Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity

Article abstract of DOI:10.1124/dmd.111.043505

CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential.

Full text of DOI:10.1124/dmd.111.043505

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2012/02/10/dmd.111.043505
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1521-009X/12/4005-943–951$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:943–951, 2012
Vol. 40, No. 5
43505/3764247
Identifying a Selective Substrate and Inhibitor Pair for the
□
S
Evaluation of CYP2J2 Activity
Caroline A. Lee, J. P. Jones III, Jonathan Katayama, Ru¨ diger Kaspera, Ying Jiang,
Sascha Freiwald, Evan Smith, Gregory S. Walker, and Rheem A. Totah
Department of Drug Metabolism, Pfizer Global Research, La Jolla, California (C.A.L., Y.J., S.F., E.S.); Department of Medicinal
Chemistry, University of Washington, Seattle, Washington (J.P.J., J.K., R.K., R.A.T.); and Department of Drug Metabolism,
Pfizer Global Research, Groton, Connecticut (G.S.W.)
Received October 25, 2011; accepted February 10, 2012
ABSTRACT:
CYP2J2, an arachidonic acid epoxygenase, is recognized for its
role in the first-pass metabolism of astemizole and ebastine. To ␮M, but inhibition was substrate-dependent. Of these, danazol
fully assess the role of CYP2J2 in drug metabolism, a selective was a potent inhibitor of both hydroxylation of terfenadine (IC50
CYP2J2. Forty-two drugs inhibited CYP2J2 activity by >50% at 30
؍
substrate and potent specific chemical inhibitor are essential. In 77 nM) and O-demethylation of astemizole (Ki ؍ 20 nM), and inhi-
this study, we report amiodarone 4-hydoxylation as a specific bition was mostly competitive. Danazol inhibited CYP2C9,
CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metab- CYP2C8, and CYP2D6 with IC50 values of 1.44, 1.95, and 2.74 ␮M,
olizing enzyme, involvement. Amiodarone 4-hydroxylation enabled respectively. Amiodarone or astemizole were included in a seven-
the determination of liver relative activity factor and intersystem probe cocktail for cytochrome P450 (P450) drug-interaction
extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation cor- screening potential, and astemizole demonstrated a better profile
related with astemizole O-demethylation but not with CYP2J2 pro- because it did not appreciably interact with other P450 probes.
tein content in a sample of human liver microsomes. To identify a Thus, danazol, amiodarone, and astemizole will facilitate the ability
specific CYP2J2 inhibitor, 138 drugs were screened using terfena- to determine the metabolic role of CYP2J2 in hepatic and extrahe-
dine and astemizole as probe substrates with recombinant patic tissues.
Introduction
cle, and angiogenesis (Kroetz and Zeldin, 2002; Spector et al., 2004).
CYP2J2 is also highly expressed in tumor tissues and promotes tumor
growth and proliferation (Jiang et al., 2005, 2009; Chen et al., 2011).
Several drugs are metabolized by CYP2J2 including astemizole, ebas-
tine, terfenadine, albendazole, amiodarone, and most recently vora-
paxar (Matsumoto et al., 2003; Lee et al., 2010; Ghosal et al., 2011).
Most substrates identified for CYP2J2 are also metabolized by
CYP3A4 and other isozymes (Lee et al., 2010); therefore, a specific
reaction catalyzed by CYP2J2 is necessary to determine the contri-
bution of CYP2J2 to overall P450-mediated drug metabolism. In our
previous work, we identified amiodarone side chain hydroxylation as
a CYP2J2-specific metabolic pathway based on P450 reaction phe-
notyping, which indicated that no other P450 appreciably contributed
to the formation of this metabolite (Lee et al., 2010). A specific
substrate/inhibitor pair for CYP2J2 may reveal a role for CYP2J2 in
drug metabolism that may be underestimated, especially in extrahe-
patic tissues. CYP2J2-selective inhibition has been studied previ-
ously, and several compounds mostly related to the backbone struc-
ture of terfenadine have been identified (Lafite et al., 2006, 2007;
Chen et al., 2009). However, selectivity of these agents against most
CYP2J2 is the only member of the human 2J subfamily, and, unlike
other cytochrome P450 (P450) isozymes, it is predominantly ex-
pressed in extrahepatic tissues including the heart, skeletal muscle,
placenta, small intestine, kidney, lung, pancreas, bladder, and brain
(Zeldin et al., 1996, 1997; Enayetallah et al., 2004). In the liver and
intestine, CYP2J2 constitutes 1 to 2% of total P450 content (Gaedigk
et al., 2006; Paine et al., 2006). CYP2J2 is mostly known for its ability
to convert arachidonic acid to regioselective epoxyeicosatrienoic ac-
ids, which play significant roles in maintaining the homeostasis of the
kidney, heart, and lung by controlling crucial biological processes
such as anti-inflammation, vasodilatation, relaxation of smooth mus-
This work was supported by the National Institutes of Health National Heart,
Lung, and Blood Institute [Grant R01-HL096706]; and the National Institutes of
Health National Institute of General Medical Sciences [P01-GM32165].
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
http://dx.doi.org/10.1124/dmd.111.043505.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: P450, cytochrome P450; RAF, relative activity factor; ISEF, intersystem extrapolation factor; HLM, human liver micro-
somes; HPLC, high-performance liquid chromatography; 1D, one dimensional; COSY, correlation spectroscopy; TOCSY, total correlation
spectroscopy; HSQC, heteronuclear single quantum correlation; IS, internal standard; LC-MS/MS, liquid chromatography-tandem mass
spectrometry; Clint, intrinsic clearance; DMSO, dimethyl sulfoxide; MRM, multiple reaction monitoring; CE, collision energies; DP, declus-
tering potentials; NCE, new chemical entities; PF-05218881, (E)-3-(4-((2S,3S,4S,5R)-5-1-(3-chloro-2,6-difluorobenzyloxyimino)ethyl)-3,4-
dihydroxytetrahydrofuran-2-yloxy)-3-hydroxyphenyl)-2-methyl-N(3aS,4R,5R,6S,7R,7aR)-4,6,7-trihydroxyhexahydrobenzo[d][1,3]dioxol-5-yl) acrylamide.
943

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LEE ET AL.
otherwise noted, for 2D experiments, a relaxation delay of 1.5 s was used
between transients.
drug-metabolizing enzymes has not been determined. In addition,
these compounds were all obtained through several steps of synthesis.
The aims of this study are as follows: 1) to fully characterize
amiodarone hydroxylation as a CYP2J2 probe reaction, as well as the
relative activity factor (RAF) and intersystem extrapolation factor
(ISEF) for several individual preparations and one pooled human liver
microsomal preparation; 2) to identify a readily available selective
and potent CYP2J2 inhibitor by screening a panel of 138 marketed
drugs; and 3) to refine a P450 cocktail assay in HLM that enables the
evaluation of drug interactions incorporating potential involvement of
CYP2J2.
Formation of 4-Hydroxyamiodarone by Recombinant P450s. Assays
were performed on a Biomek FX system (Beckman Coulter, Fullerton, CA). Amio-
darone was incubated at a final concentration of 1 ␮M with 11 different recombinant
P450 isoforms (CYP1A2, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -2J2, -3A4, and
-3A5) at final P450 concentration of 50 pmol/ml in 100 mM potassium phosphate
buffer (pH 7.4) at 37°C with 3 mM MgCl2. The reaction mixture was preincubated at
37°C before adding the NADPH-regenerating solution (10 mM NADP, 55 mM
isocitric acid, 55 unit/ml isocitrate dehydrogenase). The final concentration of NADPH
was 1 mM. A 50-␮l aliquot of the reaction mixture was removed at 0, 5, 10, 20, 30,
and 45 min. Aliquots were quenched with 100 ␮l of acetonitrile containing 500 ng/ml
(E)-3-(4-((2S,3S,4S,5R)-5-1-(3-chloro-2,6-difluorobenzyloxyimino)ethyl)-3,4-
dihydroxytetrahydrofuran-2-yloxy)-3-hydroxyphenyl)-2-methyl-N(3aS,4R,5R,
6S,7R,7aR)-4,6,7-trihydroxyhexahydrobenzo[d][1,3]dioxol-5-yl) acrylamide (PF-
05218881) [internal standard (IS)] and centrifuged at 2000 rpm for 10 min. Control
incubations with each of the recombinant P450 isoforms were conducted
without NADPH to monitor non-P450-mediated substrate disappearance.
4-Hydroxyamiodarone was quantified by liquid chromatography-tandem mass
spectrometry (LC-MS/MS).
Relative Activity Factor and Intersystem Extrapolation Factor Deter-
mination. The CYP2J2 relative activity factor (RAF) was determined by
monitoring the formation rate of 4-hydroxyamiodarone in HLM and recom-
binant CYP2J2 enzyme systems. The RAF value is the ratio of the activity of
the probe substrate in HLM divided by the activity in recombinant P450, with
HLM activity expressed as pmol ꢀ minϪ1 ꢀ mgϪ1 and recombinant P450
expressed as activity pmol ꢀ minϪ1 ꢀ pmol CYP2J2Ϫ1 such that the units are
pmol CYP2J2/mg microsomal protein. The ISEF incorporates CYP2J2 content
or abundance in the liver microsomal preparations and expressed as picomoles
of CYP2J2 per milligram of microsomal protein. The ISEF value is determined
by normalizing the RAF value to the CYP2J2 content in each HLM prepara-
tion, and therefore the ISEF value is unitless.
For recombinant CYP2J2, Clint was defined as the ratio of Vmax and Km
determined by monitoring 4-hydroxyamiodarone formation rate under linear
kinetic conditions. The kinetic parameters for CYP2J2 were determined under
the following conditions: 0, 0.06, 0.12, 0.23, 0.46, 0.92, 1.9, 3.8, 7.5, 15, 30,
and 60 ␮M amiodarone, 40 pmol/ml recombinant CYP2J2, and 1 mM NADPH
in 100 mM potassium phosphate buffer (pH 7.4) at 37°C. The reaction mixture
was preincubated at 37°C for 5 min before adding the NADPH-regenerating
solution (10 mM NADP, 55 mM isocitric acid, 55 unit/ml isocitrate dehydro-
genase). Fifty microliters of the reaction mixture was removed after 20 min.
The HLM Clint value was generated as the ratio of the formation rate of
4-hydroxyamiodarone divided by amiodarone concentration in the incubation.
The HLM Clint value under linear conditions is similar to the ratio of Vmax
divided by Km obtained by a complete Michaelis-Menten kinetic study. For the
determination of HLM Clint value, individual prepared HLM or pooled HLM
were incubated with amiodarone (at a concentration approximating its Km
value of 5 ␮M), 0.4 mg/ml HLM and 1 mM NADPH in 100 mM potassium
phosphate buffer (pH 7.4) at 37°C. A 50-␮l reaction mixture was removed
after 20-min incubation time. 4-Hydroxyamiodarone formation was monitored
by LC-MS/MS.
Determination of CYP2J2 Content in HLM. Mouse anti-human CYP2J2
antibody (Abnova, Walnut, CA) was used to detect and quantitate human
CYP2J2 in HLM samples from the University of Washington, School of
Pharmacy human tissue bank. Liver microsomal protein (50 ␮g), and BD
Gentest CYP2J2ϩORϩB5 Supersomes as standards (0.1, 0.05, and 0.025
pmol/␮l), were electrophoresed in NuPage Bis-Tris 12-well gels (gradient
8–12%) and transferred to polyvinylidene difluoride membranes. Blots were
incubated with primary antibody for 4 h followed by secondary antibody (goat
anti-mouse). Protein bands were visualized using an Odyssey infrared imager
(Li-Cor Biosciences, Lincoln, NE), following the manufacturer’s instructions,
Materials and Methods
General Chemicals and Reagents. All chemicals evaluated as inhibitors
were purchased from Sigma-Aldrich (St. Louis, MO) and were used without
further purification. Human CYP2J2 Supersomes (containing human CYP2J2,
cytochrome P450 reductase and cytochrome b5) and pooled human liver
microsomes were purchased from BD Gentest (Woburn, MA). Terfenadine
alcohol and terfenadine carboxylate were purchased from Ultrafine Chemical
Co. (Manchester, England). High-performance liquid chromatography
(HPLC)-grade ammonium acetate was purchased from Mallinckrodt Baker,
Inc. (Phillipsburg, NJ). HPLC-grade acetonitrile and methanol were purchased
from Honeywell Burdick & Jackson (Muskegon, MI).
Isolation of 3- and 4-Hydroxyamiodarone. Amiodarone was incubated at
37°C to a final concentration of 30 ␮M in 100 mM potassium phosphate buffer
(pH 7.4) containing 50 pmol/ml CYP2J2 Supersomes, 10 mM MgCl2, and 1
mM NADPH. The total incubation volume was 60 ml, and total reaction time
was 60 min. The incubation was quenched with 36 ml of acetonitrile and
centrifuged at approximately 2000 relative centrifugal force for 10 min. The
supernatant was removed and diluted to 600 ml with water containing 0.1%
formic acid. The resulting solution was then centrifuged at approximately
40,000 relative centrifugal force for 30 min. Initial isolation of 4- and 3-hy-
droxyamiodarone was achieved using an Aqua C18 column (5 ␮m, 10 ϫ 250
mm; Phenomenex, Torrance, CA). The mobile phases consisted of 0.1%
formic acid in water (mobile phase A) and acetonitrile (mobile phase B). The
sample was introduced into the column via a loading pump (300 ml/run), and
the metabolites were eluted with a linear gradient from 6% mobile phase B to
60% mobile phase B in 40 min at a flow rate of 4 ml/min. One-minute fractions
were collected over the course of the run, and metabolite elution was moni-
tored by UV detection (254 nm). Fractions containing 3- and 4-hydroxyamio-
darone were combined and diluted with water containing 0.1% formic acid
until the acetonitrile content was approximately 10%. Final isolation of the
individual metabolites was achieved using a Luna C8 (2) column (5 ␮m, 4.6 ϫ
250 mm; Phenomenex). The mobile phases used were the same as those
described above. The sample was introduced via a loading pump, and the
metabolites were eluted using a linear gradient from 10% mobile phase B to
70% mobile phase B in 50 min at a flow rate of 1 ml/min. Metabolite elution
was monitored as described above, and metabolites were collected manually.
Structural Characterization of 4- and 3-Hydroxyamiodarone by NMR.
NMR spectra were recorded on a Bruker Avance 600 MHz system controlled
by TOPSPIN (version 2.0), equipped with a 5-mm TCI CryoProbe (Bruker
BioSpin Corporation, Billerica, MA). The 1D spectra were recorded using a
sweep width of 12,000 Hz and a total recycle time of 7.2 s. The resulting
time-averaged free induction decays were transformed using an exponential
line broadening of 1.0 Hz to enhance signal to noise. Samples were dissolved
in 0.15 ml of dimethyl sulfoxide-d6 “100%” (Cambridge Isotope Laboratories,
Andover, MA) and placed in 3-mm diameter tubes. All spectra were referenced
using residual dimethyl sulfoxide-d6 (1H ␦ ϭ 2.5 ppm and 13C ␦ ϭ 39.5
relative to tetramethylsilane, ␦ ϭ 0.00). Phasing, baseline correction, and
integration were all performed manually. If needed, the BIAS and SLOPE
functions for the integral calculations were adjusted manually. The final and quantified using a calibration curve generated from CYP2J2ϩORϩb5
concentration of the isolated metabolites 4- and 3-hydroxyamiodarone were Supersomes.
0.31 and 0.22 mM, respectively, determined using the Sicco method (Walker
et al., 2011). COSY, TOCSY, and multiplicity-edited HSQC data were re-
corded using the standard pulse sequences provided by Bruker. The 2D
Correlation Analysis of 4-Hydroxyamiodarone and Astemizole O-
Demethylation Activity in Individually Prepared HLM and Pooled HLM.
Amiodarone (5 ␮M) or astemizole (0.3 ␮M) (both at Km) were incubated with
experiments were typically acquired using a 1K ϫ 128 data matrix with 16 various individual, prepared HLM and pooled HLM at a final concentration of
dummy scans. The data were zero-filled to a size of 1K ϫ 1K. Unless 0.1 mg/ml (astemizole) or 1 mg/ml (amiodarone) in 100 mM potassium

CYP2J2-SELECTIVE SUBSTRATE AND INHIBITOR
945
diclofenac, 40 ␮M S-mephenytoin, 5 ␮M dextromethorphan, 0.3 ␮M astemi-
zole, and 2 ␮M midazolam. IC50 values were determined for danazol, pimo-
zide, miconazole, or terfenadine using the seven P450 probe cocktail or
astemizole (0.3 ␮M) alone. The final inhibitor concentration range was 0.1,
0.3, 1, 3, 10, and 30 ␮M. The 300-␮l reaction mixture containing inhibitor and
seven P450 probe substrate or astemizole alone were preincubated at 37°C for
15 min before adding the NADPH-regenerating solution (10 mM NADP, 55
mM isocitric acid, 55 unit/ml isocitrate dehydrogenase, and final concen-
tration of NADPH was 1 mM). The reaction proceeded for an additional 8
min before being quenched with 600 ␮l of acetonitrile containing 100
ng/ml IS, PF-05218881. Metabolite formation of astemizole was monitored
by LC-MS/MS.
Mechanism of Inhibition and Ki for Danazol Using Astemizole As Probe
Substrate. Determination of Ki was performed with recombinant CYP2J2 (2
pmol/ml), diluted in 100 mM potassium phosphate buffer (pH 7.4), supple-
mented with astemizole (final concentration 0.15, 0.3, 0.6, 1.2, 2.4 ␮M) and
inhibitor danazol (0, 0.05, 0.1, 0.2, 0.4 ␮M), and preincubated for 5 min at
37°C in a shaking water bath. Reactions were initiated by adding NADPH (1
mM final concentration) and allowed to proceed for 5 min. Incubations were
quenched by adding an equal volume of ice-cold acetonitrile supplemented
with internal standard (0.05 ␮M terfenadine). Samples were vortexed then
centrifuged for 10 min. Calibration standards were performed under assay
conditions with heat-inactivated recombinant CYP2J2. Solvent concentrations
were corrected for and are equal in each assay (0.01% DMSO, 0.4% ethanol, as
lowest possible solvent concentrations due to low solubility of danazol).
For the determination of potential time and mechanism-based inhibition,
recombinant CYP2J2 (0.5 pmol ꢀ mlϪ1) was mixed with danazol (0.0003–10
␮M) and preincubated with and without 1 mM NADPH for 30 min. The
reactions were initiated by the addition of 0.3 ␮M astemizole. Quenching and
analysis of incubations were similar to those described in the previous para-
graph. To confirm time and mechanism-based inhibition, a dilution assay was
also performed. Recombinant CYP2J2 (10 pmol/ml) in 100 mM potassium
phosphate buffer (pH 7.4) was supplemented with 20 nM danazol only (time
dependent), 20 nM danazol and 1 mM NADPH (mechanism-based), and with
or without NADPH (controls). After each 0- and 30-min preincubation time,
incubations were diluted 10-fold into an activity assay mixture containing 1
mM NADPH and 0.3 ␮M astemizole (final concentration) in 200 ␮l of 100
mM potassium phosphate buffer (pH 7.4). Incubation time for the activity
assays was 5 min, and samples were processed as described in the paragraph
above. Analysis of astemizole metabolites is described under Analytical
Methods.
TABLE 1
Ionization mode, m/z transitions, and retention times for 4- and
3-hydroxyamiodarone
m/z Ratios for 4- and 3-Hydroxyamiodarone
Analyte
Q1 m/z
Q3 m/z
DP
CE
Retention Time (min)
4-Hyroxyamiodarone
3-Hyroxyamiodarone
662.4
662.4
100
100
60
60
40
40
12.7
13.2
phosphate buffer (pH 7.4) at 37°C. The reaction mixture was preincubated at
37°C before adding the NADPH-regenerating solution (10 mM NADP, 55 mM
isocitric acid, and 55 unit/ml isocitrate dehydrogenase). Final concentration of
NADPH was 1 mM. A 50-␮l aliquot of reaction mixture was removed at 5 or
20 min for astemizole or amiodarone, respectively. The aliquots were
quenched with 100 ␮l of acetonitrile containing 500 ng/ml of IS, PF-
05218881. The amount of 4-hydroxyamiodarone or astemizole O-demethy-
lated metabolites formed was monitored by LC-MS/MS.
Incubation Conditions for CYP2J2 Inhibition Screen. Recombinant
CYP2J2 Supersomes (0.1 pmol/incubation), 0.2 ␮M terfenadine (in methanol)
or 1 pmol recombinant CYP2J2/incubation and 0.3 ␮M astemizole (in ethanol)
(both substrates at Km), and potassium buffer (100 mM, pH 7.4, 200 ␮l final
volume) were incubated with each inhibitor [30 ␮M final concentration in
dimethyl sulfoxide (DMSO)] in a 96-well polypropylene plate (Nunc, Naper-
ville, IL) and prewarmed at 37°C for 5 min. The reactions were initiated by
addition of NADPH (1 mM final concentration in water). The final DMSO
concentration in the incubations was 1% (v/v). Control incubations with
DMSO and no inhibitor with or without the addition of methanol or ethanol to
investigate solvent effects were compared with reactions without DMSO,
methanol, or ethanol, and no significant differences in CYP2J2 activity were
observed. After a 5-min reaction time, incubations were quenched with 200
␮l of ice-cold acetonitrile containing internal standard (clemizole for
terfenadine incubations and norastemizole for astemizole incubations),
immediately vortexed, and placed on ice. After cooling for 10 min,
quenched samples were centrifuged at 14,000g for 5 min at room temper-
ature. The supernatant was directly analyzed by LC-MS (terfenadine) or
LC-MS/MS (astemizole).
Development of the Seven P450 Probe Substrate Cocktail for Drug
Interactions Screening. The rate of metabolite formation of the cocktail probe
substrates was monitored in the presence and absence of astemizole (0.3 ␮M)
or amiodarone (5 ␮M) to evaluate whether the CYP2J2 probe would alter their
metabolism. The final concentration of the P450 probe cocktail consisted of
phenacetin (10 ␮M, CYP1A2), paclitaxel (5 ␮M, CYP2C8), diclofenac (5 ␮M,
CYP2C9), S-mephenytoin (40 ␮M, CYP2C19), dextromethorphan (5 ␮M,
CYP2D6), and midazolam (2 ␮M, CYP3A4) with either astemizole or amio-
darone in 100 mM potassium phosphate buffer (pH 7.4) at 37°C. The 300-␮l
reaction mixture was preincubated at 37°C for 15 min before adding the
NADPH-regenerating solution (10 mM NADP, 55 mM isocitric acid, 55
unit/ml isocitrate dehydrogenase, and final concentration of NADPH was 1
mM). The reaction proceeded for an additional 8 min and was quenched with 600 ␮l
of acetonitrile containing 100 ng/ml IS, PF-05218881. The amount of metabolite
formed from each probe substrate was monitored by LC-MS/MS.
Analytical Methods. Quantification of 4- and 3-hydroxyamiodarone. A 10-␮l
aliquot of quenched incubation sample was injected onto a Phenomenex
Kromasil C4 (150 ϫ 2 mm 3.5 ␮; Phenomenex) HPLC column with a CTC
PAL autosampler (LEAP Technologies, Carrboro, NC) and an integrated
HPLC pumping system (Shimadzu Scientific Instruments, Columbia, MD).
These compounds were then eluted and detected by an API 4000-triple
quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City,
CA) fitted with a TurboIonSpray interface. Mobile phase A was 0.1% formic
acid, mobile phase B was acetonitrile with 0.1% formic acid, and the flow rate
was 0.2 ml/min. The starting condition for the HPLC gradient was 80:20
(A/B), and this condition was held for 0.3 min. From 0.3 to 9 min, the
mobile phase composition changed linearly to 60:40 (A/B). This condition
was held for 11 min. The gradient was returned in a linear fashion to 80:20
(A/B) from 11 min to 13.9 min and re-equilibrated until 15 min. Multiple
Evaluation of Single Probe Versus Cocktail P450 Probe Substrate
Assay. To confirm the robustness of the seven P450 probe substrate assay, a
comparison of IC50 values was determined using the seven probe cocktail
assay and the single probe substrate astemizole in pooled HLM. The seven
P450 probe cocktail consisted of 10 ␮M phenacetin, 5 ␮M paclitaxel, 5 ␮M
TABLE 3
MRM transitions, collision energies, and declustering potentials for each P450
probe
TABLE 2
Metabolite
P450
MRM
CE
DP
m/z transitions and ionization mode for O-desmethyl astemizole and internal
standard, PF-05218881
Acetaminophen
CYP1A2
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP2J2
m/z 152.0 Ͼ 110.0
m/z 870.5 Ͼ 525.4
m/z 312.3 Ͼ 231.1
m/z 235.2 Ͼ 150.1
m/z 258.1 Ͼ 199.1
m/z 445.1 Ͼ 121.1
m/z 342.3 Ͼ 203.1
m/z 687 Ͼ 320
20
25
30
25
40
50
40
30
61
80
60
71
80
40
60
60
6␣-Hydroxypaclitaxel
4-OH-diclofenac
4-OH-S-Mephenytoin
Dextrorphan
Desmethyl astemizole
1-OH-Midazolam
Internal standard
m/z Ratios for Desmethyl Astemizole and Internal Standard
Analyte
Q1 m/z
Q3 m/z
DP
CE
CXP
Desmethyl astemizole
PF-05218881
445.1
687
121.1
320
40
60
50
30
15
15
CYP3A4
PF-5218881
CXP, collision cell exit potential.

946
LEE ET AL.
TABLE 4
Proton and 13C NMR chemical shifts for amiodarone, M1, and M2
Amiodarone HCl
M1 (4-Hydroxy Amiodarone)
M2 (3-Hydroxy Amiodarone)
HO
CH
3
CH
4 but3yl
HO
3 butyl
1 butyl
I
2 butyl
O
I
2'
O
I
O
O
O
O
6'
O
O
2 DEAE
I
4
5
1 DEAE
O
I
N
4 DEAE
H C
N
I
3
7
CH
H C
3
N
3
6
CH
H C
3
3
5 DEAE
CH
3
1H Chemical shift in ppm 13C Chemical shift 1H Chemical shift in ppm 13C Chemical shift 1H Chemical shift in ppm 13C Chemical shift
(multiplicity, coupling
constants, integration)
7.47 (d, J ϭ 7.8, 1H)
7.29 (t, J ϭ 7.5, 1H)
7.37 (t, J ϭ 7.5, 1H)
7.67 (d, J ϭ 8.6, 1H)
8.19 (s, 2H)
in ppm
(multiplicity, coupling
constants, integration)
7.42 (d, J ϭ 7.8, 1H)
7.28 (t, J ϭ 7.6, 1H)
7.36 (t, J ϭ 7.8, 1H)
7.66 (d, J ϭ 8.2, 1H)
8.15 (s, 2H)
2.75 (t, J ϭ 7.7, 2H)
1.73 (cm, 2H)
1.39 (cm, 2H)
3.36 (cm, 1H)
4.39 (t, J ϭ 5.2, 1H)
4.04 (bs, 2H)
2.99 (bs, 2H)
2.64 (bs, 4H)
in ppm
(multiplicity, coupling
constants, integration)
7.47 (d, J ϭ 7.4, 1H)
7.29 (t, J ϭ 7.6, 1H)
7.35 (t, J ϭ 7.4, 1H)
7.67 (d, J ϭ 8.2, 1H)
8.14 (s, 2H)
2.71/2.86 (cm, 2H)
1.74 (cm, 2H)
3.52 (bs, 1H)
1.00 (d, J ϭ 6.2, 3H)
4.55 (s, 1H)
4.02 (t, J ϭ 6.7, 2H)
2.96 (t, J ϭ 6.7, 2H)
2.60 (q, J ϭ 7.4, 4H)
1.00 (t, J ϭ 7.1, 6H)
in ppm
4
5
6
7
120.9
121.1
120.3
124.0
124.9
111.2
139.7
27.6
29.4
22.0
13.2
NA
67.2
49.9
47.1
8.6
124.4
125.4
111.5
140.0
28.1
24.6
32.3
60.5
124.1
124.4
110.9
139.7
24.5
36.3
65.5
23.6
2Ј/6Ј
1 bubButyl 2.73 (t, J ϭ 7.3, 2H)
2 Butyl
3 Butyl
4 Butyl
OH
1 DEAE
2 DEAE
4 DEAE
5 DEAE
1.69 (cm, 2H)
1.26 (cm, 2H)
0.84 (t, J ϭ 7 .4, 3H)
NA
4.38 (t, J ϭ 4.9, 2H)
3.67 (q, J ϭ 4.6, 2H)
3.37 (cm, 4H)
NA
a
71.6
51.6
47.5
12.4
a
a
a
1.33 (t, J ϭ 7.2, 6H)
1.02 (t, J ϭ 7.1, 6H)
d, doublet; t, triplet; q, quartet; s, singlet; cm, complex multiplet; bs, broad singlet; NA, not applicable; DEAE, diethyl amino ethanol.
a No 1H/13C cross peak was observed.
reaction monitoring (MRM) was used to monitor the compounds. Table 1
lists the ionization mode, m/z transitions, and retention times for 4- and
3-hydroxyamiodarone.
to 99:1 (A/B) from 1.9 to 1.95 min and re-equilibrated until 2 min. MRM was
used to monitor the compounds. Table 2 lists the m/z transitions and ionization
mode for O-desmethyl astemizole and internal standard, PF-05218881. The
retention times were 0.94 and 0.97 min for O-desmethyl astemizole and
internal standard, respectively. The peak area ratio of the analyte to the internal
standard was determined for each injection and used to quantify the amount of
metabolite formed.
Astemizole inhibition screening assay. Metabolites and standards were mea-
sured by LC-MS/MS, performed on a Waters Quattro Premier XE Micromass
system coupled to a Waters ACQUITY Sample Manager and Binary Solvent
Manager (UPLC; Waters, Milford, MA), and detected by electrospray ionization
(source temperature 350°C, capillary 3.5 kV, cone 45V, extractor 5V, cone gas
flow 2 l ꢀ hϪ1, desolvation gas flow 800 l ꢀ hϪ1). Twenty-five microliters of sample
was loaded onto an ACQUITY UPLC BEH Phenyl (1.7 ␮m, 2.1 ϫ 50 mm,
column heat 50°C) column at a flow rate of 3.5 ml/min. Mobile phase A was
ammonium formate (20 mM, pH 9.4) and mobile phase B was acetonitrile. The
initial condition for the HPLC gradient was 90:10 (A/B). This was held for 0.5
min. From 0.5 to 1.5 min, the mobile phase composition changed linearly to 100%
B and was maintained for 3.5 min. From 3.5 to 4.0 min, the gradient was returned
to 90:10 (A/B). Mass ions were identified by fragmentation of 445.1 to 120.9 m/z
(desmethyl astemizole: dwell 0.05 s, cone 50.0 V, collision 40.0 V) and 472.13 to
436.16 (terfenadine 3: 0.05 s, 35.0 V, 30.0 V).
Analysis of astemizole O-demethylated metabolite formation. A 20-␮l ali-
quot of the quenched incubation sample containing O-desmethyl astemizole
was injected onto a Synergi Polar-RP (2 ϫ 30 mm 4 ␮; Phenomenex) HPLC
column with a CTC PAL autosampler (LEAP Technologies) and an integrated
HPLC pumping system (Shimadzu Scientific Instruments). The metabolite was
eluted and detected by an API 4000-triple quadrupole mass spectrometer
(Applied Biosystems/MDS Sciex) fitted with a TurboIonSpray interface. Mo-
bile phase A was 0.1% formic acid, mobile phase B was acetonitrile with 0.1%
formic acid, the flow rate was 0.8 ml/min. The initial condition for the HPLC
gradient was 99:1 (A/B). This condition was held for 0.3 min. From 0.3 to 1.2
min, the mobile phase composition changed linearly to 1:99 (A/B). This
condition was held for 1.9 min. The gradient was returned in a linear fashion
Terfenadine hydroxylation inhibition screening assay. Analysis of terfena-
dine hydroxylation was performed on an HP 1100 LC/MSD system (Agilent
Technologies, Santa Clara, CA) quadrupole mass spectrometer coupled to an
HPLC system. Positive ions were generated from an electrospray ionization at
350°C. A 40-␮l aliquot of quenched sample was loaded onto a reverse-phase
HPLC system using a Zorbax Extend C-18 column (5 ␮M, 2.1 ϫ 50 mm;
Agilent Technologies) at a flow rate of 300 ␮l/min. Chromatographic separa-
tion of terfenadine alcohol and potential terfenadine carboxylate was attained
with a three-step linear gradient. Mobile phase A was 10 mM ammonium
acetate, pH 5.5, and mobile phase B was methanol. The initial condition for the
HPLC gradient was 50:50 (A/B) and increased linearly to 10:90 (A/B) to 2
FIG. 1. Time-dependent formation of 4-hydroxyamiodarone metabolite (M1) by
recombinant CYP2J2 and pooled HLM. No other isozyme had any appreciable
formation of 4-hydroxyamiodarone.

CYP2J2-SELECTIVE SUBSTRATE AND INHIBITOR
947
Statistical Analysis. The general screen samples were conducted in dupli-
cate and reported as the average, whereas IC50 and Ki experiments were
performed in triplicate and reported as the average Ϯ S.D. IC50 and Ki data
analyses were performed by nonlinear regression using GraphPad Prism (ver-
sion 5.02; GraphPad Software Inc., San Diego, CA).
TABLE 5
Formation of CYP2J2-mediated metabolites of astemizole and amiodarone in
various HLM preparations
HLM
CYP2J2 Content Astemizole Desmethylation
4-Hydroxyamiodarone
pmol/mg
pmolϪ1 ꢀ min ꢀ pmolϪ1
CYP2J2 (mean Ϯ S.D.)
pmolϪ1 ꢀ min ꢀ pmolϪ1
CYP2J2 (mean Ϯ S.D.)
Results
130
128
118
119
153
133
149
144
109
128
159
129
146
0.047
0.165
0.900
0.567
0.941
1.578
0.912
1.131
2.20
1.20
2.40
3.50
7.60
1357 Ϯ 77
254 Ϯ 18
38 Ϯ 3
172 Ϯ 12
80 Ϯ 34
39 Ϯ 2
80.7 Ϯ 0.6
77 Ϯ 1
98 Ϯ 1
196 Ϯ 7
87 Ϯ 17
93 Ϯ 3
44 Ϯ 6
32 Ϯ 1
82 Ϯ 2
18.8 Ϯ 0.9
7.2 Ϯ 0.2
12.7 Ϯ 0.3
7.9 Ϯ 0.1
2.8 Ϯ 0.1
4.5 Ϯ 0.2
3.2 Ϯ 0.2
0.9 Ϯ 0.2
2.1 Ϯ 0.2
0.9 Ϯ 0.1
1.6 Ϯ 0.1
0.5 Ϯ 0.0
3.1 Ϯ 0.1
Structural Characterization of 4 and 3-Hydroxyamiodarone
Metabolites. In a previous study that screened for CYP2J2 substrates,
amiodarone was oxidized by CYP2J2 to a hydroxylated metabolite
minimally formed by CYP3A4. Further analysis of the hydroxylated
metabolite of amiodarone showed that this product was a mixture of
two metabolites, 4- and 3-hydroxyamiodarone (Table 4). Comparison
of the 1H spectrum of the M1 isolate with a similarly acquired spectrum
of amiodarone revealed the absence of the terminal methyl of the butyl
side chain and the presence of a new set of resonances at ␦3.36 and ␦4.39.
COSY and TOCSY data of the M1 isolate indicated connectivity between
Pooled HLM
2.59
1
the two new resonances and the other H resonances of the butyl side
chain (see Supplemental Figs. 1–3). All other acquired NMR and mass
min, followed by a 2-min hold and then back to 50:50 (A/B) over 1 min. spectral data are consistent with the structure of M1 as 4-hydroxyamio-
ChemStation Rev. A. 10.02 (Agilent Technologies) was used to set the
selection of ion windows for single ion monitoring data acquisition. The mass
ions monitored were m/z 488.0, 502.0, and 326.6 corresponding to terfenadine
alcohol, terfenadine carboxylate, and clemizole (the internal standard), respec-
tively. Dwell times were set at 889 ms per ion.
Six versus seven P450 probe cocktail assay. The reaction monitoring
(MRM) LC-MS/MS analysis was conducted on an ABI 4000 Q TRAP Mass
Spectrometer (Applied Biosystems, Foster City, CA) using a turbo ion spray
source in positive ionization mode. MRM transitions, collision energies (CE),
and declustering potentials (DP) are listed in Table 3.
Samples were separated using a Phenomenex Onyx Monolithic C18, 4.6 ϫ
50 mm HPLC column with a CTC PAL autosampler (LEAP Technologies) and
an integrated HPLC pumping system (Shimadzu Scientific Instruments). Mo-
bile phase A was 0.1% formic acid in water and mobile phase B was
acetonitrile with 0.1% formic acid. At the beginning of the injection, the
primary gradient pump flow rates were 0.2 ml/min 99:1 (A/B), and the dilution
pump flow rate was 2.8 ml/min of A (100%). After the analytes were loaded
onto the column, the dilution pump was stopped (minimal flow was maintained
at 0.01 ml/min flow to prevent back flow) and the primary gradient pumps
were ramped to 3.0 ml/min to initiate the gradient. The primary gradient was
changed to 90:10 (A/B) and held for 0.42 min. From 0.42 to 0.6 min, the
mobile phase composition changed linearly to 75:25 (A/B). From 0.6 to 1.45
min, the mobile phase composition changed linearly to 35:65 (A/B). This
condition was held to 1.57 min. The gradient was returned in a linear fashion
to 90:10 (A/B) from 1.57 to 1.58 min and re-equilibrated for 1.9 min. The
injection volume was 20 ␮l. The metabolite concentrations were calculated
using Analyst 1.4 software (Applied Biosystems, Carlsbad, CA).
darone analog of amiodarone.
The M2 isolate was identified as the 3-hydroxybutyl analog of
amiodarone. Comparison of the 1H spectrum of the M2 isolate with a
similarly acquired spectrum of amiodarone revealed the absence of
the terminal methyl of the butyl side chain (triplet, ␦0.84, J ϭ 7.4 Hz)
and the presence of a new doublet (␦1.00, J ϭ 6.2 Hz) (Table 4). In
addition, COSY and TOCSY data of the M2 isolate indicated con-
nectivity between the new doublet and a broad singlet (not observed
in amiodarone) at ␦3.52. Multiplicity-edited HSQC data contain a
correlation between this broad singlet and a 13C resonance at ␦65.5.
The above data and all other acquired NMR data are consistent with
the structure of M2 as the 3-hydroxybutyl analog of amiodarone
(Supplemental Figs. 4–6).
4-Hydroxyamiodarone Is Specifically Formed by CYP2J2.
P450 reaction phenotyping studies conducted with human liver mi-
crosomes (pooled HLM) and a panel of recombinant P450s (CYP1A2,
-2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -2J2, -3A4, and -3A5) revealed
that the formation of 4-hydroxyamiodarone was CYP2J2-specific and
was not formed by other drug-metabolizing enzymes, whereas 3-hy-
droxyamiodarone was formed by both CYP2J2 and CYP3A4 (data not
shown). For CYP2J2, the formation of 4-hydroxyamiodarone was
linear to 20 min as shown in Fig. 1 and metabolite formation followed
Michaelis-Menten kinetics (Supplemental Fig. 7). Small levels of the 4-
and 3-hydroxyamiodarone were detected in HLM (4-hydroxyamiodarone
is shown in Fig. 1, but 3-hydroxyamiodarone data are not shown).
Examination of CYP2J2-Mediated Astemizole Demethylation
and Amiodarone 4-Hydroxylation in Various HLM Preparations.
CYP2J2 protein levels were quantified by Western blot analysis for 13
TABLE 6
Relative activity factor and intersystem extrapolation factor for CYP2J2 in HLM
HLM
CYP2J2 Content in HLM
RAF (CLint
)
CYP2J2 ISEF
pmol/mg
0.047
0.002
0.165
0.900
0.567
0.941
1.578
0.912
1.131
2.590
pmol/mg
0.0008
0.0016
0.0007
0.0014
0.0015
0.0016
0.0009
0.0009
0.0008
0.0017
130
165
128
118
119
153
133
149
144
0.01760
1.03472
0.00401
0.00154
0.00271
0.00169
0.00060
0.00096
0.00068
0.00066
FIG. 2. Correlation of astemizole O-demethylation and amiodarone 4-hydroxyla-
tion in a panel of individual HLM preparations.
Pooled HLM

948
LEE ET AL.
correlation; however, there is no scientific rationale to exclude HLM
TABLE 7
130 from the correlation analysis.
Percentage of inhibition of terfenadine hydroxylation (terfenadine) and
astemizole O-demethylation (astemizole)
RAF and ISEF for CYP2J2. The kinetic studies for recombinant
CYP2J2 and HLM were conducted under linear conditions. Studies
were conducted using recombinant CYP2J2, nine individually pre-
pared HLM samples, and a single pooled HLM sample consisting of
60 livers. For recombinant CYP2J2, the Clint value was determined by
taking the ratio of the Vmax for 4-hydroxyamiodarone rate to its Km
value. For the HLM studies, the 4-hydroxyamiodarone rate was de-
termined at an amiodarone substrate concentration approximating the
Km (5 ␮M). For HLM, the Clint value was determined by taking
the ratio of the 4-hydroxyamiodarone rate to substrate concentration.
The CYP2J2 RAF values were determined as the ratio of the Clint in
HLM to Clint in recombinant CYP2J2, with the units of pmol
CYP2J2/mg HLM (Table 6). In the individually prepared HLM sam-
ples, the RAF based on Clint ranged from 0.0007 to 0.0017 and the
RAF value determined using pooled HLM value was 0.0017. The
ISEF values ranged from 0.0006 to 1.03 for the individually prepared
HLM samples and 0.00066 for pooled HLM.
Screening 138 Drugs as CYP2J2 Inhibitors. From the 138 com-
pounds screened for CYP2J2 inhibitors (Supplemental Table 1), 42
compounds were identified that inhibit terfenadine hydroxylation by
50% or more at 30 ␮M, whereas eight compounds (danazol, keto-
conazole, lansoprazole, loratadine, miconazole, nicardipine, or-
phenadrine, and verapamil) markedly reduced CYP2J2 activity by
90% or more (Table 7).
To identify a specific CYP2J2 inhibitor, the top 40 chemicals
identified that inhibited terfenadine hydroxylation by 50% or more
were also screened for inhibition of astemizole O-demethylation ac-
tivity. Twenty-four compounds inhibited astemizole metabolism by
50% or more, whereas danazol, ketoconazole, loratadine, miconazole,
and nicardipine inhibited terfenadine or astemizole metabolism by
90% or more (Table 7). To further refine the selection of a potent
substrate-independent CYP2J2 inhibitor, an inhibition screen was
conducted in pooled human liver microsomes with six selective probe
substrates to test the inhibitory potential of the top 20 drugs identified
Values are average of duplicates.
% Activity Remaining
% Activity Remaining
Terfenadine
Drug
Terfenadine
Drug
(Astemizole)
(Astemizole)
Lansoprazole
Orphenadrine
Verapamil
Danazol
0.7 (71.1)
1.2 (102.2)
1.3 (69.1)
1.6 (8.0)
3.2
Albendazole
Quercetin
Paroxetine HCl
Mevinolin
29.3 (65.5)
29.8 (44.7)
31.6 (39.0)
37.7 (13.6)
39.0 (18.7)
43.0 (123.4)
43.5 (50.5)
44.2 (108.8)
44.3 (87.0)
45.1 (204.1)
45.3 (70.1)
45.7 (123)
46.1 (70.5)
48.7 (54.4)
48.9 (65.9)
50.8
Cisapride
Amiodarone
Methadone
Domperidone
Clomiphene
Fluoxetine
Ranolazine
Nortriptyline
Methadone
Omeprazole
Fluvoxamine
Cyclobenzaprine
Budesonide
Clozapine
Miconazole
Ketoconazole
Astemizole
Nicardipine
Loratadine
Tamoxifen
Clotrimazole
Simvastatin
Haloperidol
Mefloquine
Amodiaquine
Isradipine
3.4 (10.1)
5.8 (5.4)
8.3
8.9 (5.8)
11.4 (4.0)
11.5 (9.1)
14.1 (5.6)
14.3 (5.0)
15.6 (14.7)
22.4 (44.6)
22.5 (7.7)
23.7 (13.6)
26.2 (20.9)
27.29 (6.6)
51.3 (25.6)
51.4 (64.1)
51.7 (12.9)
Ivermectin
Pimozide
Quinapril
Thioridazine
individually prepared HLM and one pooled HLM samples. The
CYP2J2 content (pmol CYP2J2/mg microsomal protein) was variable
and ranged from 0.047 to 7.60 pmol/mg in the individual HLM
samples (obtained from the School of Pharmacy human tissue bank).
The pooled HLM sample contained 2.59 pmol/mg CYP2J2 (Table 5).
The CYP2J2 protein content did not correlate with CYP2J2-catalytic
activity assessed by either astemizole demethylation or 4-hydroxya-
miodarone, as seen in Table 5. However, the catalytic activity mea-
sured for the two CYP2J2 probe substrates correlated, r2 ϭ 0.97
(Fig. 2). It is acknowledged that HLM 130 generates high CYP2J2
activity despite very low levels quantified by Western blot analysis,
which greatly influenced the correlation analysis. Attempts were made
to add additional liver samples to the correlation analysis, but no HLM
samples were found to have activity between the cluster of HLM as inhibitors of CYP2J2-mediated terfenadine and astemizole metab-
samples and HLM 130. It is duly noted that if one were to remove olism against CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and
HLM 130, the r2 value falls to 0.47, which indicates a lower level of CYP3A4 to determine respective IC50 values (Table 8). Five com-
TABLE 8
IC50 values of potential CYP2J2 inhibitors against other P450s
IC50 (␮M)
Drug
CYP1A2
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A4
Albendazole
Astemizole
Bepridil
Clotrimazole
Danazol
Ͼ30
Ͼ30
Ͼ30
1.25
Ͼ30
Ͼ30
28.3
Ͼ30
26
6.46
Ͼ30
Ͼ30
Ͼ30
Ͼ30
5.33
Ͼ30
1.55
Ͼ30
Ͼ30
Ͼ30
Ͼ30
Ͼ30
Ͼ30
0.803
1.95
Ͼ30
5.00
Ͼ30
2.45
5.75
2.95
Ͼ30
1.56
Ͼ30
Ͼ30
Ͼ30
1.39
3.70
14.3
Ͼ30
Ͼ30
Ͼ30
Ͼ30
0.136
1.44
Ͼ30
3.66
Ͼ30
8.94
19.2
Ͼ30
Ͼ30
0.378
Ͼ30
Ͼ30
Ͼ30
10.2
Ͼ30
Ͼ30
Ͼ30
Ͼ30
13.7
Ͻ0.1
7.41
Ͼ30
2.87
Ͼ30
Ͼ30
10.1
13.4
2.80
14.9
3.30
24.2
Ͼ30
Ͼ30
10.5
Ͼ30
Ͼ30
Ͼ30
Ͼ30
Ͼ30
Ͼ30
0.883
2.74
Ͼ30
4.79
Ͼ30
16.3
2.90
Ͻ0.1
Ͼ30
1.59
Ͼ30
0.96
Ͼ30
18.8
Ͼ30
13.7
Ͼ30
Ͼ30
10.6
Ͼ30
Ͻ0.1
Ͼ30
8.20
Ͼ30
Ͼ30
Ͻ0.1
Ͼ30
Ͼ30
0.667
Ͼ30
Ͼ30
24.1
Ͼ30
2.11
Ͼ30
Ͼ30
17.6
Haloperidol
Isradipine
Ivermectin
Ketoconazole
Lansoprazole
Loratadine
Mefloquine
Nicardipine
Orphenadrine
Paroxetine
Pimozide
Quercetin
Simvastatin
Tamoxifen
Verapamil

CYP2J2-SELECTIVE SUBSTRATE AND INHIBITOR
949
(Fig. 4A) but could not be confirmed in dilution experiments commonly
used for the determination of mechanism-based inhibition.
Discussion
In this study, we report 4-hydroxyamiodarone as a specific CYP2J2
metabolite, devoid of contributions by other drug-metabolizing iso-
forms. Moreover, 4-hydroxyamiodarone formation correlated with
O-desmethyl astemizole, a previously reported metabolite of CYP2J2
(Matsumoto et al., 2002), in a panel of individually prepared HLM.
CYP2J2 enzyme activity did not appear to correlate with protein
content determined by Western blotting, as similarly observed by
other investigators (Yamazaki et al., 2006). We also identified danazol
as a selective inhibitor of CYP2J2, as assessed by inhibition of
terfenadine hydroxylation (IC50 ϭ 77 nM) and astemizole O-demeth-
ylation (Ki ϭ 20 nM). Danazol is specific for CYP2J2 at low con-
centrations (ϳ0.5 ␮M), but higher concentrations can inhibit other
isoforms such as CYP2C9, CYP2C8, and CYP2D6 with IC50 values
of 1.44, 1.95, and 2.74 ␮M, respectively.
We previously observed that amiodarone is hydroxylated on the
butyl side chain predominantly by CYP2J2 (Lee et al., 2010). In this
study, NMR analysis revealed that the hydroxylated metabolite is a
mixture of two distinct products, 4- and 3-hydroxyamiodarone. These
two metabolites have been recently identified in biliary excreta in
human subjects after amiodarone dosing (Deng et al., 2011). Deng et
al. (2011) identified 33 metabolites derived from amiodarone excreted
in human bile by mass spectral fragmentation patterns or confirmation
by comparison of chromatographic retention times and mass spectra
with available reference standards. Our work confirms the formation
of the 4- and 3-hydroxyamiodarone by CYP2J2 in HLM, and it is
likely that the former metabolite can be made in extrahepatic tissues
that may contribute to the overall in vivo formation.
Most substrates identified for CYP2J2 are also substrates for
CYP3A4 or CYP2D6 (Lee et al., 2010). CYP2J2 catalyzes the for-
mation of both 4- and 3-hydroxyamiodarone, whereas CYP3A4 and
other isozymes were only capable of forming 3-hydroxyamiodarone.
From reaction phenotyping data, 4-hydroxyamiodarone is a unique
CYP2J2-mediated metabolite, providing a tool to characterize
CYP2J2 inhibition potential in tissue microsomal preparations. Other
probe substrates such as terfenadine and astemizole are good CYP2J2
probe substrates when studying recombinant CYP2J2. The formation
of hydroxy-terfenadine (Rodrigues et al., 1995; Lafite et al., 2007) is
predominantly formed by CYP3A4, whereas O-desmethyl astemizole
is also formed by CYP2D6 (Matsumoto and Yamazoe, 2001). The
identification of 4-hydroxyamiodarone as a CYP2J2-specific reaction
will aid the assessment of CYP2J2 inhibitory potential of new chem-
ical entities (NCE) in drug discovery and development, especially as
we understand its role in extrahepatic tissues.
FIG. 3. Structure of danazol.
pounds (danazol, ketoconazole, loratadine, miconazole, and nicardi-
pine) significantly reduced terfenadine or astemizole metabolism.
However, only danazol (Fig. 3) seemed selective against CYP2J2 and
modestly inhibited CYP2C8, CYP2D6, and CYP2C9 with IC50 values
of 1.95, 2.74, and 1.44 ␮M, respectively, and did not appreciably
inhibit CYP3A4, CYP1A2, or CYP2C19 at concentrations as high as
30 ␮M. The remaining compounds were either not potent inhibitors
against both terfenadine and astemizole oxidation or inhibited at least
one other cytochrome P450 in the low nanomolar range and were
excluded from further consideration.
Seven Probe Substrate Cocktail. A seven probe cocktail that
included CYP2J2 contribution was established containing phenacetin
(CYP1A2), paclitaxel (CYP2C8), diclofenac (CYP2C9), S-mepheny-
toin (CYP2C19), dexamethorphan (CYP2D6), astemizole (CYP2J2),
and midazolam (CYP3A4). Initial evaluation of the seven cocktail
probes was conducted with amiodarone as the CYP2J2 probe sub-
strate; however, addition of amiodarone resulted in substantial inhi-
bition of other P450-mediated pathways. More specifically, amioda-
rone (5 ␮M) inhibited CYP2C8, CYP2C9, CYP2C19, and CYP2D6,
resulting in the decrease in metabolite formation from these isozymes
by 33, 68, 29, and 37%, respectively (Table 9). When astemizole (0.3
␮M) was included as the seventh probe substrate, only marginal
inhibition of CYP3A4 activity (20%) was observed with minimal
effect on the other isoforms (Table 9). Four compounds (miconazole,
pimozide, danazol, and terfenadine) were used to validate the seven
probe cocktail in which CYP2J2-related IC50 values generated were
similar to values measured in the single probe astemizole assay only
(Table 10).
Recombinant CYP2J2 Inhibition Constants (IC50 and Ki) for
Danazol. IC50 values were ascertained using both inhibition of terfe-
nadine hydroxylation (0.077 Ϯ 0.001 ␮M) and astemizole O-demeth-
ylation (0.019 Ϯ 0.006 ␮M) (Fig. 4A) using recombinant CYP2J2
enzyme. A Ki of 20 nM for danazol was calculated based on astemizole
O-demethylation activity (Fig. 4B), and the kinetics fit to a model of
mixed inhibition with a high component of competitive inhibition (␣ ϭ
18.3). However, a precise characterization of the mechanism was se-
verely hampered by 1) the assay’s limit of quantitation (nanomolar range)
and the distortion caused by substrate depletion, which was unavoidable
With the use of 4-hydroxyamiodarone formation, it is now possible
at low substrate concentrations (astemizole-O-demethylation Km value of to determine the RAF and ISEF values for this isoform to the overall
0.3 ␮M); 2) a potential allosteric component of astemizole-O- P450-mediated metabolism in the liver. Because this isoform is
demethylation; and 3) a possible residual solvent effect. A slight IC50 largely found in extrahepatic tissues, it will be important to determine
shift from 0.019 to 0.012 ␮M using a 30-min preincubation in the in the future a physiological based pharmacokinetic model that incor-
presence of inhibitor and NADPH, but in absence of substrate, was found porates hepatic and extrahepatic tissue metabolism to depict overall
TABLE 9
Effect of amiodarone or astemizole on probe substrate activity
% Inhibition of Metabolite Formation (Mean Ϯ S.D.)
CYP2J2 Probe Substrate
CYP1A2
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A4
Amiodarone, 5 ␮M
Astemizole, 0.3 ␮M
Ϫ0.6 Ϯ 9.4
Ϫ0.6 Ϯ 4.3
32.7 Ϯ 1.9
1.1 Ϯ 11.7
68.3 Ϯ 1.7
Ϫ2.7 Ϯ 2.6
28.6 Ϯ 20
6.0 Ϯ 8.2
36.5 Ϯ 2.8
8.5 Ϯ 1.6
Ϫ18.4 Ϯ 10
20.3 Ϯ 3.0

950
LEE ET AL.
30 ␮M. It is possible that some of the inhibitors are also substrates that
TABLE 10
have very low clearance and were not identified as CYP2J2 substrates.
Danazol was also identified as a substrate, which supports the finding that
it is mostly a competitive inhibitor of astemizole and terfenadine oxida-
tion. This observation is also consistent with previous findings (Lee et al.,
2010) in which the isoxazole ring is the site of metabolism, suggesting
that this moiety is oriented toward the heme instead of the ethinyl moiety,
which is a structural alert for potential mechanism-based inhibition.
Inhibition of CYP2J2 in the cardiac tissue may partially be responsible
for the cardiotoxicity observed by some of these agents. Work is currently
underway to measure CYP2J2 inhibition in cardiac tissue and to deter-
mine whether this inhibition can lead to cardiac toxicity.
Based on structure activity studies using terfenadine as a back-
bone, Lafite et al. (2006, 2007) synthesized a series of compounds
and tested their ability to inhibit CYP2J2 hydroxylation of ebas-
tine. Two compounds were identified as selective potent inhibitors
of CYP2J2, with compound 4 having a Ki of 160 nM. Chen et al.
(2009) used the synthetic compounds designed by Lafite et al.
(2006, 2007) to inhibit CYP2J2 in tumor cell lines. Whereas these
compounds appear to be selective inhibitors of CYP2J2, a few
caveats should be noted in that they were not tested against a large
panel of P450s, especially CYP2D6, and they are not readily
available.
To facilitate screening the large number of NCE synthesized in
drug discovery programs as potential inhibitors of CYP2J2, evaluation
of amiodarone and astemizole as a seventh probe substrate to include
in the high-throughput P450 inhibition probe cocktail for drug inter-
action assessments was investigated. Whereas amiodarone and
astemizole are ideal as single probe substrates to evaluate the potential
of an NCE to inhibit recombinant CYP2J2, we found that only
astemizole was suitable in a P450 cocktail assay setting because it had
limited interactions with the other P450 isoforms. Unfortunately,
amiodarone inhibited several P450s, namely CYP2C8, CYP2C9,
CYP2C19, and CYP2D6, at 29% or greater, whereas astemizole only
inhibited CYP3A4 to a minor extent. Moreover, the cocktail drug-
drug interaction probes did not alter astemizole O-demethylation
activity because similar IC50 values were generated for miconazole,
pimozide, danazol, and terfenadine in the seven probe cocktail versus
single probe assay evaluation.
Comparison of IC50 values generated in single vs. multiple probe cocktail
Single probe substrate evaluated with astemizole O-demethylation.
Inhibitor
IC50 (␮M) Single Probe
IC50 (␮M) Seven Probe
Miconazole
Pimozide
0.58
Ͼ30
0.64
Ͼ30
Danazol
Terfenadine
Ͻ0.1
1.13
Ͻ0.1
1.83
contribution of various P450s. Among the various HLM preparations
evaluated, the RAF value was rather consistent, varying only 2.6-fold.
However, the value was quite low and reflected low overall CYP2J2
activity in the HLM. It is noteworthy that the ISEF value that accounts
for potential variation in the amount of CYP2J2 in the HLM per
milligram of protein is highly variable in the different HLM prepara-
tions ranging from 0.0006 to 1.03, a ϳ1700 fold variation among the
individual preparations with pooled HLM ISEF value near the lower
end of that range. The large variation in ISEF values is likely attrib-
uted to variability of P450 content or abundance in HLM because the
recombinant P450 content is constant in this analysis. Moreover, the
antibody binding to an epitope to CYP2J2 in the various HLM
preparations cannot distinguish functional from nonfunctional pro-
tein. Taken together, these factors likely contribute to the large
variation.
4-Hydroxyamiodarone correlated well with astemizole O-
demethylation in a set of nine individually prepared HLM. It is
noteworthy that correlation of either 4-hydroxyamiodarone or O-
desmethyl astemizole with CYP2J2 protein content in the HLM
was very low. The ability of the CYP2J2 antibody to recognize
functional enzyme as well as apo-inactive enzyme will contribute
to the lack of correlation. However, the high correlation between
these two probe substrates suggests that the contribution of
CYP2D6 to astemizole O-demethylation is relatively minor (Ma-
tsumoto and Yamazoe, 2001). CYP3A4 contributes mostly to the
hydroxylation of astemizole and will not interfere with O-demeth-
ylation catalyzed by CYP2J2.
Danazol emerged as a specific inhibitor of CYP2J2 among the 138
drugs approved for clinical use in the United States that were screened.
Inhibition screens were performed with terfenadine and astemizole be-
cause 4-hydroxyamiodarone was discovered after screens were con-
ducted. Because CYP2J2 is the main cardiac isozyme, the list of drugs
tested included several drug classes including those that modulate cardiac
function or have known cardiac toxicity. When a similar list of drugs was
screened to identify substrates of CYP2J2 (Lee et al., 2010), only eight
In conclusion, 4-hydroxyamiodarone and danazol are specific
CYP2J2 probe reaction and inhibitor, respectively. The utility of
danazol as a specific CYP2J2 inhibitor will reveal the contribution
of this isoform toward the overall P450-mediated clearance of an
NCE. The presence of CYP2J2 activity in HLM will facilitate
substrates were identified. The list of potential inhibitors identified was awareness of the potential extrahepatic contributions in total sys-
much larger after 42 drugs met the initial criteria of Ͼ50% inhibition at temic clearance that may be underestimated using in vitro systems.
FIG. 4. A, IC50 determination for danazol inhi-
bition of astemizole O-demethylation with and
without a 30-min preincubation of the inhibitor
with enzyme and NADPH. B, kinetic plots for
velocity versus substrate concentration while
varying danazol concentrations.

CYP2J2-SELECTIVE SUBSTRATE AND INHIBITOR
951
Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2.
Bioorg Med Chem Lett 16:2777–2780.
Lafite P, Dijols S, Zeldin DC, Dansette PM, and Mansuy D (2007) Selective, competitive and
mechanism-based inhibitors of human cytochrome P450 2J2. Arch Biochem Biophys 464:
155–168.
Lee CA, Neul D, Clouser-Roche A, Dalvie D, Wester MR, Jiang Y, Jones JP 3rd, Freiwald S,
Zientek M, and Totah RA (2010) Identification of novel substrates for human cytochrome
P450 2J2. Drug Metab Dispos 38:347–356.
Matsumoto S, Hirama T, Kim HJ, Nagata K, and Yamazoe Y (2003) In vitro inhibition of human
small intestinal and liver microsomal astemizole O-demethylation: different contribution of
CYP2J2 in the small intestine and liver. Xenobiotica 33:615–623.
Matsumoto S, Hirama T, Matsubara T, Nagata K, and Yamazoe Y (2002) Involvement of
CYP2J2 on the intestinal first-pass metabolism of antihistamine drug, astemizole. Drug Metab
Dispos 30:1240–1245.
Matsumoto S and Yamazoe Y (2001) Involvement of multiple human cytochromes P450 in the
liver microsomal metabolism of astemizole and a comparison with terfenadine. Br J Clin
Pharmacol 51:133–142.
Furthermore, astemizole can be added to existing P450 probe
cocktail assays to screen for CYP2J2 inhibition in drug discovery.
Authorship Contributions
Participated in research design: Lee, Jones, Kaspera, and Totah.
Conducted experiments: Jones, Katayama, Kaspera, Jiang, Freiwald, Smith,
and Walker.
Contributed new reagents or analytic tools: Jones, Katayama, Kaspera, and
Jiang.
Performed data analysis: Lee, Jones, Kaspera, Smith, Walker, and Totah.
Wrote or contributed to the writing of the manuscript: Lee, Jones, Kaspera,
Smith, Walker, and Totah.
Paine MF, Hart HL, Ludington SS, Haining RL, Rettie AE, and Zeldin DC (2006) The human
intestinal cytochrome P450 “pie.” Drug Metab Dispos 34:880–886.
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Address correspondence to: Dr. Rheem A. Totah, Department of Medicinal
Chemistry, Box 357610, University of Washington, Seattle, WA. E-mail:
rtotah@uw.edu
Lafite P, Dijols S, Buisson D, Macherey AC, Zeldin DC, Dansette PM, and Mansuy D (2006)