Identification of a suitable and selective inhibitor towards aldehyde oxidase catalyzed reactions

Article abstract of DOI:10.3109/00498254.2013.819594

1.Aldehyde oxidase (AO) is a liver cytosolic molybdoflavoprotein enzyme whose importance in drug metabolism is gaining in the recent. The objective of this work is to find a potent and selective inhibitor for AO activity using phthalazine oxidation as a marker reaction. 2. Among organic solvents tested, it was identified that methanol was not a suitable choice for AO activity even at concentrations less than 0.2% v/v. Acetonitrile and DMSO did not show any effect till 0.5% v/v but thereafter activites tend to decrease. 3. For selectivity, 23 compounds were selected and evaluated for their effects on AO and nine CYP450 enzymes. Among the tested compounds chlorpromazine, estradiol, hydralazine, quetiapine and raloxifene were selected based on their potency of inhibition towards AO activity. 4. Raloxifene was found to be a non-specific inhibitor of all major tested CYP450 enzymes and was excluded as a selective inhibitor for AO. Quetiapine also showed a degree of inhibition towards the major CYP450 tested. Hydralazine used as a specific inhibitor during the past for AO activity demonstrated a stimulation of AO activity at high and low concentrations respectively and the inhibition noted to be time dependent while inhibiting other enzymes like monoamine oxidase. 5. Estradiol showed no inhibition towards the tested CYP450 enzymes and thus proved to be a selective and specific inhibitor for AO activity with an uncompetitive mode of inhibition.

Full text of DOI:10.3109/00498254.2013.819594

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Xenobiotica, 2014; 44(3): 197–204
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RESEARCH ARTICLE
Identification of a suitable and selective inhibitor towards aldehyde
oxidase catalyzed reactions
1
Ramakrishna Nirogi , Vishwottam Kandikere , Raghava Choudary Palacharla , Gopinadh Bhyrapuneni ,
2
2
2
2
Vijaya Bhargava Kanamarlapudi , Ranjith Kumar Ponnamaneni , and Arun Kumar Manoharan
2
2
1 2
Discovery Research and Pharmacokinetics and Drug Metabolism, Suven Life Sciences Ltd, Serene Chambers, Banjara Hills, Hyderabad, India
Abstract
Keywords
1
2
3
. Aldehyde oxidase (AO) is a liver cytosolic molybdoflavoprotein enzyme whose importance Aldehyde oxidase, cytochrome P450,
in drug metabolism is gaining in the recent. The objective of this work is to find a potent and
selective inhibitor for AO activity using phthalazine oxidation as a marker reaction.
. Among organic solvents tested, it was identified that methanol was not a suitable choice
for AO activity even at concentrations less than 0.2% v/v. Acetonitrile and DMSO did not
show any effect till 0.5% v/v but thereafter activites tend to decrease.
. For selectivity, 23 compounds were selected and evaluated for their effects on AO and nine
CYP450 enzymes. Among the tested compounds chlorpromazine, estradiol, hydralazine,
quetiapine and raloxifene were selected based on their potency of inhibition towards AO
activity.
drug–drug interaction, uncompetitive
History
Received 6 May 2013
Revised 19 June 2013
Accepted 23 June 2013
Published online 22 October 2013
4
5
. Raloxifene was found to be a non-specific inhibitor of all major tested CYP450 enzymes and
was excluded as a selective inhibitor for AO. Quetiapine also showed a degree of inhibition
towards the major CYP450 tested. Hydralazine used as a specific inhibitor during the past for
AO activity demonstrated a stimulation of AO activity at high and low concentrations
respectively and the inhibition noted to be time dependent while inhibiting other enzymes
like monoamine oxidase.
. Estradiol showed no inhibition towards the tested CYP450 enzymes and thus proved to be a
selective and specific inhibitor for AO activity with an uncompetitive mode of inhibition.
Introduction
an important role to play in the metabolism of drugs or
substrates with different chemical structures and functional-
ities, demonstrated by an increase of its role for metabolizing
drugs from a proportion of 0.13 to 0.45 (Garattini & Terao,
Majority of focus for drug–drug interactions was done on
cytochrome P450 (CYP450) enzyme systems, which are
majorly involved in metabolizing many of the administered
drugs (Chauret et al., 1998). With the advances in the
structural activity relationships and drug discovery programs
shifting towards compounds with minimal CYP450 liability,
results in compounds cleared by alternative enzymatic
pathways typically characterized as non-cyp mediated drug
disposition (Obach, 2004; Sahi et al., 2008). Non-cyp
enzymes include the aldehyde oxidase (AO), monoamine
oxidases (MAOs), xanthine oxidase (XO), flavin-containing
monooxygenases (FMOs), aldehyde dehydrogenase (ALDH)
whose involvement in the drug metabolism and precipitation
of drug–drug interactions is under evaluation (Benedetti et al.,
2012). AO is a molybdenum cofactor containing soluble
enzyme present in the liver and other tissues of several
preclinical species (Pryde et al., 2010). It is involved in the
phase I metabolism of the aldehyde group containing
compounds, aza and oxo heterocycle compounds (Garattini
&
Terao, 2012), play a role in the metabolic activation of
certain drugs to their active metabolites like famciclovir to
penciclovir (Clarke et al., 1995; Rashidi et al., 1997). It is also
involved in the clearance of certain agents like zaleplon
(
(
Kawashima et al., 1999; Lake et al., 2002), methotrexate
Jordan et al., 1999), zoniporide (Dalvie et al., 2010),
zonisamide (Sugihara et al., 1996), investigational drugs
Akbane et al., 2011; Connor et al., 2006) and is further
2
006; Panoutsopoulos et al., 2004).
Among all the non-CYP mediated pathways, aldehyde
(
inhibited by many drugs (Obach et al., 2004) which are in use
today. Although direct involvement in metabolism of drugs is
less, AO plays a greater role in the intermediary metabolism
of compounds, since aldehydes are frequent metabolic
intermediates in metabolic reactions (Obach et al., 2004).
Significant species differences are accounted for this
enzyme (Pryde et al., 2010; Sahi et al., 2008) and until date
no sufficient literature unlike the CYP450, to extrapolate
oxidase mediated metabolism of substrates, and its inhibition
by drugs is gaining importance in the recent. AO, through its
unique structure, distribution, and substrate recognition, has
Address for correspondence: Ramakrishna Nirogi, Suven Life Sciences
Ltd, Serene Chambers, Road – 5, Avenue 7, Banjara Hills, Hyderabad –
5
00 034, India. E-mail: ramakrishna_nirogi@yahoo.co.in

1
98 R. Nirogi et al.
Xenobiotica, 2014; 44(3): 197–204
the inhibition potency of a NCE compound under develop-
ment exists. The interest of the scientific community on
this enzyme continues to grow in the recent (Garattini &
Terao, 2011), resulting in a method to scale up the intrinsic
clearance obtained in vitro to in vivo in humans (Zientek
et al., 2010).
standard (IS) 4-phenyl-1-1-phthalazone. The plates were
ꢀ
then centrifuged at 4000 g for 10 min at 4 C. An aliquot of
the supernatant fraction was withdrawn and 10 mL of the
sample was injected on the LC-MS/MS for quantitation
purpose.
Microsomes and hepatocytes are used for various reaction
phenotyping assays wherein certain specific chemical inhibi-
tors are used to identify the major enzymes involved in the
metabolism of the compound under interest. However, for an
accurate assessment of the enzymes involved, the inhibitors
chosen are to be enough selective to the particular enzyme
being tested. Hence, for the proposed work, the selectivity
of such inhibitors used for phenotyping assays with respect
to aldehyde oxidase activity was determined. Twenty-three
compounds were tested for their selectivity towards AO
activity based from research published on single point
inhibition assay for AO (Obach et al., 2004). Prior to the
selectivity assay, we emphasized the possible role of organic
solvents, which are routinely used to solubilize NCE’s for
in vitro drug metabolism studies on AO activity. The
conversion of phthalazine, a specific substrate of human
liver cytosolic aldehyde oxidase isoform to its metabolite 1-
phthalazone was monitored in the presence of various
concentrations of organic solvents.
Determination of selectivity of inhibitors towards
AO activity
Assay was conducted in a total volume of 0.2 mL. Initial
concentration of the inhibitor (5 mM in DMSO: acetonitrile
1:1 v/v) was suitably diluted (first row of the column 1 in
a 96-well microtitre plate) in 0.150 mL of 50 mM phos-
phate buffer to produce 0.050 mM of the inhibitor. For the
remaining columns 2 to 7, 100 mL of 50 mM potassium
phosphate buffer was added. For each row, 50 mL of the initial
inhibitor-buffer mixture from first well was diluted serially till
7th well to produce the concentration range of 0.03 to 25 mM
of each inhibitor selected. To this, 0.050 mL of 12 mM
substrate in phosphate buffer was added and pre-incubated at
ꢀ
3
7 C for 10 min. Reaction was commenced with the addition
of 0.050 mL of 0.2 mg/mL human liver cytosol, so that final
protein concentration and substrate concentration in the
incubation mixture becomes 3.0 mM and 0.05 mg/mL.
Incubation was progressed for 10 min after which 0.150 mL
of the incubation mixture was precipitated in 0.3 mL of
acetonitrile containing the internal standard, 4-phenyl-1-
phthalazone and 0.1% formic acid v/v. Thereafter filtered
through a 0.45 mM Millipore filtration apparatus into a
receiver plate containing 0.1% formic acid in water and
Materials and methods
The substrate, (phthalazine), metabolite (1-phthalazone), and
the internal standard (4-phenyl-1-2(H)-phthalazinone) were
purchased from Sigma Aldrich (St Louis, MO). The solvents
acetonitrile, methanol, and dimethyl sulfoxide (DMSO) were
purchased from Merck chemical Ltd (Mumbai, India). Human
liver cytosolic fraction (Xtreme pool of 200 donors) and
human liver microsomes, (mixed gender, pool of 50) were
purchased from Xenotech, LLC (Lenexa, KS). N-Benzyl
nirvanol, sulfaphenazole were from BD Gentest, alphanaptha-
flavone ticlopidine, ketoconazole and quinidine from Sigma
Aldrich (St Louis, MO). All other chemicals and reagents
were purchased from standard chemical suppliers.
ꢀ
stored at 4 C until analysis.
Selectivity of AO inhibitors towards CYP450
enzyme-mediated reactions
Specific aspects pertaining to incubation condition for each
assay (e.g. protein concentration, incubation time, internal
standard, analytical parameters) instrumentation used to
assess the enzyme activities of CYP1A2 (phenacetin O-
deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6
(
Bupropion hydroxylation), CYP2C8 (amodiaquine N-deethy-
lation), CYP2C9 (diclofenac 4-hydroxylation), CYP2C19
mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan
Effect of organic solvents on AO-mediated
phthalazine oxidation
(
O-demethylation), CYP2E1 (chlorzoxazone 6-hydroxylation)
and CYP3A4 (midazolam 1-hydroxylation) are shown in
Tables 1 and 2.
Initial enzyme kinetics experiments revealed that phthalazine
oxidation was linear with 0.05 mg/mL cytosolic protein and
linear up to 10.0 min of incubation. Therefore, all incubations
were conducted at a protein concentration of 0.05 mg/mL for
All the incubations were conducted in 96 deep well plates.
In brief, microsomes were prepared at the specific concen-
trations as described in Table 2; in 100 mM potassium
phosphate buffer to which the respective CYP isoform probe
substrate was added. The inhibitor in DMSO or acetonitrile
was added and diluted serially to produce the desired working
concentration range of 0.00–45 mM. The reactions were
commenced with the addition of NADPH and at end of
specified incubation period mentioned in Table 2; reactions
were terminated with acetonitrile containing the internal
standard (IS). For all the CYP450 assays, the percentage of
organic solvent in the incubation mixture was kept very low
(not more than 0.1% for DMSO and not more than 1% for
acetonitrile). The plates were centrifuged at 4000 rpm for
10 min and the supernatants were mixed with equal volumes
1
0.0 min. 3.0 mM of substrate (final concentration) prepared
in phosphate buffer was added to a total incubation volume
of 0.2 mL containing 50 mM potassium dihydrogen phos-
phate, (pH 7.4, 0.1 mM EDTA) and 0.05 mg/mL of cytosolic
ꢀ
protein (pre-incubated at 37 C for 15 min) and allowed
the reaction to proceed for 10 min. An appropriate volume
of organic solvent (DMSO, acetonitrile and methanol) was
added to get the desired percentage (0.2, 0.5, 1 and 2% v/v) of
organic solvent and control incubations contained no organic
solvent. The reactions were initiated by the addition of
substrate. At pre-determined time intervals, 150 mL of the
incubation mixture was removed and quenched with 300 mL
of 0.1% v/v formic acid in acetonitrile containing the internal

DOI: 10.3109/00498254.2013.819594
Selective inhibitor towards aldehyde oxidase 199

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00 R. Nirogi et al.
Xenobiotica, 2014; 44(3): 197–204
Table 2. Incubation conditions (protein concentration, incubation time and substrate concentration) for the major CYP450
reactions in liver microsomes and AO in cytosol.
Incubation conditions
Incubation
CYP450/Non-CYP
Enzyme
Cytochrome P450
marker reaction
Protein
(mg/mL)
Substrate
time (min)
concentration (mM)
1
2
2
2
2
2
2
2
3
A2
A6
B6
C8
C9
C19
D6
E1
Phenacetin deethylation
0.025
0.025
0.050
0.025
0.025
0.200
0.025
0.050
0.025
0.050
18
8
20
8
50
1.0
40
1.0
10
75
5
150
2.5
3
Coumarin hydroxylation
Bupropion hydroxylation
Amodiaquine deethylation
Diclofenac hydroxylation
Mephenytoin hydroxylation
Dextromethorphan demethylation
Chlorzoxazone hydroxylation
Midazolam hydroxylation
Phthalazine oxidation
8
40
12
20
2
A4
AO
10
of 0.1% formic acid in water. All samples were analyzed by
LC-MS/MS.
LC-MS/MS analysis
All analytical methods were conducted by HPLC-MS/MS
comprising API-4000 Qtrap mass spectrometer (Applied
Biosystems MDS Sciex, Toronto, Canada) equipped with
a turbo ion spray ionization source. For the analysis of 1-
Phthalazone, separation was achieved on a HPLC system
(
Shimadzu Corporation, Kyoto, Japan) with two LC-20AD
prominences pumps, a SIL-HTc auto sampler unit, CTO-10AS
VP thermo stated column oven and DGU20A3 degasser unit.
1
0 mL of centrifuged samples was injected onto an Atlantis d
C-18 reverse phase column (150 ꢁ 4.6 mm, 5.0 mm; Waters)
pre-equilibrated in a mobile phase consisting of 10 mM
ammonium formate (pH 3.0 with formic acid) and acetonitrile
in the ratio of 85:15% v/v (Mobile phase-A) and 15:85% v/v
Figure 1. Effect of organic solvents on 1-phthalazone formation in
human liver cytosol.
(
Mobile phase-B). Initially mobile phase-B was started with
0% for 0.10 min, followed by a linear gradient from 95%
Results
4
Effect of organic solvents on AO-mediated
pthalazine oxidation
within 1.5 min, maintained until 3.50 min and returned to
initial 40% within 3.51 min and maintained up to 4.0 min
for re-stabilization of column. The retention times of analyte
and the internal standard are 2.31 and 3.32 min at a flow
rate of 1 mL/min. The mass transitions for 1-phthalazone and
The effects of various organic solvents on aldehyde oxi-
dase mediated conversion of phthalazine to its metabolite
1-phthalazone were summarized in Figure 1. As shown
4
-phenyl-1-2(H)-phthalazinone were 147.10 ! 90.10 and
in the graph, the presence of DMSO and acetonitrile at
concentrations of 50.5% v/v did not affect the conversion of
phthalazine to 1-phthalazone with respect to control. There
was a slight increase in the activity of enzyme at lower
concentrations of acetonitrile at 0.2% v/v. At concentrations,
greater than 0.5% v/v of acetonitrile and DMSO, a decrease
in the activity of aldehyde oxidase was observed. However,
under the same experimental conditions, all concentrations
of methanol tested, showed a decrease in the activity of the
enzyme. A mixture of DMSO:acetonitrile in the ratio of
2
23.20 !130.10. Data was collected and processed using
Analyst 1.4.2 software (MDS-SCIEX, Concord, Ontario,
Canada). The analytical conditions followed for each
CYP450 and AO enzyme were given in Tables 1 and 2.
Data analysis
For the effect of organic solvents on AO activity, results
are obtained as an average of triplicate determinations and
are represented as mean ꢂ standard deviation. The ratio of the
averaged activity in the incubations containing organic
solvent to the average activity in the controls containing no
organic solvent was expressed as percent of enzyme activity
remaining with respect to control.
1:1 (v/v) did not show any effect on the aldehyde oxidase
mediated phthalazine oxidation.
Inhibition of AO-mediated pthalazine oxidation
by standard drugs
For the determination of IC50 values, percent activity
remaining of pthalazine oxidation was plotted as a function
of concentration of the inhibition. The data were typically fit
to a sigmoidal dose response variable slope using GraphPad
Prism 4.0. Similar procedure was followed for determining
the IC50 values against the cytochrome P450 enzymes.
Among the compounds tested, raloxifene, hydralazine,
b-estradiol, chlorpromazine, quetiapine were found to be
potent inhibitors of AO-mediated 1-phthalazone formation.
The dose response curves were depicted in Figure 2. The
inhibitory effects of other compounds tested on aldehyde

DOI: 10.3109/00498254.2013.819594
Selective inhibitor towards aldehyde oxidase 201
oxidase activity were moderate to low with IC50 values
ranging from 1.5 mM to >25 mM, as given in Table 3.
CYP1A2 activity: Alphanapthaflavone (IC ¼ 0.03 mM),
50
was included as a positive control inhibitor for phenacetin-
o-deethylation assay. Amlodipine, chlorpromazine and nifedi-
pine showed mild inhibition of phenacetin-O-deethylase
activity while other compounds tested were weak inhibitors
of CYP1A2 activity as found with their IC50 values. Estradiol,
quetiapine did not show any inhibition at the entire concen-
tration range tested. Hydralazine was found to be a weak
inhibitor of CYP1A2 activity
Selectivity of AO inhibitors towards
CYP450 enzymes
All the inhibitors tested for aldehyde oxidase activity were
subjected to their selectivity for individual CYP450-mediated
reaction using human liver microsomes by means of marker
or probe substrate reactions as mentioned above under
materials and methods section. The IC50 values of the
inhibitors were summarized in Table 3. The effect of organic
solvents on cytochrome P450 was evaluated during the
validation of CYP inhibition studies. The organic solvents
DMSO and acetonitrile did not show any inhibition on
cytochrome P450 activities except phenacetin O-deethylation
where more than 0.05% of DMSO inhibited CYP1A2 activity
CYP2A6 activity: All the compounds tested did not show any
inhibition for CYP2A6 mediated coumarin hydroxylation.
Tranylcypromine was employed as a positive control and the
IC50 is consistent with historical values (IC ¼ 0.08 mM).
50
CYP2B6 activity: Bupropion-4-hydroxylation was employed
as the marker reaction for CYP2B6 activity. Amlodipine,
ketoconazole, raloxifene and sertraline showed an inhibition
towards the enzyme activity with IC50 values of 0.63, 1.90,
(
Nirogi et al., 2011).
1.90 and 1.0 mM, respectively. Other compounds tested
showed minimal inhibition of CYP2B6 activity.
a
Ticlopidine (IC50 of 0.07 mM) was included as a standard
diagnostic inhibitor for CYP2B6 activity. Estradiol and
hydralazine were found to be devoid of inhibition potency
towards bupropion hydroxylation but moderate inhibition was
observed with quetiapine and chlorpromazine.
CYP2C8 activity: N-desethyl amodiaquine formation was
monitored for CYP2C8 activity and zafirlukast (IC50 of
0.014 mM) was used as a positive control to monitor the
reaction. None of the compounds tested show any inhibition
towards CYP2C8 mediated enzyme catalysis. Estradiol,
chlorpromazine and raloxifene posses weak to moderate
inhibition towards CYP2C8 activity.
CYP2C9 activity: The formation of 4-hydroxy diclofenac in
human liver microsomes was monitored using sulfaphenazole
(IC50 of 0.165 mM) as probe inhibitor for CYP2C9 activity.
Figure 2. Inhibition of aldehyde oxidase by chlorpromazine, estradiol,
quetiapine and raloxifene.
Table 3. Selectivity of AO inhibitors towards CYP450 enzymes.
IC50 (mM)
Drug
AO
CYP1A2
CYP2A6
CYP2B6
CYP2C8 CYP2C9 CYP2C19
CYP2D6
CYP2E1
CYP3A4
Amlodipine
Chlorpromazine
Cyclobenzaprine
Doxepine
Erythromycin
Estradiol
Hydralazine
Imipramine
Ketoconazole
Loperamide
Nifedipine
Olanzapine
Ondansetron
Phenytoin
Promethazine
Propafenone
Quetiapine
Quinacrine
Raloxifene
Sertraline
Tacrine
Tamoxifene
Verapamil
8.8 ꢂ 4.6
0.27 ꢂ 0.04
6.77 ꢂ 0.3
17 ꢂ 12
4.5 ꢂ 2.9
3.2 ꢂ 0.5
>45
>45
>45
>45
>45
>45
>45
>45
>45
>22.5
>45
>5
>45
>45
>45
>45
>45
>45
>45
>45
>45
>7.5
15 ꢂ 4.8
>45
0.63 ꢂ 0.1
18 ꢂ 2.2
>45
>45
>45
>45
>22.5
>45
1.9 ꢂ 0.4
16 ꢂ 2.7
13 ꢂ 5.4
>45
9.4 ꢂ 5
20 ꢂ 9
>45
>45
>45
19 ꢂ 0.2
>22.5
>45
3.0 ꢂ 0.2
>45
>45
>45
>45
6.8 ꢂ 0.9
15 ꢂ 0.4
18 ꢂ 3
40 ꢂ 5.4
1.9 ꢂ 0.5
15 ꢂ 5.8
2 ꢂ 0.3
>45
>45
33 ꢂ 2.7
>45
>45
>45
35 ꢂ 3.2
>22.5
33 ꢂ 3.8
4.2 ꢂ 0.4
>45
6.3 ꢂ 1.4
12 ꢂ 5.4
15 ꢂ 7.3
27 ꢂ 6.3
30 ꢂ 1.9
>45
29 ꢂ 0.8
19 ꢂ 14
>45
6.4 ꢂ 2
>22.5
0.62 ꢂ 0.14
>45
>45
>45
>22.5
NC
>22.5
>45
>5
>22.5
>45
>5
>22.5
9.6 ꢂ 7.6
7.1 ꢂ 0.88
17 ꢂ 3
29 ꢂ 9
8.8 ꢂ 2.1
31 ꢂ 6.6
0.02 ꢂ 0.01
1.4 ꢂ 0.05
6.9 ꢂ 2.3
>45
>5
24 ꢂ 15
2.1 ꢂ 0.3
8.6 ꢂ 0.8
4.0 ꢂ 2.5
>45
>5
>45
1.0 ꢂ 0.4
>45
3.3 ꢂ 0.5
>45
>45
6.6 ꢂ 0.24
12 ꢂ 3.4
6.5 ꢂ 2.1
>22.5
3.5 ꢂ 0.4 2.4 ꢂ 1.2
15 ꢂ 7.5
>45
4.8 ꢂ 1.7
>45
>45
>45
>45
>45
>45
>45
>45
>45
35 ꢂ 4.5
35 ꢂ 4.5
36 ꢂ 4.7
17 ꢂ 0.2
15 ꢂ 2.6
>45
0.8 ꢂ 0.1
6.4 ꢂ 2.9
>45
18 ꢂ 12
>45
>45
>45
23 ꢂ 9
30 ꢂ 16
>45
>45
31 ꢂ 4.9
3.2 ꢂ 2.1
7.3 ꢂ 5.3
0.94 ꢂ 0.3
5.92 ꢂ 2.1
0.09 ꢂ 0.01
29 ꢂ 7.5
5.31 ꢂ 3.1
3.3 ꢂ 2.9
8.8 ꢂ 5.6
5.3 ꢂ 0.01
17 ꢂ 9
>45
30 ꢂ 0.6
>22.5
18 ꢂ 6
8.7 ꢂ 5.3
>45
27 ꢂ 15
>45
>45
1.8 ꢂ 1.2
0.07 ꢂ 0.0
39 ꢂ 10
0.04 ꢂ 0.0
4.0 ꢂ 1.3
2.4 ꢂ 0.0
15 ꢂ 4.1
12 ꢂ 3.0
>45
19 ꢂ 2.0
>45
9.5 ꢂ 0.1
>45
>45
20 ꢂ 0.2
>45
>45
18 ꢂ 2.8
>45
*
>45
>45
9.8 ꢂ 1.3
10 ꢂ 7
1.9 ꢂ 0.2
1.0 ꢂ 0.5
>45
3.1 ꢂ 2.5 2.7 ꢂ 1.3
5.8 ꢂ 0.91
16 ꢂ 2.8
40 ꢂ 3.2
18 ꢂ 2.2
>45
3.2 ꢂ 2.5
3.4 ꢂ 1.7
>45
15 ꢂ 4.7
>45
>45
>45
24 ꢂ 9.3 3.4 ꢂ 0.34 2.3 ꢂ 1.5
11 ꢂ 8
10 ꢂ 1.6
2.1 ꢂ 0.3
>45
>45 >45 >45
>45
26 ꢂ 1.6
4.6 ꢂ0.2
NA – not applicable; NC – not calculated.
Same transition as 1-hydroxy midazolam.
*

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02 R. Nirogi et al.
Xenobiotica, 2014; 44(3): 197–204
Amlodipine, nifedipine, raloxifene and tamoxifene showed
moderate inhibition of the enzyme while the other compounds
showed a weak inhibition towards CYP2C9 activity.
Except raloxifene, the other four inhibitors selected did not
show inhibition towards CYP2C9 mediated diclofenac
hydroxylation.
activity. Better alternatives and no compromised activity for
enzymes exist as long as the content of these solvents are kept
at a relatively low level. From the results of the study, the
content of DMSO and acetonitrile in the incubation mixture
should be kept lower than 0.5%. Methanol, among the three
solvents tested, showed a significant decrease in the activity
of AO even at the lowest concentration tested (0.2% v/v). This
suggests that methanol would not be a solvent of choice
during assays involving the determination of aldehyde oxidase
activity.
CYP2C19 activity: Raloxifene and N-3-benzylnirvanol
were identified as the potent inhibitors of CYP2C19 mediated
S-mephenytoin hydroxylation with an IC50 of 0.80 and
0.16 mM. Inhibitors other than these showed moderate to
weak inhibition of mephenytoin hydroxylation as exemplified
The objective of the current investigation was to identify
and characterize a selective and potent chemical inhibitor for
AO-mediated enzyme activity in human liver cytosol for its
application as a specific inhibitor for AO activity in
phenotyping assays using hepatocytes. The following study
was carried out since, the inhibitors used for phenotyping
studies in hepatocytes should be selective and potent enough
for the specific enzyme under study and to avoid any bias in
the interpretation of data or prediction of clearance due to that
specific enzyme. However, as the contribution of non-cyp
mediated pathways became evident in the recent literature,
inhibitors selected in phenotyping studies should have no
inhibition towards other CYP450-mediated pathways. One
among such non-CYP450 enzymes involved in the metabol-
ism of drugs either primarily or secondarily is AO. AO,
implication in influencing the drug clearance has been cited
in the literature in recent past, so the selectivity of chemical
inhibitors and drugs used were evaluated to highlight whether
there would be, if possible any inhibition of AO activity.
A data set of 23 compounds was tested for their inhibition
on AO activity and selectivity over other CYP450 enzymes in
human liver cytosol and in microsomes. Among the com-
pounds tested, chlorpromazine, estradiol, hydralazine, que-
tiapine and raloxifene appear to be more potent inhibitors of
AO activity with an IC50 of less than 1.0 mM. Raloxifene was
found to be a very potent inhibitor of phthalazine oxidation
and the mechanism of inhibition is a uncompetitive mode,
thus making a low risk of drug–drug interaction by raloxifene
through an AOX mediated pathway (Barr & Jones, 2011;
Obach et al., 2004). However, it inhibited all the tested
CYP450 enzymes non-selectively and so cannot be used as a
specific probe inhibitor for AO activity even though it has
nanomolar inhibition potency towards AO.
by their IC50 values.
CYP2D6 activity: Inhibition of dextromethorphan hydroxyl-
ation in the presence of CYP2D6 enzyme was monitored in
the presence of quinidine as a probe inhibitor of the reaction.
Propafenone and quinacrine also showed a potent inhibition
towards the enzyme mediated catalysis equipotent to that of
quinidine (IC50 of 0.07 and 0.04 versus 0.04 mM) indicating
that these compounds cannot be a selective inhibitor for AO
mediated activity. Promethazine was also found to be a strong
inhibitor of CYP2D6 with its IC50 value of 1.8 mM.
chlorpromazine and raloxifene inhibited CYP2D6 quite
potently (IC50 1.9 and 4.0 mM). No inhibition was observed
with hydralazine, estradiol and quetiapine.
CYP2E1 activity: Chlorzoxazone-6-hydroxylation was used
as a CYP2E1 marker in both in vitro and in vivo studies
(
Kim et al., 1994; Peter et al., 1990). 4-methyl pyrazole was
included as diagnostic inhibitor for this reaction and with the
exception of ketoconazole wherein a mild inhibition was
observed (IC50 of 4.2 mM) all other tested compounds were
classified as weak inhibitors of CYP2E1.
CYP3A4 activity: Midazolam 1-hydroxylation was markedly
inhibited by the standard inhibitor ketoconazole (IC50 of
0
.02 mM). Along with ketoconazole, loperamide, raloxifene
and tamoxifen were the other inhibitors which showed
inhibition of CYP3A4 activity (IC50 1.0 to 3.0 mM). Other
inhibitors tested showed a weak to no inhibition at the entire
concentration range tested. Propafenone inhibition towards
CYP3A4 activity could not be tested as it had the same
transition with that of 1-hydroxy midazolam and thus
excluded from consideration. Estradiol and hydralazine
failed to inhibit CYP3A mediated midazolam hydroxylation
at the concentration tested unlike chlorpromazine and
quetiapine wherein moderate inhibition was observed towards
midazolam hydroxylation.
Chlorpromazine, a potent inhibitor of AO also inhibited
dextromethorphan hydroxylation catalyzed by the poly-
morphic enzyme CYP2D6. Its inhibition potency towards
other tested enzymes was minimal but its inhibition over
CYP2D6 precludes its usage as a specific inhibitor for AO.
However, in a recent study using I/ki approach (Zientek et al.,
Above all, only five compounds, chlorpromazine, hydra-
lazine, estradiol, quetiapine and raloxifene were proved to be
potent enough for AO activity.
2
010), the potential for in vivo drug–drug interactions for
Discussion
chlorpromazine mediated inhibited phthalazine oxidation was
assessed and the ratio was found to be greater than 0.1
indicating a moderate risk of drug interactions in the clinic
especially when substrates, like zaleplon or carbazeran,
metabolized by AO are administered.
The results of the present study emphasize the role of organic
solvents in influencing the metabolic activities of enzymes to
be studied in a routine preclinical drug discovery program
(
Chauret et al., 1998). It may be desirable to examine two
solvent concentrations to verify that the selected conditions
do not affect the enzyme activity and results thereafter (Busby
et al., 1999). DMSO and acetonitrile represent the solvents
of choice for solubilization of chemical libraries for high-
throughput screening applications for pharmacological
Quetiapine, used as anti-psychotic was also found to be a
potent inhibitor of AO activity. It is not inhibitory towards
the polymorphic CYP450 enzymes but inhibited CYP3A4
enzyme quite potently. However, the IC50 value against
CYP3A4 is 11 fold greater than obtained with AO. This

DOI: 10.3109/00498254.2013.819594
Selective inhibitor towards aldehyde oxidase 203
information may be worthwhile to note when this compound
is used as a diagnostic inhibitor or when a particular enzyme
activity needs to be suppressed.
in hepatocytes experiments, making the way for estradiol as a
diagnostic marker inhibitor.
The arterial vasodilator drug hydralazine, used for the
treatment of hypertension was also found to be a potent
inhibitor of AO activity in human liver cytosol but with an
inhibition potency of less than 10-fold to that of raloxifene.
However, in the concentration range tested, we could observe
a stimulation of AO activity particularly with the higher
concentrations used. The mechanism behind the increase
in phthalazine oxidation is unknown. An IC50 curve was
generated excluding those concentrations where a stimulation
rather than inhibition was found. Hydralazine showed no
inhibition potency towards CYP3A4 and other enzymes tested
Acknowledgements
Authors wish to acknowledge the support received from
Mr. Venkateswarlu Jasti, CEO, Suven Life Sciences Ltd,
Hyderabad.
Declaration of interest
The authors report no conflict of interest. The authors alone
are responsible for the content and writing of this paper.
References
(
IC50 greater than >22.5 mM). Hydralazine, activity towards
inhibiting AO was found to be time dependent (Johnson et al.,
985; Lake et al., 2002) but rather specific to AO than other
Akbane T, Tanaka K, Irie M, et al. (2011). Case report of extensive
metabolism by aldehyde oxidase in humans: pharmacokinetics and
metabolite profile of FK3453 in rats, dogs, and humans. Xenobiotica
41:372–84.
1
CYP450 enzymes tested. In a study (Johnson et al., 1985;
Critchley et al., 1994) similar AO inhibitory activities were
found in vitro, with the potent AO inhibitor hydralazine in
guinea pig, baboon and rabbit liver preparations, and in vivo
with carbazeran in guinea pig and human indicating the lack
of species differences in the inhibition of AO by hydralazine
and the mechanism of inhibition towards AO in human liver
cytosol remains to be explored yet. Further hydralazine was
also shown to be an inhibitor of various enzymes including
monoamine oxidase enzymes with similar inhibition potency
towards AO (Johnson et al., 1985) making its usage
questionable in phenotyping assays.
Barr TJ, Jones PJ. (2011). Inhibition of human liver aldehyde oxidase:
implications for potential drug–drug interactions. Drug Metab Dispos
3
9:2381–6.
Benedetti MS, Whomsley R, Baltes E. (2006). Involvement of enzymes
other than CYPs in the oxidative metabolism of xenobiotics. Expert
Opin Drug Metab Toxicol 2:895–921.
Busby Jr WF, Ackermann JM, Crespi CL. (1999). Effect of methanol,
ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of
cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27:
2
46–9.
Clarke SE, Harrell AW, Chenery RJ. (1995). Role of aldehyde oxidase in
the in vitro conversion of famciclovir to penciclovir in human liver.
Drug Metab Dispos 23:251–4.
Chauret N, Gauthier A, Nicoll-Griffith DA. (1998). Effect of common
organic solvents on in vitro cytochrome P450-mediated metabolic
activities in human liver microsomes. Drug Metab Dispos 26:1–4.
Critchley DJP, Rance DJ, Beedham C. (1994). Biotransformation of
carbazeran in guinea pig: effect of hydralazine pretreatment.
Xenobiotica 24:37–47.
Among all the compounds tested beta estradiol, an
endogenous hormone was found to be potent and a specific
inhibitor of AO activity as found by the compound no
inhibitory effect over the CYP450 enzymes tested. The mode
of inhibition of estradiol towards AO-mediated phthalazine
oxidation was found to be an uncompetitive or a mixed mode
of inhibition and the propensity to produce drug-drug
interactions was found to be less as evidenced by its I/Ki
value in a recent study by (Barr & Jones, 2011) Estradiol
thus became the only inhibitor among the tested compounds
to have absolutely no major CYP450 inhibition and can be a
selective inhibitor of AO.
Dalvie D, Zhang, C, Chen W, et al. (2010). Cross-species comparison
of the metabolism and excretion of zoniporide: contribution of
aldehyde oxidase to interspecies differences. Drug Metab Dispos 38:
6
41–54.
Garattini E, Terao M. (2011). Increasing recognition of the importance
of aldehyde oxidase in drug development and discovery. Drug Met
Rev 43:374–86.
Garattini E, Terao M. (2012). The role of aldehyde oxidase in drug
metabolism. Expert Opin Drug Metab Toxicol 8:487–503.
Jordan MCG, Rashidi RM, Laljee H, et al. (1999). Aldehyde oxidase-
catalysed oxidation of methotrexate in the liver of guinea-pig, rabbit
and man. J Pharm Pharmacol 51:411–18.
Johnson C, Beedham CS, Stell JGP. (1985). Hydralazine: a potent
inhibitor of aldehyde oxidase activity in vitro and in vivo. Biochem
Pharmacol 34:4251–6.
Kawashima K, Hosoi K, Naruke T, et al. (1999). Aldehyde oxidase-
dependent marked species difference in hepatic metabolism of the
sedative-hypnotic, zaleplon, between monkeys and rats. Drug Metab
Dispos 27:422–8.
Kim RB, O’Shea D, Wilkinson GR. (1994). Relationship in healthy
subjects between CYP2E1 genetic polymorphisms and the 6-
hydroxylation of chlorzoxazone: a putative measure of CYP2E1
activity. Pharmacogenetics 4:162–5.
Conclusion
To conclude, the presence of organic solvent in an incubation
assay can affect the outcome and thereby the interpretation of
the data. So before performing any assay, it would be obvious
to determine whether if any, effect of solvents used on the
activities of the enzyme under study. For AO activity, DMSO
and acetonitrile at a low concentration of50.5% would be an
appropriate choice than methanol.
Both, estradiol and hydralazine can be used as a specific
inhibitor of AO activity in phenotyping assays to assess
the possible role or contribution of AO in the clearance of
drug under investigation. Both the drugs have been found
to be inhibitors of AO activity and no inhibitory towards
the CYP450 enzyme systems tested. But stimulation at high
concentrations and time dependent inhibitory effect of
hydralazine (Strelevitz et al., 2012) over AO activity may
preclude its usage as a specific inhibitor of AO activity
Lake BG, Ball SE, Ka J, et al. (2002). Metabolism of zaleplon by human
liver: evidence for involvement of aldehyde oxidase. Xenobiotica 32:
8
35–47.
Nirogi R, Kandikere V, Bhyrapuneni G, et al. (2011). Effect of dimethyl
sulfoxide on in vitro cytochrome P450 mediated phenacetin
O-deethylation in human liver microsomes. Drug Metab Dispos 39:
2
162–4.
Obach RS, Huynh P, Allen CM, Beedham C. (2004). Human liver
aldehyde oxidase: inhibition by 239 drugs. J Clin Pharmacol 44:7–19.
Obach RS. (2004). Potent inhibition of human liver aldehyde oxidase
by raloxifene. Drug Metab Dispos 32:89–97.

2
04 R. Nirogi et al.
Xenobiotica, 2014; 44(3): 197–204
O’Connor D, Jones P, Chambers MS, et al. (2006). Aldehyde oxidase
and its contribution to the metabolism of a structurally novel,
functionally selective GABAA a5-subtype inverse agonist.
Xenobiotica 36:315–30.
Panoutsopoulos GI, Kouretas D, Beedham C. (2004). Contribution of
aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase on
the oxidation of aromatic aldehydes. Chem Res Toxicol 17:1368–76.
Peter R, Bocker R, Beaune PH, et al. (1990). Hydroxylation of
chlorzoxazone as a specific probe for human liver cytochrome
P450IIE1. Chem Res Toxicol 3:566–73.
from human, guinea pig, rabbit and rat liver. Drug Metab Dispos 25:
805–13.
Sahi J, Kishore KK, Black CB. (2008). Aldehyde oxidase activity and
inhibition in hepatocytes and cytosolic fractions from mouse, rat,
monkey and human. Drug met let 2:176–83.
Sugihara K, Kitamura S, Tatsumi K. (1996). Involvement of mammalian
liver cytosols and aldehyde oxidase in reductive metabolism of
zonisamide. Drug Metab Dispos 24:199–202.
Strelevitz TJ, Orozco CC, Obach RS. (2012). Hydralazine as a selective
probe inactivator of aldehyde oxidase in human hepatocytes: estima-
tion of the contribution of aldehyde oxidase to metabolic clearance.
Drug Metab Dispos 40:1441–8.
Pryde DC, Dalvie D, Hu Q, et al. (2010). Aldehyde oxidase: an
enzyme of emerging importance in drug discovery. J Med Chem 53:
8441–60.
Rashidi MR, Smith JA, Clarke SE, Beedham C. (1997). In vitro
oxidation of famciclovir and 6-deoxypenciclovir by aldehyde oxidase
Zientek M, Jiang Y, Youdim K, Obach RS. (2010). In vitro–in vivo
correlation for intrinsic clearance for drugs metabolized by human
aldehyde oxidase. Drug Metab Dispos 38:1322–7.