Zebularine metabolism by aldehyde oxidase in hepatic cytosol from humans, monkeys, dogs, rats, and mice: Influence of sex and inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.07.053

To aid in the clinical evaluation of zebularine, a potential oral antitumor agent, we initiated studies on the metabolism of zebularine in liver cytosol from humans and other mammals. Metabolism by aldehyde oxidase (AO, EC 1.2.3.1) was the major catabolic route, yielding uridine as the primary metabolite, which was metabolized further to uracil by uridine phosphorylase. The inhibition of zebularine metabolism was studied using raloxifene, a known potent inhibitor of AO, and 5-benzylacyclouridine (BAU), a previously undescribed inhibitor of AO. The Michaelis-Menten kinetics of aldehyde oxidase and its inhibition by raloxifene and BAU were highly variable between species.

Full text of DOI:10.1016/j.bmc.2005.07.053

Bioorganic & Medicinal Chemistry 14 (2006) 62–66
Zebularine metabolism by aldehyde oxidase in hepatic cytosol
from humans, monkeys, dogs, rats, and mice:
Influence of sex and inhibitors
Raymond W. Klecker,* Richard L. Cysyk and Jerry M. Collins
Laboratory of Clinical Pharmacology, Center for Drug Evaluation and Research, Food and Drug Administration,
Silver Spring, MD 20993, USA
Molecular Therapeutics Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
Received 14 July 2005; revised 27 July 2005; accepted 27 July 2005
Available online 6 September 2005
Abstract—To aid in the clinical evaluation of zebularine, a potential oral antitumor agent, we initiated studies on the metabolism of
zebularine in liver cytosol from humans and other mammals. Metabolism by aldehyde oxidase (AO, EC 1.2.3.1) was the major cat-
abolic route, yielding uridine as the primary metabolite, which was metabolized further to uracil by uridine phosphorylase. The inhi-
bition of zebularine metabolism was studied using raloxifene, a known potent inhibitor of AO, and 5-benzylacyclouridine (BAU), a
previously undescribed inhibitor of AO. The Michaelis–Menten kinetics of aldehyde oxidase and its inhibition by raloxifene and
BAU were highly variable between species.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
among species, strains of species, and even between male
and female of the same strain, and sex differences in AO
activity can be regulated by hormone levels.4,8–10
Zebularine is a pyrimidinone ribonucleoside that targets
epigenetic modulation of DNA methylation.1,2 It is
being proposed for clinical evaluation as an oral antitu-
mor agent. However, in a recent preclinical pharmacoki-
netic study of zebularine, the authors found poor oral
bioavailability in rhesus monkey, Fisher 344 rat, and
CD2F1 mouse.3 In this study, only parent zebularine
was measured, leaving open the possibility that metabo-
lism might account for its poor oral bioavailability.
Raloxifene (Rlx) was developed as a selective estrogen
receptor modulator and is recognized with antiosteopo-
rotic and other beneficial properties.11,12 Raloxifene is
an oral drug, clinically available and a potent inhibitor
of AO.13,14 5-Benzylacyclouridine (BAU) was first used
as a laboratory tool for inhibition of uridine phos-
phorylase.15–17 It was studied in animals to reduce
5-fluorouracil toxicity and potentiate the antitumor
activity 5-fluoro-20-deoxyuridine and subsequently
Phase I clinical trials were performed.18–20
Zebularine is a cytidine analog that lacks the amino
group normally found on the 4-position of the cytosine
base. The 2-hydroxypyrimidine base of zebularine,
which does not have a ribose sugar, has been identified
The results of our study, contained in this report, show
that zebularine is an excellent substrate for hepatic AO,
indicating that the poor oral bioavailability of zebular-
ine may be due to rapid hepatic metabolism of absorbed
zebularine to uridine with further hepatic degradation to
uracil, Figure 1. No added cofactors are required for
these enzyme activities. In a search for inhibitors of
AO to enhance oral bioavailability of zebularine clini-
cally, we investigated a known potent inhibitor, raloxif-
ene, and discovered a previously unknown inhibitor,
5-benzylacyclouridine.11,12 Moreover, results obtained
with human liver cytosol were compared to those from
as
a substrate for aldehyde oxidase (AO, EC
1.2.3.1).4,5 Additionally, reports have described the
activation by AO of closely related compounds 5-iodo-
2-pyrimidinone-20-deoxyribose (IPdR) and 5-fluoro-2-
pyrimidinone to 5-iodo-20-deoxyuridine (IUdR) and
5-fluorouracil, respectively.6,7 The activity of AO differs
Keywords: Aldehyde oxidase; Zebularine; Raloxifene; 5-Benzylacyclo-
uridine; Hepatic metabolism; Inhibition.
*
Corresponding author. Tel.: +1 301 796 0114; fax: +1 301 796
9818; e-mail: klecker@cder.fda.gov
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.053

R. W. Klecker et al. / Bioorg. Med. Chem. 14 (2006) 62–66
63
O
O
N
O
HN
HN
O
N
O
N
H
O
N
Uridine
Phosphorylase
O
Aldehyde
Oxidase
HO
HO
OH
OH
OH
OH
Zebularine
Uridine
Uracil
Figure 1. Enzymatic pathway of zebularine. Zebularine is metabolized by AO to uridine in hepatic cytosol. Uridine is metabolized further to uracil
by uridine phosphorylase. No added cofactors are required for these enzyme activities.
160
Zebularine
140
120
100
Uridine
80
60
Uracil
12.5
2.5
5
7.5
10
15
17.5
Retention Time (min
Figure 2. HPLC radio-chromatogram of zebularine and metabolites. Sample was from rat hepatic cytosol, 120 lg protein, and 40 lM
[
14C]zebularine, and subjected to 15 min incubation. Chromatogram was acquired by on-line radioactivity detection. Zebularine was metabolized by
AO to uridine (6.5% of radioactivity) and uridine was metabolized further by uridine phosphorylase to uracil (1.1% of radioactivity).
3
hepatic cytosolic preparations from mammalian species
used in the preclinical development of zebularine.
2
2. Results
Female
Two HPLC columns in series were required to separate
uridine from zebularine when using radioactivity detec-
tion with a 0.5 ml flow cell. The amount of cytosolic
protein was varied to keep the total metabolism in each
sample less than 25% of the parent. Figure 2 shows the
chromatogram of a typical separation of zebularine and
metabolites. The peaks labeled uracil and uridine eluted
with and had the same spectra as uracil and uridine ref-
erence standards (data not shown). If zebularine was a
substrate for uridine phosphorylase, its product would
be the 2-hydroxypyrimidine base. The reference stan-
dard for the base elutes near uracil and no evidence
was found to suggest that zebularine was a direct sub-
strate for uridine phosphorylase.
1
0
Male
0
100
200
300
400
500
Zebularine (µM)
Figure 3. Velocity (nmol/min/mg protein) vs zebularine concentration
(lM). Velocity of zebularine metabolism was determined from
triplicate samples of pooled hepatic cytosol from male (n = 28) and
female (n = 14) humans. The lines are the plots generated using the
estimated Km and Vmax values from the Lineweaver–Burk analysis.
The symbols are the mean and standard deviation of triplicate samples.
ley rat. The CD-1 mouse had the greatest differences in
values of Km and Vmax between the sexes. In fact, the
male mouse had the highest Km and Vmax values for
all the species. The Km value in the female was 4-fold
lower than in the male and the Vmax value was 50-fold
lower than the value in the male mouse. The female
mouse had the lowest measurable Vmax of all the cyto-
solic fractions, except the Beagle dog. Despite using
3 mg protein in the cytosol incubation, there was no evi-
dence of enzymatic activity in the male Beagle dog.
The metabolic rate for different concentrations of zebul-
arine in both male and female human liver cytosol is
shown in Figure 3 and the Lineweaver–Burk in Figure
4. These analyses were performed for each species and
segregated by sex where possible.
The Vmax and Km values in the male and female human
liver cytosol were similar (Table 1). Likewise, there were
no sex differences for these values in the Sprague–Daw-

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R. W. Klecker et al. / Bioorg. Med. Chem. 14 (2006) 62–66
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
10
Male
Female
8
Mouse, male
0.2
-
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
1/C (µM)
6
4
2
0
Figure 4. Lineweaver–Burk plot. Lineweaver–Burk analysis, 1/v
(nmol/min/mg protein) vs 1/C (lM zebularine), of the human data
presented in Figure 3. The slope and y-intercept were used to
determine the Km and Vmax for metabolism of zebularine by AO.
The lines are the linear regression fit of the data and the symbols are
the mean and standard deviation of triplicate samples.
Monkey, male
Human, female
Table 1. Km (lM) and Vmax (nmol/min/mg protein) for aldehyde
oxidase in hepatic cytosol of different species
a
a
Human, male
Sex
Km
Vmax
Human
Male
7.3
8.4
2.1
2.7
4.6
Rat, female
Rat, male
Female
Male
Male
Cynomolgus monkey
Beagle dog
Sprague–Dawley rat
15
Mouse, female
No activity
Male
11
11
0.85
0.90
0
100
200
300
400
500
Female
Male
Female
CD-1 mouse
102
27
11
0.22
Zebularine (µM)
Figure 5. Metabolism of zebularine by AO in hepatic cytosol. Lines
show the plots obtained when the estimated Km and Vmax values are
applied to the Michaelis–Menten equation for enzyme metabolism.
Solid lines were used for males and dashed lines for females of each
species. Symbols showing the data and standard deviation were
omitted to improve the clarity of the plot. A representative goodness of
fit can be seen in the male and female human data shown in Figures 3
and 4.
a Values are determined from the slope and intercept of double reci-
procal plots obtained from triplicate samples ranging over six or
more concentrations of zebularine.
Figure 5 compares the velocity versus substrate concen-
tration curves for each species. For human and rat, there
was little difference in the curves for the male and fe-
male. As previously noted, the greatest difference was
observed in the data for the male and female mice. At
low concentrations, <15 lM, the velocity of metabolism
in the male mouse was less than in the male and female
human as well as the male monkey.
Table 2. Inhibition of zebularine metabolism in male hepatic cytosol
of different species
a
Approximate IC50
BAU (lM)
Rlx (nM)
Human
250
800
8
500
Inhibition of zebularine metabolism by raloxifene was
most sensitive in human liver cytosol with an IC50 value
<10 nM (Table 2). The IC50 values for monkey, mouse,
and rat are greater than 50-fold higher than in human.
The second inhibitor, BAU, was discovered while using
it as a potential tool to simplify the analysis of metabo-
lites by blocking uracil formation through inhibition of
uridine phosphorylase (Fig. 1). In addition, it was no-
ticed that zebularine metabolism was also inhibited.
The IC50 value for BAU is lowest in the human cytosol
(200–300 lM) and only 3- to 4-fold more potent than in
monkey and mouse. The IC50 value for rat liver cytosol
is about the same as for human.
Cynomolgus monkey
Sprague–Dawley rat
CD-1 mouse
300
>800
>1100
500
a Approximate IC50 value determined from percent inhibition in
duplicate samples using two concentrations of zebularine and three
concentrations of inhibitor.
prehensive surveys performed at the same time using the
same substrate in mammalian species that are used for
preclinical testing. We found that the male CD-1 mouse
had the highest values of Vmax and Km for AO in the
cytosol fractions tested. The female CD-1 mouse had a
50-fold lower value of Vmax but still a higher value for
Km than the other species. No other strains of mice were
tested. The Beagle dog had no detectable AO activity
using zebularine as a substrate, in agreement with other
reports of little or no AO activity in the dog.21,22 Care
must be taken when evaluating chemotherapeutic agents
3. Discussion
The amount of AO activity is variable among species,
strains, and sex.4,8–10 Our study is one of the most com-

R. W. Klecker et al. / Bioorg. Med. Chem. 14 (2006) 62–66
65
with primary elimination routes of enzymatic degrada-
4. Conclusion
tion by AO. There may be differences in activity or tox-
icity when evaluating these agents in different species,
different strains of species, and even different sexes of
the same strain, especially in the mouse.
Zebularine is metabolized by aldehyde oxidase in hepat-
ic liver cytosol from most mammals. The amount of
activity can vary among species, between sexes of the
same species, especially CD-1 mouse, and is absent in
at least one species, the Beagle dog. The metabolism
of zebularine by aldehyde oxidase can be inhibited by
raloxifene and BAU. Both inhibitors might be used clin-
ically to improve the oral bioavailability of zebularine
and other pyrimidinone nucleosides, such as IPdR.
Toxicity studies of chemotherapeutic agents under devel-
opment are performed in various mammalian species.
Due to the variable AO activity among species, it will be
more difficult to predict a safe starting dose for humans
based on the preclinical testing of zebularine. On the other
hand, differences in AO activity in the different sex of the
same strain of mouse may be helpful in distinguishing tox-
icity caused by the parent zebularine molecule.
5. Experimental
Yoshihara and Tatsumi described differences in rates of
2-hydroxypyrimidine metabolism by purified hepatic
AO from male and female mice.4 In that study, there
were no sex-related differences in inhibition of AO by
several inhibitors. Hence, we studied the inhibition of
zebularine metabolism in only the males of each species.
The Lineweaver–Burk analysis at the different inhibitor
concentrations for each species suggested a mixed non-
competitive inhibition for both inhibitors.23 Obach dem-
onstrated that raloxifene could be an uncompetitive or a
noncompetitive inhibitor depending on substrate.14
Zebularine and [2-14C]zebularine, 18.3 mCi/mmol, were
obtained through the Drug Synthesis and Chemistry
Branch, Developmental Therapeutics Program, NCI,
Rockville, MD. Raloxifene (Rlx) was acquired commer-
cially. 5-Benzylacyclouridine (BAU) was a generous gift
from Shih Hsi Chu, PhD, Brown University, Provi-
dence, RI.
5.1. Kinetic and inhibition studies
The hepatic cytosolic fractions prepared from human,
Cynomolgus monkey, Beagle dog, Sprague–Dawley
rat, and CD-1 mouse were obtained from CellzDirect,
Los Angeles, CA. The human liver cytosol was pooled
from 28 male or 14 female individuals. Each 300 ll en-
zyme assay consisted of 50 ll of 500 mM potassium
phosphate, pH 7.5, containing 50 mM EDTA, 150 ll
water inhibitor, 50 ll diluted cytosol, and 50 ll
[14C]zebularine (varying concentrations) to start the
incubation. The amount of cytosolic protein used per
sample ranged from 0.02 to 0.2 mg for each species, ex-
cept the male Beagle dog (up to 3 mg protein). Incuba-
tion was performed at 37 ꢁC for 15 min and stopped
by placing the samples in boiling water for >2 min.
Samples were centrifuged for 5 min at ꢀ20,000g. The
supernatant was injected directly into the HPLC.
Our data suggest that hepatic AO may be the limiting fac-
tor in the oral bioavailability of zebularine. Preclinical
studies in monkey, rat, and mouse indeed describe a low
oral bioavailability.3 One means to increase bioavailabil-
ity may be coadministration of an inhibitor of AO.
Raloxifene, an antiosteoporotic, is clinically available
and a potent inhibitor of AO.13,14 The concentration re-
quired to inhibit zebularine metabolism by 50% in human
liver cytosol is less than 10 nM. In healthy volunteers giv-
en a single 185 mg oral dose of raloxifene, plasma concen-
trations reached 25 nM.12 Higher concentrations of
raloxifene are likely along its initial path of absorption.
Thus, modulation of zebularine catabolism in humans is
feasible.
BAU was first used as a laboratory tool for inhibition of
uridine phosphorylase.15–17 When given to animals and
as an investigational drug in humans, BAU reached suf-
ficient concentrations to inhibit uridine phosphory-
lase.18–20 BAU can be given orally and may provide an
alternate choice for clinical inhibition of AO activity.
Metabolic studies were performed in triplicate at zebul-
arine concentrations that were approximately <0.3 to
>10 times the value of Km for each species. Inhibition
studies were performed in duplicate at 10 and 40 lM
zebularine. Three inhibitor concentrations were chosen
in an attempt to bracket the IC50 value for each species
and were compared to uninhibited controls.
Moreover, inhibition of hepatic AO may be beneficial to
the clinical effectiveness of other pyrimidinones. IPdR is
a prodrug that is activated by AO to yield IUdR.6 IUdR
is metabolized by hepatic thymidine phosphorylase and
much of it may be inactivated to IUra during absorption
without reaching the systemic circulation. Despite the
fact that AO is required for activation of IPdR, it may
be beneficial to initially inhibit this activation until the
maximum amount of IPdR can be absorbed. For orally
absorbed drugs that are activated by AO and then
quickly inactivated in the liver by different enzymes,
an inhibitor of AO may increase the absorption of the
prodrug, allowing an increased circulation of the desired
activated drug to reach its target in the cell.
The samples were analyzed by HPLC with on-line radio-
activity detection with a 0.5 ml flow cell and a scintilla-
tion cocktail rate of 4 ml/min. The mobile phase
consisted of 10 mM sodium acetate, pH 5, at a flow rate
of 0.6 ml/min through two Zorbax SB300, C8, 5lm,
4.6 · 250 mm, columns (Agilent Technologies, Palo
Alto, CA) in series.
5.2. Lineweaver–Burk analysis
The values for Km and Vmax were determined from the
linear regression fit of the double reciprocal plot of 1/ve-
locity (nmol/min/mg protein) vs 1/concentration (lM).

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Vmax is Km/slope.
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