Evaluation of Cytochrome P450 Selectivity for Hydralazine as an Aldehyde Oxidase Inhibitor for Reaction Phenotyping

Article abstract of DOI:10.1016/j.xphs.2018.11.007

Hydralazine has been reported as a selective mechanism-based inactivator of aldehyde oxidase (AO) and it is widely used in the pharmaceutical industry for reaction phenotyping to estimate fraction metabolized by AO and to identify AO substrates. In this study, however, hydralazine was found to inhibit CYP1A2, 2B6, 2D6, and 3A in human suspension hepatocytes under reaction phenotyping assay conditions, at concentrations that chemically knocked out most of the AO activities (≥50 μM). Furthermore, hydralazine is a time-dependent inhibitor of CYP1A2. Based on these findings, precautions need to be taken when using hydralazine as an AO inhibitor for in vitro studies because fraction metabolized by AO is likely to be overestimated and the likelihood of false positives in identifying AO substrates increases.

Full text of DOI:10.1016/j.xphs.2018.11.007

Journal of Pharmaceutical Sciences xxx (2019) 1-4
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences
journal homepage: www.jpharmsci.org
Note
Evaluation of Cytochrome P450 Selectivity for Hydralazine as an
Aldehyde Oxidase Inhibitor for Reaction Phenotyping
Xin Yang, Nathaniel Johnson, Li Di*
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc, Groton, Connecticut 06340
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydralazine has been reported as a selective mechanism-based inactivator of aldehyde oxidase (AO) and
it is widely used in the pharmaceutical industry for reaction phenotyping to estimate fraction metab-
olized by AO and to identify AO substrates. In this study, however, hydralazine was found to inhibit
CYP1A2, 2B6, 2D6, and 3A in human suspension hepatocytes under reaction phenotyping assay condi-
Received 10 August 2018
Revised 16 October 2018
Accepted 2 November 2018
tions, at concentrations that chemically knocked out most of the AO activities (ꢀ50
mM). Furthermore,
hydralazine is a time-dependent inhibitor of CYP1A2. Based on these findings, precautions need to be
taken when using hydralazine as an AO inhibitor for in vitro studies because fraction metabolized by AO
is likely to be overestimated and the likelihood of false positives in identifying AO substrates increases.
© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords:
cytochrome P450 (CYP)
hepatocyte(s)
human liver microsomes
inhibition
metabolism
liquid chromatography-mass spectrometry
(LC-MS)
drug metabolizing enzyme(s)
drug design
in vitro model(s)
metabolic clearance
Introduction
differences in AO expression and activity further complicate our
ability to translate the impact of AO on drug metabolism and
Aldehyde oxidase (AO) has gained significant attention in
recent years due to a number of clinical drug candidate failures
caused by underprediction of human clearance and toxicity.1-5
With the current high throughput metabolic stability screening
paradigm using liver microsomes to identify metabolic liabilities,
the impact of AO on drug metabolism is often overlooked in drug
discovery, as AO is a cytosolic enzyme and not present in liver
microsomes. Screening of AO metabolic stability must, therefore,
be run in liver cytosols or hepatocytes. Another challenge to
measure AO contribution to clearance is that common drug dis-
covery animal models often do not express AO (e.g., dogs) or have
quite variable AO activities (e.g., rats and mice).5-8 Species
pharmacokinetics from preclinical species to humans. Significant
advances have been made over the years to understand AO
in vitroein vivo extrapolation, species differences, animal models,
drug-drug interaction potential, interindividual variability, tissue
distribution, and genetic polymorphism.1-3
Determination of fraction metabolized by AO (Fm,AO) is the first
step toward building in vitroein vivo extrapolation and under-
standing AO victim drug-drug interaction potential. A selective AO
inhibitor is critical for the identification of AO substrates, the
evaluation of AO-mediated metabolic pathways, reaction pheno-
typing, and Fm,AO determination. Raloxifene, a selective estrogen
receptor modulator, has been shown to be a potent, reversible, and
noncompetitive AO inhibitor.9 However, raloxifene also inhibits
many major cytochrome P450s (CYPs) making it unsuitable as a
selective AO inhibitor in human hepatocytes for reaction pheno-
typing studies.10 Raloxifene may still be used in liver cytosols to
differentiate the contribution between AO and XO (xanthine oxi-
dase) because CYPs are not present in cytosols.11 An alternative
selective AO inhibitor is therefore needed for Fm,AO determination
and for identifying AO substrates in hepatocytes. Hydralazine, an
antihypertensive agent, has recently been reported as a selective
mechanism-based inactivator of AO, but the detailed mechanism
Abbreviations used: AO, aldehyde oxidase; CYP, cytochrome P450; Fm, fraction
metabolized; Fm,AO, fraction metabolized by aldehyde oxidase; IC50, half maximal
inhibitory concentration; IVIVE, in vitroein vivo extrapolation; Ki, inhibition con-
stant; Km, substrate concentration at half the maximum velocity; kobs, inactivation
rate; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MBI, mecha-
nism-based inactivator; MRM, multiple reaction monitoring; rpm, revolution per
min; [S], substrate concentration; TDI, time-dependent inhibitor; XO, xanthine
oxidase.
* Correspondence to: Li Di (Telephone: þ1 860 715 6172).
E-mail address: Li.Di@pfizer.com (L. Di).
https://doi.org/10.1016/j.xphs.2018.11.007
0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

2
X. Yang et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-4
of inactivation is not well understood.12 Although hydralazine
appears to be quite selective against various CYPs based on pub-
lished data using reversible inhibition assay conditions,10,12 studies
on internal drug discovery compounds using the hepatocyte relay
reaction phenotyping assay conditions13 indicate that hydralazine
might have an inhibitory effect on CYPs. Other group also found
more pronounced CYP2D6 inhibition by hydralazine.14 To under-
stand the broader applications of hydralazine as a selective AO
inhibitor in hepatocytes for reaction phenotyping studies, selec-
tivity against 7 CYPs was evaluated in this study. The results can
help to define the potential limitations of using hydralazine for AO
reaction phenotyping, Fm,AO determination, and AO substrate
identification.
CYP Time-Dependent Inhibition Study of Hydralazine
The CYP1A2 time-dependent inhibition study by hydralazine was
conducted using the “Add Method” described in the literature15
Hydralazine (50
tocytes in 100
shaker (150 rpm) in an incubator (95% air/5% CO2 and 75% relative
humidity) at 37ꢁC. At various time points within 2 h, 10
L of
phenacetin (1A2 substrate, 50 M final concentration) or solvent
mM) was incubated with human suspension hepa-
mL of 0.5 million cells/mL cell density on an orbital
m
m
control was added to incubation mixture and continued to incubate
for another 5 min. The reactions were quenched with cold acetoni-
trile containing internal standard, centrifuged, and the supernatant
was transferred to a new plate for LC-MS/MS analysis. A similar
time-dependent inhibitor (TDI) experiment was also conducted in
human liver microsomes at 0.5 mg/mL in the presence of nicotin-
amide adenine dinucleotide phosphate collecting time points within
1 h with phenacetin or 6 substrate cocktails (1A2, 2C8, 2C9, 2C19,
2D6, and 3A).16 In all experiments, organic solvent content was
<0.1%. Percentage activity remaining was calculated and kobs (inac-
tivation rate) values were obtained using the initial slope based on
the Ln (% activity remaining) versus time curve.17 A parallel line test
was performed to evaluate the difference with and without in-
hibitors (Prism 6 for Windows, Version 6.03; GraphPad Software,
Inc.).
Materials and Methods
Materials
All chemicals were obtained from Pfizer Global Material Man-
agement (Groton, CT) or purchased from Sigma-Aldrich (St. Louis,
MO) unless specified. Cryopreserved human hepatocytes (Lot DCM)
consisting of 10 donors with 3 men and 7 women were custom-
pooled and prepared by BioreclamationIVT (Baltimore, MD). Hu-
man liver microsomes (Lot HLM103) of 50 donors containing 36
men and 14 women were purchased from Xenotech (Kansas City,
KS). Polystyrene plates were purchased from Corning (Corning,
NY).
LC-MS/MS Conditions
The LC mobile phases were (A) high performance liquid chroma-
tographygradewatercontaining0.1%formicacid; and (B) acetonitrile
containing 0.1% formic acid. The following solvent gradient or
equivalent was used: 95%(A)/5%(B) for 0.3 min, 95%(A)/5%(B)-5%(A)/
95%(B) from 0.3 to 1.0 min, 5%(A)/95%(B) from 1.0 to 1.7 min, 5%(A)/
95%(B)-95%(A)/5%(B) from 1.7 to 2.0 min. A flow rate of 0.5 mL/min
was used to elute the compounds from the column (Kinetex C18, 30ꢂ
CYP Selectivity Study of Hydralazine
Hepatocytes were thawed and resuspended in Williams me-
dium E (WEM GIBCO-BRL, custom formula supplemented with 50
mM HEPES and 26 mM Na2CO3). The cells were counted and
viability was determined using the trypan blue exclusion method.
Hydralazine at final concentrations of 25, 50, 100, and 150
added to suspended hepatocytes at a density of 0.5 million cells/mL
in 200
L total volume and preincubated for 30 min at 37ꢁC to
inactivate AO.13 Media (170
L) containing hydralazine were
mM was
3 mm, 2.6 mm; Phenomenex, Torrance, CA). A sample aliquot of 3 mL
was injected for analysis using a CTC PAL autosampler (Leap Tech-
nology, Carrboro, NC). Shimadzu high performance liquid chroma-
tography AD30 pumps (Columbia, MD) connected to an AB Sciex
(Foster City, CA) 5500 triple quadrupole mass spectrometer equipped
with a TurboIonSpray source using MRM mode was also used. Ana-
lyst™ 1.5.2software (AppliedBiosystems, FosterCity, CA) wasapplied
to data collection, processing, and analysis.
m
m
removed from the hepatocytes, and fresh media containing sub-
strates were added to the cells with a final cell density of 0.5 million
cells/mL, 1 mM substrate concentration, and 200 mL final incubation
volume. The preincubation hydralazine concentrations were
approximately 7-fold higher than those in the final incubation
(3.75, 7.5, 15 and 22.5 mM). The specific substrate reactions were
Results and Discussion
midazolam (3A) to 10-OH-midazolam, dextromethorphan (2D6) to
dextrorphan, diclofenac (2C9) to 40-OH-diclofenac, paclitaxel (2C8)
The percentage inhibition data of hydralazine against 7 CYP en-
zymes and AO at 4 concentrations are summarized in Table 1 with
hydralazine removed from media after 30 min preincubation.13
Hydralazine showed ꢀ85% inhibition of AO at concentrations ꢀ50
to 6a
-OH-paclitaxel, S-mephenytoin (2C19) to 40-OH-S-meph-
enytoin, phenacetin (1A2) to acetaminophen, bupropion (2B6) to
OH-bupropion, and zaleplon (AO) to oxozaleplon.12,13 Metabolite
formation was monitored to detect the specific metabolic reaction
by each of the enzymes. Detailed mass transitions of the metabo-
mM in the suspension human hepatocytes suggesting 50 mM
lites have been described previously12,13 and the 6
a-OH-paclitaxel
Table 1
MRM transition m/z was 870/286. The plates were put on an
orbital shaker (150 rpm; VWR, Radnor, NJ) in a 37ꢁC incubator (95%
air/5% CO2 and 75% relative humidity). At various time points (0, 5,
Selectivity of Hydralazine at 4 Concentrations Against CYP Enzymes in Suspension
Human Hepatocytes With 30 min Preincubation
Enzymes
Substrate
% Inhibition Standard Deviation
25 50 100 150 mM
10, 20, 30, 45, and 60 min), 10
mL of hepatocyte suspension was
m
M
m
M
m
M
removed from the incubation and added to 100
m
L of cold aceto-
nitrile containing internal standard (10 ng/mL terfenadine). The
solution was centrifuged (Eppendorf, Hauppauge, NY) at 3000 rpm
for 10 min at room temperature and the supernatant was trans-
ferred for liquid chromatography-tandem mass spectrometry (LC-
MS/MS) analysis. Percentage inhibition was calculated using the
initial slope of the metabolite formation time course using peak
area ratio from LC-MS/MS (Prism 6 for Windows, Version 6.03;
GraphPad Software, Inc., La Jolla, CA).
CYP1A2
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A
AO
phenacetin
bupropion
paclitaxel
38 13
15 14
56 22
22 14
67 15
33 14
74 11
36 12
5
7
2
6
7
5
3
9
5
4
7
4
7
4
6
8
diclofenac
13
7
4
10
8
6
S-mephenytoin
dextromethorphan
midazolam
zaleplon
7
7
14 12
31
25
85
5
4
4
49 11
28 14
65
37
93
9
4
1
11
71
9
7
94
2
Data were generated from 2-3 runs in different days with duplicates in each day.

X. Yang et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-4
3
hydralazine inhibits most AO activities and can be used for reaction
phenotyping studies. At 25 M, hydralazine had less than 80% AO
inhibition, which was too lowtoknockoutmostof the AO activity for
reaction phenotyping. At concentrations ꢀ50 M (concentrations
m
m
suitable for reaction phenotyping), hydralazine is rather selective
against the CYP2C family of enzymes with <20% inhibition (CYP2C8,
CYP2C9, and CYP2C19). However, it showed moderate to strong in-
hibition of CYP1A2 (56%-74%), CYP2B6 (22%-36%), CYP2D6 (31%-
65%), and CYP3A (25%-37%). The inhibition is likely to be more
pronounced if the media containing inhibitors are not removed after
preincubation. Inhibition of CYP2D6 and CYP3A by hydralazine has
been previously demonstrated to be ~25% at 50 mM, but inhibition of
other CYPs has not yet been reported.12 The more potent inhibition
observed in this study compared to that reported in the literature
could potentially be due to lower substrate concentrations (1 mM vs.
K
m of 2-40
m
M) according to the following equations: IC50/Ki ¼ 1þ
[S]/Km for competitive inhibition; ICt50/KI ¼ (1þ[S]/Km)*0.693/(kin-
act*t) for mechanism-based inactivation.18 Substrate concentration
of 1 mM is selected instead of Km since 1 mM is the typical concen-
tration used in reactionphenotyping studies invitroand it is closer to
physiologic exposure concentrations in vivo for most drug
candidates.
TDI of hydralazine was also investigated for 6 CYP enzymes (1A2,
2C8, 2C9, 2C19, 2D6, and 3A) using a cocktail approach up to 300
m
M16 in human liver microsomes supplemented with nicotinamide
adenine dinucleotide phosphate. Hydralazine was identified as a TDI
for CYP1A2 from the initial screen, but the other CYPs (2C8, 2C9,
2C19, 2D6, and 3A) were not (data not shown). This suggests that
hydralazine inhibition of other CYPs is reversible. The TDI of hy-
dralazine for CYP1A2 was further studied in both human hepato-
Figure 1. Time-dependent inhibition of CYP1A2 by hydralazine at 50
suspension hepatocytes and human liver microsomes.
m
M in human
cytes and human liver microsomes at 50 mM, a lowest concentration
knocks down most AO activities and is suitable for reaction pheno-
typing. The results are shown in Figure 1. The kobs values for CYP1A2
lupus-like syndrome.28 After oral administration in humans, hy-
dralazine undergoes extensive first-pass metabolism by N-acetyl-
transferases.29,30 For intravenous dosing, hydralazine combines with
endogenous aldehydes and ketones to form hydrazine metabolites
that are equipotent as hydralazine.29 The acute liver injury appears
to be due to hydralazine metabolism to an immunologic adduct that
can result in an immunoallergic hepatitis or a more delayed lupus-
and an autoimmune hepatitis-like syndrome.28 Because hydralazine
is structurally similar to dihydralazine, it is possible that protein
adduct formation due to covalent modification of CYP1A2 might also
link to its immune responses and hepatic toxicity.
When evaluating enzyme selectivity, it is best to consider using
conditions that closely mimic those in the reaction phenotyping
assays, including substrate concentration. At high substrate con-
centrations, assays are less sensitive in detecting inhibition due to
concentration dependency of reversible inhibition and substrate
protection effects of irreversible inhibition. Both reversible and
time-dependent inhibition need to be evaluated for selectivity of an
inhibitor. Selectivity can also be substrate-dependent and, there-
fore, monitoring the selectivity for the actual drug discovery com-
pound during reaction phenotyping experiments can also be
helpful to inform Fm estimation. At the concentrations that knock
inactivation of hydralazine at 50
and 0.012 min-1 in liver microsomes. The parallel line test shows that
the slopes are significantly different for 50 M hydralazine versus
m
M are 0.0072 min-1 in hepatocytes
m
solvent control (p < 0.0001 for both hepatocytes and microsomes).
The reason that previous studies10,12 did not observe TDI may be due
to substrate protection caused by high substrate concentration un-
der the assay conditions.19-22 These data indicate that hydralazine is
a TDI of CYP1A2 at the effective AO inhibitory concentration (50 mM)
in both hepatocytes and microsomes, and that the inactivation might
be CYP-mediated. A structurally similar analog of hydralazine,
dihydralazine, has been reported to be a mechanism-based inacti-
vator of CYP1A2 and CYP3A4.23 The mechanism of inactivation has
been proposed to be covalent modification of CYPs. The chemically
reactive metabolite(s) of dihydralazine generated by CYP1A2 and
CYP3A4 through metabolic activations bind and inactivate the
enzyme themselves.23 It has also been postulated that covalent
binding of a reactive metabolite of dihydralazine to CYP1A2 might be
involved in the formation of antiliver microsome antibodies in
dihydralazine hepatitis.23 CYP1A2 inactivation by hydralazine might
have similar mechanisms as dihydralazine due to structural simi-
larity. Whether covalent modification of CYP1A2 by hydralazine has
any relevance to its immune responses and hepatotoxicity like
dihydralazine remains to be seen. Although there are no reports in
the literature that hydralazine is a CYP1A2 substrate, in silico pre-
diction with StarDrop™ 6.524-26 shows that CYP1A2 has high prob-
ability of being involved in hydralazine metabolism. Radicals
generated by other mechanisms (metal ions, peroxidases, and syn-
thase)27 might also be potential mechanisms of inactivation. Hy-
dralazine was one of the first oral antihypertensive medications
introduced into the clinic in the late 1950s, but was officially
approved for use in the United States in 1984.28 Hydralazine has
been linked to several forms of acute liver injury as well as a
out most AO activity (ꢀ50
mM), hydralazine shows significant in-
hibition of a number of CYPs. Therefore, precautions need to be
made when using hydralazine as an inhibitor to identify AO sub-
strates and for reaction phenotyping to estimate Fm,AO in hepato-
cyte systems in vitro.
Acknowledgments
The authors thank Karen Atkinson for editing the article and
Amit Kalgutkar for useful discussion.

4
X. Yang et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-4
15. Chen Y, Liu L, Monshouwer M, Fretland AJ. Determination of time-dependent
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