+H]+

ion at m/z 391 (=419–28) resulted from N-desethylation

of AG-024322. The product ion mass spectrum of M6

gave a major fragment ion of 374. M12 had a retention

time of 19.2 min and an [M+H]+ ion at m/z 567 (=419–

28+176) consistent with a glucuronide of M6. The

product ion mass spectrum of M12 gave fragment ions

of 550, 391 and 374. M15 showed a retention time of

49.2 min, and exhibited [M+H]+ at m/z 392 (=419-27),

which is likely formed through desethyl amination of

AG-024322. The product ion mass spectra gave frag-

ment ions of 392 and 374. M13 exhibited [M+H]+ at

m/z568 (=419-27+176) consistent with a glucuronide of

M15. The retention time of M13 was 27.0 min and the

MS/MS mass spectrum yield fragment ions of 568 and

392. M16 exhibited [M+H]+ at m/z 433 (=419+14), re-

NH2

Gluc

NH2

Gluc

NH2C2H5

NH3

NH2C2H5

OH2

AG-024322

(m/z 419)

(m/z 391)

M12

(m/z 567)

M13

(m/z 568)

M14

(m/z 609)

M15

(m/z 392)

- 2H

-2H

Parent Glucuronide

(m/z 595)

550 419

374

550 391

374 -

392

433

550 374

415 433

374

391

M16

(m/z 433)

Scheme 3. Proposed metabolic scheme of AG-024322 in rats.

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

259

Table 6. Metabolite Profiles of AG-024322 in plasma, urine, feces, and bile from rats administered [14C] AG-024322 in-

travenously.

Mean % Radiochromatogram for Plasma and Mean % Dose for Urine, Feces and Bile

Matrix

(Time) Gender Parent Gluc M6 M12M13 M14UnkAG-024322 M15 M16

Pooled male 4.52      87.70  

Plasma

Pooled female2.61      80.06  

Male average1.19 0.19 0.090.35 0.09 1.21 0.13

Urine

Female average0.55 0.05 0.05

  1.07



Male average11.61 2.80 2.0421.343.161.439.44 3.16 4.36

Feces

Female average28.98 1.04 1.669.39 1.202.9718.97 1.86 1.15

Bile Male average 8.99 2.62 1.148.28 1.19 16.13  4.13

: not detectable using either LC-MS or RAM; : Trace metabolites detected using LC-MS only; Parent Gluc: parent glucuronide, M1,

M2 or M3 (M.W. 594); M6: N-desethylated metabolite of AG-024322 (M.W. 386); M12: Glucuronide of M6 (M.W. 566); M13: Glu-

curonide of M15 (M.W. 567); M14: Glucuronide of M16 (M.W. 608); M15: Desethyl amination metabolite of AG-024322 (M.W. 391);

M16: Combined mono-oxygenation and dehydrogenation product of AG-024322 (M.W. 432); Unk: Unknown/unidentified metabolite.

sulted from a combined mono-oxygenation and dehy-

drogenation of AG-024322. The retention time of M16

was 50 min, and its MS/MS mass spectra gave fragment

ions at 433, 415 and 374. M14 demonstrated [M+H]+ at

m/z 609 (= 419 + 14 + 176) consistent with a glucuron-

ide of M16. M14 had a retention time of 28.3 min and its

mass spectra gave major fragment ions of 609, 550, 433

and 374. The chemical structure of unknown (Unk) can-

not be determined with no mass spectrum data available.

4. DISCUSSIONS

The in vitro metabolism of AG-024322 was initially in-

vestigated in liver microsomes incubated with [14C]AG-

024322 to assess the metabolic stability across species.

The experiments were performed with NADPH only to

reflect Phase 1 metabolic activity. Subsequent studies

were also conducted using hepatocytes, which has a

complete ensemble of metabolizing enzymes of all iso-

forms, cofactors, and cellular components, providing an

alternate assessment of in vivo clearance particularly for

compounds that are mainly cleared via glucuronidation

[10,11]. AG-024322 exhibited the highest metabolic

stability in both human hepatocytes and liver micro-

somes as compared to other nonclinical species (Table

1). Extensive microsomal metabolism in the rat and dog

was observed, which is consistent with the high in vivo

clearance observed in these species. The metabolites

identified in human liver microsomes and hepatocytes

were also observed in the nonclinical species evaluated.

Several in vitro metabolites of AG-024322 were tenta-

tively identified from microsome and hepatocyte incuba-

tions using chromatographic resolution, and concurrent

radiometric and mass-selective quantification (Figure 1;

Scheme 2). These metabolites including the unidentified

metabolites as well as the parent drug account for all the

major peaks of radioactivity in microsomes and hepato-

cytes. The N-desethyl metabolite and glucuronides of the

parent drug were major metabolites observed across

species. The monkey exhibited the highest extent of glu-

curonidation in hepatocytes, while dog and rat demon-

strated more extensive N-desethylation and methylation/

N-acetylation in hepatocytes, and produced novel uni-

dentified metabolites in microsomes. Metabolism of AG-

024322 in human hepatocytes and HLM is more compa-

rable to those in monkeys. Both species demonstrated

glucuronodation and oxidative N-desethylation as the

predominant metabolic pathways.

The excretion mass balance study conducted in rats dem-

onstrated that the primary route of excretion of [14C]AG-

024322 was via feces (76.5%) with minimal contribu-

tions (3.2%) of urinary excretion (Table 5). The high

fecal elimination is reflected by biliary elimination that

accounted for approximately 56% of the total radioactiv-

ity within 48 hours of dosing. As proposed in Scheme 3,

the glucuronide of the parent drug and M6 identified in

vitro were also observed in rats following 30-minute IV

infusion of [14C]AG-024322. Only one parent glucuron-

ide, likely M3 based on the retention time, was observed

in rats, which was consistent with the findings that only

M3 was detected in the rat hepatocyte incubations (Fig-

ure 1 and Table 2). The parent glucuronide appeared to

be the most abundant metabolite in plasma and in all

excreta collected, representing up to 29% of dose in fe-

ces. Another major metabolite, M13, was the glucuroni-

dation product of a desethyl aminated metabolite (M15),

representing up to 21% of dose in feces, as UGTs are

primarily involved in conjugation of metabolites from

oxidation reactions. M6, M15 and M16 were oxidative

metabolites, each representing less than 5% of dose, and

were also further conjugated to its glucuronides. The

metabolite profile in bile (Table 6) is supportive to the

findings of extensive biliary excretion, which involves

conjugation of the parent drug and oxidative metabolites

with glucuronic acid prior to excretion into the bile and

direct secretion of the parent drug into bile.

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

260

Reaction phenotyping studies using the standard bat-

tery of microsomes from insect cells expressing individ-

ual human CYPs or UGTs were conducted to assess the

two principal pathways of AG-024322 biotransformation;

oxidation and glucuronidation. The formation of M6 in

human recombinant demonstrated the predominant role

of CYP3A4 enzyme in the oxidative metabolism of AG-

024322. The relative contribution of the identified CYPs

was subsequently examined by inhibition studies with

CYP-selective inhibitors. As shown in Table 2, only

ketoconazole (selective CYP 3A inhibitor) inhibited N-

desethylation of AG-024322 with up to 86% inhibition

in HLM over an 8-minute of incubation. Quinidine,

quercetin or furafyline had no inhibitory effects in the

formation rate of N-desethylation. Quinidine is also a

known CYP3A heterotropic activator [12], and in this

case yields indirect additional evidence towards pre-

dominant participation of CYP3A enzymes (Table 3).

Formation kinetics of this pathway were also examined

in a set of representative human liver microsomes and

resulted in an apparent Km of 10 µM and Vmax of 20

pmol/mg/min. The in vivo CLint and CLh extrapolated

from the in vitro kinetic data [13] were estimated to be

1.86 and 1.7 ml/min/kg, respectively. According to the

projected human clearance from interspecies scaling (12.8

ml/min/kg, data not shown), the formation of M6 was

estimated to contribute 13% of the hepatic clearance.

Glucuronidation catalyzed by UGTs is one of major

pathways for drug metabolism and elimination in hu-

mans. Identification of the UGTs responsible for glu-

curonidation of AG-024322 in vitro would assist in the

prediction of adverse reactions resulting from drug-drug

interactions or genetic polymorphism. The integrated

approaches used here for UGT reaction phenotyping

were using recombinant enzymes and human liver mi-

crosomes with primary focusing on identification of the

well-characterized hepatic UGTs, lA1, 1A3, 1A4, 1A6,

1A8, 1A10, 2B7 and 2B15. The involvement of UGT1

A1 was demonstrated from the UGT recombinant incu-

bation experiments, resulting in three glucuronides,

which were identical with the M1, M2 and M3 peaks

that also observed in human hepatocyte incubations.

UGT1A1 is a polymorphic isoform responsible for the

glucuronidation of structurally diverse drugs, non-drug

xenobiotics and endogenous compounds (e.g. bilirubin

and estrogens) [14,15]. It is the primary UGT responsi-

ble for bilirubin glucuronidation. UGT1A1 is highly

expressed in liver, gastrointestinal tract, and bladder

[16-18]. It has been reported that genetic polymorphisms

of the UGT1A1 gene were significantly related to severe

toxicity of irinotecan [19-21]. AG-024322 showed high

affinity towards UGT1A1 in vitro. Potential interaction

liability of AG-024322 with drugs metabolized by UGT1

A1 is also an area of great interest and warrants addi-

tional studies. It is of concern if glucuronidation via

UGT1A1 plays a major role in the elimination of AG-

024322 in humans as competitive inhibition of UGT1A1

may be associated with toxicity, and in patients exhibit-

ing compromised bilirubin metabolism [22].

Formation kinetics of the three glucuronides of AG-

024322 were further investigated in the same set of hu-

man liver preparation as that for the formation kinetic

study of oxidative metabolism. Both phosphate buffer

(pH 7.1) and WEM for this system were evaluated.

WEM is a carbonate-based hepatocyte media reagent.

Engtrakul [11] examined the effects of microsome incu-

bation condition on the formation of AZT-glucuronide,

including various physiological and nonphysiological

buffer systems, such as Tris, phosphate, acetate, carbon-

ate, citrate buffers and WEM. Their experiments sug-

gested that both carbonate buffer and WEM produced

significantly higher amounts of AZT-glucuronide than

other buffers. Therefore, we also included the WEM in

the formation kinetic system to compare with the com-

monly used phosphate buffer. Based on the in vitro CLint,

the extrapolated total in vivo CLint for the three glu-

curonides, M1, M2 and M3, was 1.25 and 5.25 ml/

min/kg using phosphate buffer and WEM, respectively,

contributing 10% and 37% of the predicted human

plasma clearance. In agree with the findings by Engtra-

kul et al. [11], we also found that in the case of glu-

curonidation of AG-024322, WEM appeared to be a

more physiologically relevant buffer yielded almost 4-

fold higher intrinsic clearance by glucuronidation than

the phosphate buffer. Additional studies to examine the

impact of WEM, and more importantly, its components

to probe the function of UGTs in vitro are warranted. In

combination of both contributions for CYP3A mediated

N-desethylation and UGT1A1-mediated glucuronidation,

total metabolic clearance could account at best for 50%

of the predicted human plasma clearance using WEM.

This value may be an underestimation since our calcula-

tion did not account for the biliary excretion, formation

of oxidative metabolite other than M6, and glucuronida-

tion of Phase I metabolites, which might be significant

consideration based on the findings from the excretion

study in rats. Caution should be made when extrapolat-

ing metabolic lability via glucuronidation of UGT en-

zymes from HLM, since this system appeared to under-

estimate the degree of clearance (UGT 1A1 is expressed

in human and rat and in general exhibited comparable

substrate specificities and efficiencies (Vmax/Km) of the

glucuronide formation, and thus rat and human UGT1A1

are functionally similar [23,24]. It is expected to have

comparable glucuronidation profiles between rats and

humans in vivo.

In our calculation for the well-stirred model, protein

binding value was not used. AG-024322 was highly

bound to human plasma (95%). If the plasma protein

binding of AG-024322 is included in the expression de-

scribing the well-stirred model of hepatic extraction and

if the scaling-up in vitro intrinsic clearance used without

W. Z. Zhong et al. / HEALTH 1 (2009) 249-262

261

microsome binding data, the projected in vivo clearance

is drastically lower. Obach [25] examined the in vivo

hepatic clearance predicted from the in vitro system with

the measured in vivo clearance and was found that in

some cases, the clearance predicted from in vitro using

HLM were substantially lower than those observed in

vivo. The compounds exhibiting such a discrepancy were

all characterized by high protein binding. When plasma

protein binding values were removed from the well-

stirred model for these compounds, scaled-up values for

clearance were remarkably close to those measured in

vivo [26]. Therefore, as an initial estimation of the con-

tribution of the two major metabolic pathways in AG-

024322 clearance, the protein binding value was not

used in the well-stirred model, and was found to be more

reasonable in the clearance projection.

5. CONCLUSIONS

Investigation of in vitro and in vivo metabolism of

AG-024322 demonstrates that UGT1A1 mediated glu-

curonidation and CYP3A mediated oxidation represent

the major metabolic pathways of AG-024322. The esti-

mated contribution of the glucuronidation to total plasma

clearance in humans was approximately 3-fold higher

than that from desethylation of AG-024322. Using the

WE M buffer system in human liver microsomes to as-

sess the metabolic pathway by glucuronidation improved

the clearance predictability of AG-024322. Because of

the polymorphism of UGT1A1 and the reported toxicity

associated with competitive inhibition of UGT1A1 either

by a co-administered drug or in patients exhibiting com-

promised bilirubin metabolism, this information was

used to influence the clinical study design to only in-

clude subjects having normal expression of UGT1A1 in

the first human study thereby decreasing the toxicity risk

to patients.

6. ACKNOWLEDGMENTS

Authors would like to thanks Drs. Hai-Zhi Bu and Deepak Dalvie for

helpful discussion in structure elucidation of AG-024322 metabolites,

Drs. Weiwei Tan and Rachel Courtney for the discussion of the human

study design and sharing the human pharmacokinetic data with us, and

Drs. Scott Fountain, Bill Smith, Ellen Wu and Bhasker Shetty for their

invaluable comments and support of this project.

7. ABBREVIATIONS

CDK, cyclin-dependent kinase; HLM, human liver mi-

crosome; CYP, cytochrome P450; UGT, UDP-glucurono

syltransferase; UDPGA, UDP-glucuronic acid; WEM,

Williams E medium; HPLC, high-performance liquid

chromatography; MS, mass spectrometry; min, min-

ute(s); IS, internal standard; RAM, radiochemical activ-

ity monitor.

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