Identification of Novel Substrates for Human Cytochrome
Caroline A. Lee, David Neul, Andrea Clouser-Roche, Deepak Dalvie, Michael R. Wester,
Ying Jiang, J. P. Jones III, Sascha Freiwald, Michael Zientek, and Rheem A. Totah
Pfizer Global Research & Development-La Jolla Laboratories, San Diego, California (C.A.L., D.N., A.C.-R., D.D., M.R.W., Y.J.,
S.F., M.Z.); and Department of Medicinal Chemistry, University of Washington Seattle, Washington (J.P.J., R.A.T.)
Received September 17, 2009; accepted November 17, 2009
Several antihistamine drugs including terfenadine, ebastine, and
astemizole have been identified as substrates for CYP2J2. The
overall importance of this enzyme in drug metabolism has not been
fully explored. In this study, 139 marketed therapeutic agents and
compounds were screened as potential CYP2J2 substrates. Eight
novel substrates were identified that vary in size and overall topol-
ogy from relatively rigid structures (amiodarone) to larger complex
structures (cyclosporine). The substrates displayed in vitro intrin-
sic clearance values ranging from 0.06 to 3.98 ?l/min/pmol
CYP2J2. Substrates identified for CYP2J2 are also metabolized by
CYP3A4. Extracted ion chromatograms of metabolites observed
for albendazole, amiodarone, astemizole, thioridazine, mesorid-
azine, and danazol showed marked differences in the regioselec-
tivity of CYP2J2 and CYP3A4. CYP3A4 commonly metabolized
compounds at multiple sites, whereas CYP2J2 metabolism was
more restrictive and limited, in general, to a single site for large
compounds. Although the CYP2J2 active site can accommodate
large substrates, it may be more narrow than CYP3A4, limiting
metabolism to moieties that can extend closer toward the active
heme iron. For albendazole, CYP2J2 forms a unique metabolite
compared with CYP3A4. Albendazole and amiodarone were eval-
uated in various in vitro systems including recombinant CYP2J2
and CYP3A4, pooled human liver microsomes (HLM), and human
intestinal microsomes (HIM). The Michaelis-Menten-derived intrin-
sic clearance of N-desethyl amiodarone was 4.6 greater in HLM
than in HIM and 17-fold greater in recombinant CYP3A4 than in
recombinant CYP2J2. The resulting data suggest that CYP2J2 may
be an unrecognized participant in first-pass metabolism, but its
contribution is minor relative to that of CYP3A4.
CYP2J2 is the only member of the human CYP2J subfamily known
for its role in metabolizing arachidonic acid to physiologically im-
portant epoxides. Among the CYP2 family, CYP2J2 and CYP2C9
mRNA levels were found to be highly variable in human hearts,
whereas CYP2C8 mRNA was measured in lower abundance (Delo-
zier et al., 2007). Moreover, Delozier et al. (2007) found that CYP2J2
mRNA levels in the heart were approximately 10 times higher than
those of CYP2C9 or CYP2C8. Besides high expression in heart tissue,
CYP2J2 is present in other tissues as well (Wu et al., 1996). Based on
mRNA levels, the abundance of this enzyme is greatest in the small
intestine ? heart ? skeletal muscles ? kidney ? salivary glands ?
lungs ? liver. CYP2J2 in the liver constitutes 1 to 2% of total
cytochrome P450 (P450) content, which is similar to that in the small
intestine (1.4% of total P450 content) (Paine et al., 2006).
CYP2J2 is known mostly for its role as the major human arachi-
donic acid epoxygenase in the heart and various other tissues. In
addition to its endogenous role, CYP2J2 also plays a role in drug
metabolism. A number of antihistamine drugs were identified as
substrates of CYP2J2 including terfenadine, ebastine, and astemizole
(Matsumoto and Yamazoe, 2001; Hashizume et al., 2002; Matsumoto
et al., 2002; Liu et al., 2006; Lafite et al., 2007). In fact, CYP2J2 was
shown to be the enzyme responsible for the first-pass metabolism of
ebastine. However, the overall importance of CYP2J2 in drug metab-
olism and toxicity has not been established and a broad evaluation of
potential substrates for CYP2J2 has not yet been conducted to under-
stand the role, if any, this isoform plays in overall drug metabolism.
Because CYP2J2 is predominantly expressed (based on mRNA) in the
small intestine and heart, the importance of this isoform in drug
metabolism may be appreciably underestimated because liver is the
predominate tissue typically used in drug research to profile P450s
and to estimate their contribution to the metabolism of new drug
In this study, the substrate diversity of CYP2J2 was investigated by
evaluating 139 marketed drugs from different therapeutic classes.
Mass spectral analysis was used to determine the loss of parent
compound with time when compounds were incubated with recom-
Salary support for J.P.J. was provided by the University of Washington, School
of Pharmacy Drug Metabolism, Transporter and Pharmacogenomics Research
Program funded by gifts from Abbott, Allergan, Amgen, Bend Research, Bristol-
Myers Squibb, Eli Lilly, Hoffman-La Roche, Johnson & Johnson, Merck, and
ABBREVIATIONS: P450, cytochrome P450; ClINT, Michaelis-Menten-derived intrinsic clearance; DMSO, dimethyl sulfoxide; LC, liquid chroma-
tography; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; HLM, human liver microsomes; HIM, human
intestinal microsomes; Clint, intrinsic clearance; AIC, Akaike information criterion; amu, atomic mass unit; r, recombinant.
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:347–356, 2010
Vol. 38, No. 2
Printed in U.S.A.
binant CYP2J2. The substrates metabolized by CYP2J2 were then
confirmed by metabolite identification using mass spectral analysis.
Moreover, albendazole and amiodarone Michaelis-Menten-derived
intrinsic clearances (ClINT) were determined in different in vitro
expression systems, human liver microsomes and human intestinal
microsomes, to further characterize CYP2J2 activity.
Materials and Methods
Chemicals. Compounds (Table 1) used for CYP2J2 substrate assessment,
furafylline, sulfaphenazole, quinidine, ketoconazole, methanol, buspirone, po-
tassium phosphate (monobasic and dibasic), NADPH, glucose 6-phosphate,
NADP?, glucose-6-phosphate dehydrogenase, and magnesium chloride were
purchased from Sigma-Aldrich (St. Louis, MO). Montelukast was purchased
from Cayman Chemical (Ann Arbor, MI). Benzylnirvanol, N-desethyl amio-
darone, and albendazole sulfoxide were synthesized at Pfizer Global Research
and Development (La Jolla, CA). CYP2J2 and CYP3A4 recombinant enzyme
(Supersomes) and pooled human liver microsomes and pooled human intesti-
nal microsomes were purchased from BD Gentest (Woburn, MA).
CYP2J2 Supersomes Incubations. Assays were performed on a Biomek
FX system (Beckman Coulter, Fullerton, CA). Recombinant CYP2J2 incuba-
tions contained substrate (1 ?M, final organic 0.01% DMSO, and 0.6%
acetonitrile), 100 pmol/ml recombinant CYP2J2, and 2 mM NADPH in a final
volume of 200 ?l of 100 mM potassium phosphate buffer, pH 7.4. After a
10-min preincubation period at 37°C, reactions were initiated with the addition
of NADPH. Samples (25-?l aliquots) were removed at 0, 5, 10, 20, 30, and 45
min and quenched with 75 ?l of cold methanol containing 0.1 ?M buspirone
(internal standard). Samples were then centrifuged at 3000 rpm (GH3.8A rotor;
Beckman-Coulter) for 15 min. A 50-?l aliquot of supernatant was transferred
to a clean 96-shallow well plate and combined with 50 ?l of Milli-Q water,
mixed, and analyzed with LC-MS/MS. All incubations were performed in
triplicate, and compounds metabolized by CYP2J2 were confirmed by con-
ducting triplicate experiments on three separate occasions.
Metabolite Identification of CYP2J2 Substrates. All incubations (total
volume 1.0 ml) were conducted at 37°C for 45 min in 100 mM potassium
phosphate buffer (pH 7.4) containing magnesium chloride (10 mM), recom-
binant CYP2J2 Supersomes (0.17 mg/ml of protein), and NADPH (2 mM).
Reactions were initiated by addition of substrate (15 ?M) after preincubation
of the reaction mixture for 3 min at 37°C. The final concentration of DMSO
in the incubation mixture was ?0.1%, and the final concentration of all organic
solvents in the incubation mixture was ?1%. Incubations that lacked NADPH
or Supersomes served as negative controls. Reactions were terminated with 1
ml of acetonitrile and centrifuged at 3000g for 10 min.
Danazol and Nabumetone Reaction Phenotyping with Chemical Inhib-
itors. Chemical inhibition experiments were performed using pooled human
liver microsomes (HLM) and specific chemical inhibitors for CYP1A2, 2C8,
2C9, 2C19, 2D6, and 3A4. All inhibitors were considered competitive inhib-
itors. For danazol and nabumetone, 1 ?M substrate (final organic concentra-
tion 0.01% DMSO and 0.6% acetonitrile) was mixed with 0.8 mg/ml HLM and
100 mM phosphate buffer (pH 7.4) were preincubated for 5 min at 37°C in an
incubator on the Biomek FX system. The NADPH regeneration system was
prepared fresh before the incubation at a concentration of 80 mM NADP?, 400
mM glucose 6-phosphate, and 80 units/ml glucose 6-phosphate dehydrogenase
in phosphate buffer (pH 7.4). Reactions were initiated by the addition of the
NADPH-regenerating system (one-eighth of the final volume) or an equal
volume of buffer for negative controls. The final incubation volume was 200
?l. The concentrations of inhibitors in the final reaction were 30 ?M furafyl-
line (CYP1A2), 2 ?M montelukast (CYP2C8), 5 ?M sulfaphenazole
(CYP2C9), 5 ?M benzylnirvanol (CYP2C19), 1 ?M quinidine (CYP2D6), and
1 ?M ketoconazole (CYP3A4). At 0, 10, 20, and 45 min, 25-?l aliquots were
removed and quenched with 75 ?l of cold MeOH with 0.1 ?M buspirone
(internal standard). Samples were then centrifuged at 3000 rpm (GH3.8A
rotor) for 15 min. A 50-?l aliquot of supernatant was transferred to a clean
96-shallow well plate and combined with 50 ?l of Milli-Q water, mixed, and
analyzed with LC-MS/MS.
Amiodarone and Albendazole Metabolism. Amiodarone N-deethylation
and albendazole S-oxidation kinetic assays were performed with a Genesis 150
automated system (Tecan Group Ltd., Ma ¨nnedorf, Switzerland). Reaction
mixtures (500 ?l/well) contained 470 ?l of either 0.2 mg/ml HLM, human
intestinal microsomes (HIM), 20 pmol/ml recombinant CYP3A4, or 40
pmol/ml recombinant CYP2J2 and amiodarone or albendazole (5 ?l were
added to incubation plate) to make the final concentrations ranging from 0 to
50 ?M in each experiment. The reactions were initiated with 25 ?l of 20 mM
NADPH (1 mM final). For each amiodarone concentration, samples were
collected at times of 0, 1.1, 2.6, 7.1, 11.7, 17.2, 22.7, 33.2, and 48.7 min at
37°C. Each reaction was terminated using 500 ?l of ice-cold acetonitrile
containing 0.01 ?M buspirone. For albendazole, a time course linearity study
was conducted as a pilot study, and a 20-min incubation time was optimal for
linear kinetics and ?15% substrate depletion. After a 20-min incubation,
samples were terminated with 500 ?l of ice-cold acetonitrile containing 0.01
?M buspiron. After mixing of the samples, the plates were centrifuged at 3000
rpm (GH3.8A rotor) for 15 min, and 200 ?l/well was transferred to a separate
96-well polypropylene plate for LC-MS/MS analysis for metabolite formation.
LC-MS/MS Methods. CYP2J2 metabolism study. The compounds of in-
terest were introduced onto a Synergi Polar-RP (2 ? 30 mm, 4 ?; Phenome-
nex, Torrance, CA) HPLC column with a CTC PAL autosampler (Leap
Technologies, Carrboro, NC) and an integrated HPLC pumping system (Shi-
madzu 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 TurboIon-
Spray interface. Mobile phase A was 0.1% formic acid, mobile phase B was
acetonitrile with 0.1% formic acid, and the flow rate was 0.8 ml/min. The
initial condition for the HPLC gradient was 99:1 (A:B). This was held for 0.75
min. From 0.75 to 2.5 min, the mobile phase composition changed linearly to
5:95 (A:B). This condition was held until 2.6 min. The gradient was returned
in a linear fashion to 99:1 (A:B) from 2.6 to 2.8 min and reequilibrated until
3.5 min. The injection volume was 10 ?l. Multiple reaction monitoring was
used to monitor the compounds as depicted in Table 2. The peak area ratio of
the analyte to the internal standard was determined for each injection and used
to measure substrate depletion.
Cytochrome P450 reaction phenotyping. An LC-MS/MS method similar to
that in the CYP2J2 metabolism study was used for the P450 reaction pheno-
Compounds evaluated in recombinant CYP2J2
LEE ET AL.
typing with specific chemical inhibitors. The initial condition for the gradient
was 95:5 (A:B). From 0 to 0.3 min, the mobile phase composition changed
linearly to 85:15 (A:B). From 0.3 to 1.5 min, the mobile phase composition
changed linearly to 10:90 (A:B) and was held there until 1.7 min. The gradient
was returned in a linear fashion to 95:5 (A:B) from 1.7 to 1.71 min and
reequilibrated until 2 min.
Metabolite profiling. Metabolite profiling and identification of metabolites
in the microsomal incubations was achieved by separating the metabolites on
a Kromasil C4 column (3.5 ?m, 150 ? 2.0 mm, Phenomenex) by reverse-
phase chromatography at ambient temperature. The mobile phase consisted of
0.1% formic acid (solvent A) and acetonitrile (solvent B) and was delivered at
0.200 ml/min. The initial composition of solvent B was maintained at 1% for
5 min and then increased in a linear manner as follows: 20% at 8 min; 40% at
35 min, and 90% at 42 min. It was maintained at 90% for up to 45 min and then
decreased to 1% in the next 5 min. The column was allowed to equilibrate at
1% solvent B for 5 min before the next injection. The HPLC effluent going to
the mass spectrometer was directed to waste through a divert valve for the
initial 5 min after sample injection.
Mass spectrometric analyses were performed on a ThermoFinnigan Deca
XP ion trap mass spectrometer, which was interfaced to an Agilent HP-1100
HPLC system (Agilent Technologies, Palo Alto, CA) and equipped with an
electrospray ionization source. The values for electrospray ionization were as
follows: capillary temperature 270°C; spray voltage 4.0 kV; capillary voltage
4.0 V; sheath gas flow rate 90; and auxiliary gas flow rate 30. The mass
spectrometer was operated in a positive ion mode with data-dependent scan-
ning. The ions were monitored over a full mass range of m/z 125 to 1000. For
a full scan, the automatic gain control was set at 5.0 ? 108, the maximum ion
time was 100 ms, and the number of microscans was set at 3. For MSn
scanning, the automatic gain control was 1.0 ? 108, maximum ion time was
400 ms, and the number of microscans was set at 2. For data-dependent
scanning, the default charge state was 1, default isolation width was 3.0, and
the normalized collision energy was 40 V.
Michaelis-Menten studies. Samples were injected onto a Synergi Polar-RP
(2 ? 30 mm, 4 ?) HPLC column with a CTC PAL autosampler and an
integrated HPLC pumping system. The metabolites of amiodarone and al-
bendazole were detected by an API 4000 triple quadrupole mass spectrometer
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
0.8 ml/min. The starting condition for the HPLC gradient was 99:1 (A:B). This
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 until 1.9 min. The
gradient was returned in a linear fashion to 99:1 (A:B) from 1.9 to 1.95 min
and reequilibrated until 2 min. The injection volume was 20 ?l. Multiple
reaction monitoring was used to monitor the compounds. Table 3 lists the
ionization mode, m/z transitions, and retention times for albendazole, amioda-
rone, and their metabolites. The peak area ratio of the analyte to the internal
standard (buspirone) was determined for each injection and was used to
measure substrate depletion.
CYP2J2 Homology Model. The three-dimensional model of the CYP2J2
structure was generated based on the crystal structures of CYP2B4 (Protein
Data Bank code 1po5), CYP2C8 (Protein Data Bank code 1pq2), and CYP2A6
(Protein Data Bank code 1z10). These enzymes were chosen to model the
CYP2J2 structure because they belong to the same CYP2 subfamily and were
mostly free of internal mutations or bound ligands. The sequence of CYP2J2
was obtained from SwissProt (accession number NP_000766). A multiple
sequence alignment between CYP2B4, 2C8, 2A6, and 2J2 was obtained after
the first 36 amino acid hydrophobic region of CYP2J2 was removed. This
alignment was used as input for the program Modeler 8.1 (Sali and Blundell,
1993) and a homology model was generated using a combination of molecular
dynamics, simulated annealing, and restraints based on the known structures
and the alignment. A more detailed description is available at http://salilab.org/
modeller/tutorial. The homology model was evaluated for unfavorable bond
angles and bond lengths and determined to be structurally reasonable based on
Data Analysis. In the determination of the in vitro t1/2, the analyte/internal
standard peak area ratios were converted to percentage of drug remaining,
using the T ? 0 peak area ratio value as 100%. The slope (?k) of the linear
regression from log percentage remaining versus incubation time was used in
the in vitro t1/2determination of t1/2? ?0.693/k. The t1/2is then used to
generate the intrinsic clearance (Clint) as in eq. 1:
in vitro t1/ 2?
pmol recombinant CYP2J2
Reaction phenotyping. The half-life value was determined from the loss of
parent plotted on a logarithmic scale of the natural log of the peak area ratio
versus time as described above. The half-life value is incorporated in the organ
scaled intrinsic clearance (Clint?) eq. 2:
in vitro t1/ 2?
mg microsomes?45 mg microsomes
?20 g liver
Percent contribution for each isozyme was calculated using eq. 3:
%Contribution ? 100 ?average Clint?control? ? average Clint?inhibitor?
Michaelis-Menten analysis of amiodarone and albendazole. For kinetic
parameters estimation for N-desethyl amiodarone (Vmaxand Km) and hydroxyl
amiodarone (Km), values were determined by a nonlinear least-squares algo-
rithm. Data were fitted to the Michaelis-Menten equation (eq. 4), where Vmax
is the maximum enzyme velocity, [S] is the substrate concentration, and Kmis
the Michaelis-Menten constant.
Kinetic parameters estimated for albendazole S-oxidation were determined by
nonlinear least-squares regression. Data were fitted to the Michaelis-Menten
equation as described in eq. 4 and to a substrate inhibition model (eq. 5). The
best model to fit the data was determined using the Akaike information
criterion (AIC). Ksdefines the dissociation constant for the productive enzyme
substrate complex, and Kidefines the dissociation constant for the inhibitory
enzyme substrate complex whereby two substrates can bind to the enzyme
?Ks? ?S? ? ?1 ? ?S?/Ki??
Mass spectral m/z values and retention times
Compound Q1 m/za
Q1, quadrupole 1; Q3, quadrupole 3.
am/z ratios are ?0.5 m/z.
Mass spectral conditions for albendazole, amiodarone, and metabolites
Q1, quadrupole 1; Q3, quadrupole 3.
NOVEL SUBSTRATES FOR CYP2J2
Data modeling was performed using GraphPad Prism (version 5.01; GraphPad
Software, Inc., La Jolla, CA).
Recombinant CYP2J2 Metabolism of Various Compounds. A
total of 139 drugs from different therapeutic classes (Table 1) were
evaluated for their potential to undergo metabolism by CYP2J2.
Astemizole and terfenadine served as positive controls because both
have been characterized previously as substrates for CYP2J2 (Hashi-
zume et al., 2002; Matsumoto et al., 2002, 2003; Lafite et al., 2006).
The metabolism of terfenadine by recombinant CYP2J2 is three times
faster than that of astemizole, with Clintvalues of 3.98 and 1.14
?l/min/pmol CYP2J2, respectively (corresponding half-life values
were 1.74 and 6.06 min for terfenadine and astemizole, respectively).
For the newly identified CYP2J2 substrates, Clintvalues ranged from
0.06 to 0.49 ?l/min/pmol of CYP2J2 (Table 4). Moreover, metabo-
lism by CYP2J2 was confirmed by metabolite identification via mass
Identification of Metabolites Generated from CYP3A4 and
CYP2J2. Of the 139 compounds examined, six compounds (albenda-
zole, amiodarone, astemizole, thioridazine, mesoridazine, and dana-
zol) showed relatively rapid turnover in incubations with expressed
CYP2J2. Because all compounds were also metabolized by CYP3A4,
a study to assess similarities and differences in the metabolites formed
by CYP3A4 and CYP2J2 was conducted. Figure 1 depicts the site of
metabolism by CYP2J2 and CYP3A4. Figure 2A shows the extracted
ion chromatograms of metabolites observed in incubations of albenda-
zole, amiodarone, astemizole, thioridazine, mesoridazine, and danazol
with CYP3A4. Albendazole (Fig. 2Aa) was metabolized primarily to
one oxidative metabolite, which was characterized as albendazole
sulfoxide (M1) based on its mass spectrum, fragmentation pattern, and
comparison to a synthetic standard. Likewise, metabolism of amio-
darone by recombinant CYP3A4 resulted in only one metabolite, the
N-deethylated product (M1) (Fig. 2Ab). The structure of the metabolite
was confirmed from its mass spectrum, fragmentation pattern, and com-
parison to a synthetic standard. Trace amounts of hydroxylated amioda-
rone (M2) were also detected in CYP3A4 incubations when molecular
ion was extracted from the total ion chromatogram. However, a proper
structural elucidation of this metabolite was not possible given the weak
mass spectrum of the metabolite in the MS/MS scan.
Incubation of astemizole with recombinant CYP3A4 resulted in
several metabolites (Fig. 2Ac). Metabolites (M1–M3) eluting between
25 and 26 min showed an addition of 16 amu, suggesting hydroxy-
lation of the parent compound; the exact site of hydroxylation was not
determined. The peak at 25 min (M4) gave a molecular ion at m/z 325,
suggesting N-dealkylation of the compound. A trace amount of the
O-dealkylated metabolite (M5, m/z 445) was also observed in the
incubation mixture, which coeluted with M1. Incubation of thiorida-
zine with recombinant CYP3A4 resulted in two major metabolites
(M1 and M2) (Fig. 2Ad) with molecular ions that were 16 Da greater
than the parent. The mass spectral fragmentation of both metabolites
showed a major loss of 16 amu, suggesting that both compounds were
FIG. 1. Substrate structures with arrows in-
dicating sites of metabolism by CYP2J2
(dashed arrows) and CYP3A4 (solid arrows).
Clintvalues for CYP2J2 substrates
aPreviously identified substrates serve as positive controls.
LEE ET AL.
isomeric sulfoxide metabolites or a sulfoxide and possibly an N-oxide
metabolite. Comparison of the retention time of M1 (27 min) with that
of mesoridazine indicated that M1 resulted from sulfoxidation of the
methyl sulfide moiety in thioridazine. Based on the previously pub-
lished metabolism studies of thioridazine by CYP3A4 (Berecz et al.,
2003), metabolite M2 was proposed as thioridazine 5-sulfoxide. In-
cubation of mesoridazine with recombinant CYP3A4 showed one
peak in the extracted ion chromatogram with a molecular ion that was
16 amu greater than that of the parent (Fig. 2Ae). Addition of 16 amu
in M1 suggested hydroxylation of the compound. The exact site of
oxidation could not be ascertained from mass spectral fragmentation.
However, metabolite M1 was tentatively proposed as a sulfone me-
tabolite of mesoridazine. Incubation of danazol with CYP2A4 resulted
in several hydroxylated metabolites (M1–M4) (Fig. 2Af). The mass
spectra of all metabolites were similar. However, the loss of a water
molecule in the mass spectra of all metabolites suggested that the
steroid skeleton was the site of metabolism. Hence, the exact positions
of hydroxylation could not be determined. In addition to the hydroxy-
lated metabolites, two small peaks M5 and M6 with molecular ions of
m/z 341 and 327 were also detected at retention times of 33 and 34.5
min in the total ion chromatogram of danazol incubation with
CYP3A4 (which coeluted with M1–M4). The molecular ion of M5
suggested an addition of 3 amu to the molecular ion of the parent
compound (m/z 341 versus m/z 338). Furthermore, an odd molecular
ion of the metabolite indicated a possible loss of the nitrogen atom
from the molecule. Based on the molecular ion, however, the metab-
olite was assumed to be a ?-ketoaldehyde derivative of danazol,
formed via the cleavage of the isoxazole ring. Likewise, metabolite
M6 showed a molecular ion that is 11 amu less than the parent, and
the odd molecular ion of M6 also indicated the possible loss of a
nitrogen atom. The complex fragmentation pattern and small intensity
of the metabolite made it difficult to elucidate a proper structure of the
metabolites. The isoxazole cleavage products of danazol were de-
tected previously in human urine (Rosi et al., 1977). The mechanism
of the oxidative cleavage of the isoxazole ring in danazol is unknown.
Figure 2B shows the metabolic profiles of albendazole, amio-
darone, astemizole, thioridazine, mesoridazine, and danazol after
incubation with recombinantly expressed CYP2J2 enzyme. All com-
pounds produced metabolites that were observed in CYP3A4-
mediated incubation mixtures of these compounds. As seen from the
chromatograms, however, the selectivity toward formation of these
metabolites was quite different from that observed in CYP3A4 incu-
bations. For albendazole, an additional metabolite was observed in the
CYP2J2-containing incubation mixtures (Fig. 2Ba). Metabolite M2
observed in the CYP2J2 incubation of albendazole also showed a
molecular ion of m/z 282 (addition of 16 amu like the sulfoxide, M1)
suggesting hydroxylation of the molecule. The site of hydroxylation in
M2 was the propyl group of albendazole as assessed by the mass
spectral fragmentation of the metabolite (Fig. 2). Although the exact
site of hydroxylation could not be determined from the mass spec-
trum, the terminal methyl group was proposed as the site of oxidation.
Additional work is needed to ascertain the exact position of the
hydroxyl group in the molecule.
Incubation of amiodarone with recombinant CYP2J2 gave two
0055 10 1015 152020 25 2530 3035 35 404045 45
Time (min)Time (min)
M1- M3, M5
M1 – M6
0055 10 101515 2020 252530 30 353540 40 4545 5050
Time (min)Time (min)
FIG. 2. Chromatographic profiles of substrates after incubations with recombinant CYP3A4 (A) and CYP2J2 (B).
NOVEL SUBSTRATES FOR CYP2J2
peaks (M1 and M2) (Fig. 2Bb). The molecular ions and retention
times of M1 and M2 were similar to those observed in CYP3A4
incubations. Mass spectral analysis of M2 indicated that the butyl side
chain was the possible site of metabolism (data not shown). The exact
position of hydroxylation could not be determined from the fragment
ions; hence, the metabolite cluster was assigned as M2. Incubation
of astemizole with recombinant CYP2J2 mainly resulted in the
O-dealkylated metabolite, M5 (Fig. 2Bc) and trace amounts of the
N-dealkylated metabolite (M4). This was in contrast to the CYP3A4
metabolism of the compound, which also produced hydroxylated
metabolites (Fig. 2Ac) in addition to the above metabolites. Incuba-
tion of thioridazine with CYP2J2 produced the same products as
observed in CYP3A4 incubations; comparison of the two chromato-
grams indicated that mesoridazine was also the primary product in
CYP2J2 incubations of thioridazine and metabolite M2 was produced
in very small amounts by CYP2J2 relative to CYP3A4 (Fig. 2Bd).
Similar to thioridazine, mesoridazine formed the same metabolite in
CYP2J2 incubations (M2) as the one observed with CYP3A4. As
discussed previously, the exact position of oxygen incorporation could
not be ascertained from the mass spectral data.
Incubation of danazol with recombinant CYP2J2 resulted in fewer
metabolites than with recombinant CYP3A4. In contrast with the
several hydroxylated metabolites that were observed after incubation
of danazol with CYP3A4, incubation with CYP2J2 resulted in only
two metabolites (M5 and M6) (Fig. 2Bf) in small amounts. Studies to
determine the structure of CYP2J2-mediated danazol metabolites are
For both tamoxifen and cyclosporine, recombinant CYP2J2 and
CYP3A4 formed one common metabolite, whereas CYP3A4 formed
two additional hydroxyl metabolites (Fig. 1). The metabolism of
nabumetone was also assessed by incubating the compound with
recombinant CYP2J2 and CYP3A4. However, interpreting the mass
spectral data for this compound was difficult because of poor ioniza-
tion, but the metabolites were also observed by UV (data not shown).
Comparative Metabolism of Several Substrates in HLM and
HIM. Thioridazine, mesoridazine, albendazole, and amiodarone were
incubated in HLM and HIM. The metabolic profiles were similar in
the two biological matrices, and the major metabolites mediated by
CYP3A4 were observed (data not shown). Moreover, the predominant
CYP2J2-mediated hydroxyl amiodarone metabolite was observed in
both HLM and HIM, but the unique CYP2J2-formed oxidative me-
tabolite of albendazole was not detected probably because it is a minor
P450 Reaction Phenotyping of Danazol and Nabumetone. Many
of the newly identified CYP2J2 compounds are also substrates for
CYP3A4; however, information regarding the P450-mediated dispo-
sition of danazol and nabumetone in the literature is sparse. In this
regard, P450 contributions to the metabolism of danazol and nabum-
etone were investigated in HLM as a greater number of active P450s
are represented in this system than in HIM. The reaction phenotyping
results in HLM showed that danazol is primarily a substrate for
CYP3A4 (?86% contribution) and to a lesser extent for CYP2D6
(?11% contribution) and nabumetone is a substrate for CYP1A2
(?50% contribution) and to a lesser extent for CYP3A (Table 5). In
addition to the danazol and nabumetone results, the results in Table 5
contain reaction phenotyping results for the other CYP2J2 substrates
collected from the literature.
CYP2J2 and CYP3A4 Active Site Comparison. The CYP2J2
homology model was built from multiple CYP2 crystal structures with
no internal mutations and were mostly ligand-free. The active site
cavities for CYP2J2 and CYP3A4 are depicted individually in Fig. 3,
A and B, respectively, and Fig. 3C depicts an overlay of the two active
sites with the I helix included for orientation. The cavity surfaces are
rendered with mesh to highlight the differences in the active site, with
the caveat that CYP2J2 is an approximation, given the lack of an
authentic crystal structure. The homology model suggests that the
active site volume for CYP2J2 is similar to that of CYP3A4 (1420 Å3
versus 1585 Å3); however, the geometry is quite different [solvent-
occupied active site volumes calculated using the software package
Voidoo (Uppsala University, Biomedical Centre, Uppsala, Sweden)].
It is anticipated that CYP2J2 is more constrained near the heme
prosthetic group than CYP3A4. The constraint arises because of
truncation of ?-4, and the loop preceding the truncated sheet protrudes
into the active site. The distance across the active site above the heme
from the I helix to either the loop preceding the truncated ?-4 (from
Ala277to Val346) in CYP2J2 is 9.4 Å, whereas the distance in from the
I helix to ?-4 in CYP3A4 (from Ala305to Glu374) is 15.1 Å. For many
of the newly identified substrates, there is broad overlap in metabo-
lism by CYP3A4 and CYP2J2. A single site of metabolism by
CYP2J2 was observed for several of the compounds such as cyclo-
sporine, tamoxifen, thioridazine, and mesoridazine, whereas CYP3A4
appeared to metabolize these substrates at multiple sites.
N-Desethyl Amiodarone Kinetics. The apparent kinetic parame-
ters of N-desethyl amiodarone were determined in pooled HLM,
pooled HIM, recombinant CYP2J2, and recombinant CYP3A4. For
each system, the kinetic parameters were determined by monitoring
metabolite formation over time in the presence of various substrate
concentrations. The plots showing the initial formation rates of
N-desethyl amiodarone as a function of amiodarone concentration in
the various systems are shown in Fig. 4, A to D. The Michaelis-
Menten-derived intrinsic clearance (ClINT? Vmax/Km) of N-desethyl
amiodarone was 4.6-fold greater in HLM than that observed in HIM
Overlap with CYP3A4 metabolism
Compound2J23A4 2D6 2C82C19 1A2 1A1 2A62B6 FMO References
Rodrigues et al., 1995
Matsumoto and Yamazoe, 2001; Matsumoto et al., 2002
Hashizume et al., 2002; Liu et al., 2006
Fabre et al., 1993; Ohyama et al., 2000
Rawden et al., 2000
Generated by authors
von Bahr et al., 1991; Eap et al., 1996; Berecz et al., 2003;
Wójcikowski et al., 2006
Jacolot et al., 1991; Dehal and Kupfer, 1997; Sridar et al., 2002
Kronbach et al., 1988; Dai et al., 2004
Generated by authors
Eap et al., 1996; Salih et al., 2007
FMO, flavin-containing monooxygenase.
LEE ET AL.
(Table 6). The kinetic parameters for recombinant enzymes CYP3A4
and CYP2J2 are also shown in Table 6. Recombinant CYP3A4 ClINT
was 17-fold greater than that observed in recombinant CYP2J2. An
authentic standard of the hydroxyl amiodarone metabolite was not
available; however, a Kmof 3.89 ?M was obtained for rCYP2J2 and
is within the range observed in HLM and HIM (Table 6).
Albendazole S-Oxidation Kinetics. The Michaelis-Menten kinetic
parameters of the S-oxide metabolite of albendazole were determined
at a fixed reaction time (derived from a preliminary study evaluating
initial velocity as a function of increasing substrate concentrations
experiment) in the presence of various substrate concentrations. Un-
like amiodarone, albendazole exhibited atypical or non-Michaelis-
Menten kinetics, as P450 activity decreased at higher substrate con-
centrations (concentrations greater than Ks), consistent with substrate
inhibition kinetics (Lin et al., 2001) in all reaction systems. The data
were fitted to the Michaelis-Menten and substrate inhibition models.
Selection of the best model was based on the AIC, in which the
substrate inhibition model exhibited the lowest AIC value and pro-
vided the best fit. Representative profiles of product inhibition for
albendazole S-oxidation in HLM, rCYP2J2, and rCYP3A4 systems
are depicted in Fig. 5, A to C. The apparent kinetic parameters of
albendazole S-oxidation in HLM, rCYP2J2, and rCYP3A4 are shown
in Table 7. The Kivalue was 3- to 11-fold greater than the Kmvalue
in the three enzyme systems.
The overall role of CYP2J2 in drug metabolism has not been
determined to date. Several antihistamine drugs, including terfena-
dine, ebastine, and astemizole, have been identified as good substrates
for CYP2J2, a P450 isoform predominantly expressed in the intestine
and heart tissues, with low levels in the liver (Delozier et al., 2007).
The extrahepatic contribution of CYP2J2 may result in higher than
expected in vivo clearances from in vitro liver microsome evaluations.
To investigate the substrate diversity and overall role in drug metab-
olism of CYP2J2, 139 marketed drugs from different therapeutic
classes were screened as potential substrates for CYP2J2. From the
compounds screened (Table 1), eight novel structures were identified
as substrates for CYP2J2. Moreover, these substrates were confirmed
by mass spectral metabolite identification.
The CYP2J2 substrates showed in vitro Clintvalues ranging from
0.06 to 0.49 ?l/min/pmol of CYP2J2 (or in vitro t1/2ranging from 14
to 120 min). These substrates, as shown in Fig. 1, belong to different
therapeutic classes and vary in topology from relatively rigid struc-
tures such as amiodarone to large, more complex structures such as
cyclosporine. However, compared with the antihistamine substrates,
terfenadine and astemizole, the new substrates have appreciably lower
Clintvalues, indicating a lower rate of metabolism. The limitations in
using the substrate depletion method to identify potential CYP2J2
substrates lie in the inability to detect slowly metabolized compounds
because of loss of protein activity and difficulty to ascertain true
metabolism from experimental noise. As an example, diclofenac was
evaluated in our study, in which the loss of parent compound was not
appreciable, and it was not flagged as a substrate for CYP2J2; how-
ever, Lee et al. (2005) identified it to be a substrate when they
examined metabolite formation. In the studies presented herein, me-
tabolite identification served mainly to confirm metabolism by
CYP2J2. Despite the challenges of the screening methodology, sev-
eral structurally diverse compounds were identified. The intent of this
work was not to identify all potential CYP2J2 substrates from our list
of 139 compounds, as we recognize some low clearance compounds
may have been missed.
The broad structural diversity of newly identified substrates, which
include cyclosporine, suggests that the active site volume of CYP2J2
may be large, similar to that of CYP3A4 and CYP2C8 (Johnson and
Stout, 2005). In fact, strong overlap in substrate recognition by
CYP2J2 and CYP3A4 was observed among all the newly identified
CYP2J2 substrates. Although the CYP2J2 model shown herein is a
homology model built from the crystal structures of several CYP2
family members, it is still insightful to postulate how CYP2J2 is
restrictive in its metabolism of large, bulkier substrates such as ta-
moxifen, thioridazine, mesoridazine, and cyclosporine. Unlike the
homology model of Li et al. (2008), which was built using the crystal
structure of CYP2C9 containing internal mutations and a ligand
bound to the active site, the homology model presented herein was
based on a number of CYP2 family members, with no internal
mutations and free of bound substrate. Examining the structure of the
active site of CYP2J2, ?-4 is truncated and the loop preceding the
truncated sheet protrudes into the active site, which limits access to
FIG. 3. Comparison between the active site cavity volumes for human CYP2J2
(green, based on homology model) (A), CYP3A4 (red, Protein Data Bank 1tqn)
active sites (B), and overlay of CYP2J2 and CYP3A4 (C).
NOVEL SUBSTRATES FOR CYP2J2
the heme iron for oxidation. This finding is similar to the 3D homol-
ogy CYP2J2 model constructed by Lafite et al. (2007). CYP2J2 has a
more cylinder shape and is narrower than CYP3A4 near the heme
iron, which probably limits CYP2J2 to metabolizing moieties that can
extend closer toward the active heme iron. This restricted access and
mobility around the heme iron may explain the greater selectivity of
single site-mediated CYP2J2 metabolism observed for cyclosporine,
tamoxifen, thioridazine, and mesoridazine, whereas CYP3A4 appears
to metabolize these substrates at multiple sites because of the ability
of these compounds to rotate more freely in the active site. Likewise,
Lafite et al. (2007) found that CYP2J2 was more selective than
CYP3A4 in the oxidation of terfenadone and its derivatives.
CYP2J2 forms a unique metabolite with albendazole, and the
hydroxylated metabolite of amiodarone is formed predominantly by
CYP2J2 as only trace amounts were produced by CYP3A4. In the
case of danazol, incubations with CYP2J2 formed only a single
metabolite in contrast with several metabolites formed by CYP3A4.
Danazol may enter the active site through the substrate access channel
bound by helices A and F? and ?-sheet 1 and is metabolized only
when the isoxazole ring approaches the heme iron forming a metab-
olite that is 3 amu greater than the molecular ion of the parent
compound. The odd molecular ion suggests a possible loss of nitrogen
atom. If the nitrile portion of the structure approaches the heme,
inhibition will probably occur as observed by Jones et al. (2008).
Two substrates were chosen for extensive kinetic analysis to deter-
mine differences between CYP2J2 and CYP3A4 in HLM and HIM
because the mRNA levels of CYP2J2 are fairly high in the intestine
(Bie `che et al., 2007). The Michaelis-Menten-derived intrinsic clear-
ance (ClINT) values observed for N-desethyl amiodarone in HLM and
HIM reflect differences in Vmaxvalues. The Vmaxdisparity is probably
attributed to the contribution of CYP2C8 in HLM, which is present at
lower levels in the intestine (Ohyama et al., 2000; Paine et al., 2006;
Bie `che et al., 2007). A comparison of recombinant CYP3A4 and
CYP2J2 N-desethyl amiodarone activities showed a 17-fold differ-
ence due to a significantly greater Vmaxcontribution. Although the
authentic standard for the unique hydroxyl metabolite of amiodarone
was not available, the Kmvalues of the hydroxylated metabolite were
similar in the three systems (HLM, HIM, and recombinant CYP2J2),
ranging from 3.89 to 14.9 ?M. Plasma concentrations of amiodarone
range from 0.65 to 5.7 ?M in patients (Sta ¨ubli et al., 1983), and
intestinal levels are likely to be higher than plasma levels. Although,
the contribution of CYP2J2 relative to that of CYP3A4 and CYP2C8
in HIM and HLM is minor, it may become more important in
amiodarone metabolism if these two P450s are inhibited.
In the systems evaluated, HLM, recombinant CYP3A4, and recom-
0 10 20304050
[Amiodarone] (µ µ µ µM)
[Amiodarone] (µ µ µ µM)
0 10 2030 40 50
[Amiodarone] (µ µ µ µM)
FIG. 4. Concentration-dependent metabolism of amiodarone by pooled human liver microsomes (A), human intestinal microsomes (B), recombinant CYP2J2 (C), and
recombinant CYP3A4 (D). The formation of N-desethyl amiodarone was monitored over time in the presence of increasing substrate concentrations. At each substrate
concentration, incubation samples were collected over nine time points, and the initial velocities for N-desethyl amiodarone formation were determined and used to calculate
the Michaelis-Menten kinetic parameters.
Michaelis-Menten kinetic parameters for amiodarone metabolism by HLM, HIM,
recombinant CYP2J2, and recombinant CYP3A4 to N-desethyl amiodarone and
hydroxy amiodarone by CYP3A4 and CYP2J2
? l/min/mg protein
7.41 ? 0.69
11.48 ? 3.79
2.15 ? 0.46
3.62 ? 0.43
1009 ? 34
342 ? 42
1.05 ? 0.07a
29.76 ? 1.10a
14.9 ? 2.60
5.11 ? 3.91
3.89 ? 0.79
aUnit of measure is pmol/min/pmol rP450.
bUnit of measure is ?l/min/pmol rP450.
LEE ET AL.
binant CYP2J2, the metabolism of albendazole exhibits either sub-
strate or product inhibition kinetics. The rate of albendazole S-oxida-
tion decreased when substrate concentrations exceeded 10 ?M in
HLM and recombinant CYP2J2. For recombinant CYP3A4, the me-
tabolism decreased when concentrations exceeded 20 ?M; however,
the decrease in velocity was not as pronounced as that observed in
HLM and recombinant CYP2J2. Inhibition of enzyme activity at high
concentration is one of the most common deviations from typical
Michaelis-Menten kinetics (Lin et al., 2001). In the analysis of al-
bendazole S-oxidation kinetic data, a two-site inhibition model con-
taining sites for metabolism and inhibition (Lin et al., 2001) best
modeled the kinetic data. In all systems, the Kswas similar, but the
inhibition constant, Ki, was found to be 3 to 10 times greater than
the Ks, indicating that the inhibitory site has a weaker affinity for the
substrate than the catalytic site. The decrease in albendazole S-oxida-
tion occurs around the Kivalue as shown in Fig. 5, A to C. Previous
evaluation of albendazole in HLM did not show substrate inhibition
largely because the substrate concentrations used did not exceed the
Kiconcentration (Rawden et al., 2000). However, it should be noted
that the binding of the metabolite to a second site, which may act as a
noncompetitive inhibitor, cannot be ruled out because the albendazole
S-oxidation profile will look similar to a substrate inhibition profile.
In drug discovery research, the susceptibility of a compound to
metabolism is commonly evaluated using human liver microsomes.
Low turnover compounds from HLM evaluations then progress to in
vitro and in vivo evaluation in rodents, mainly to gain insight into the
absorption, distribution, metabolism, and excretion properties of the
new drug candidate or insight for a chemical series. Despite low
metabolism observed with human and animal liver microsomes, the
compound dosed in vivo may have a systemic clearance value greater
than hepatic blood flow, indicating extrahepatic metabolism and/or
involvement of a nonhepatic route of clearance. With the knowledge
that CYP2J2 metabolizes structurally diverse substrates, this isoform
may contribute to total systemic clearance previously unaccounted
for, particularly in the intestine and heart. Hashizume et al. (2002)
showed that although CYP3A4 may be responsible for hepatic me-
tabolism of ebastine, intestinal metabolism and first-pass metabolism
seem to be CYP2J2-mediated. Moreover, CYP2J2 has been identified
as the major contributor to the first-pass metabolism of astemizole
(Hashizume et al., 2002; Matsumoto et al., 2002). It is possible that for
many of the drugs metabolized by both CYP2J2 and CYP3A4, when
CYP3A4 is implicated in both intestinal and hepatic metabolism, the
contribution of CYP2J2 in the intestine may have been overlooked
and the contribution by heart is unclear at this time. In addition, the
0.0 2.55.0 7.5
10.0 12.5 15.017.520.0
10.012.5 15.0 17.520.0
0 10 20 304050
FIG. 5. Concentration-dependent metabolism of albendazole by pooled human liver microsomes (A), recombinant CYP2J2 (B), and recombinant CYP3A4 (C). The
formation of albendazole S-oxide with increasing substrate concentration was determined at a single time point (20 min).
Michaelis-Menten kinetic parameters for albendazole S-oxidation by recombinant
rCYP2J2 and rCYP3A4 and HLM
1.89 ? 0.30
0.74 ? 0.13
74.32 ? 8.80a
4.23 ? 1.04
6.02 ? 1.82
5.29 ? 0.93
19.29 ? 6.56
68.30 ? 34.95
16.69 ? 3.91
aUnit of measure is pmol/min/mg microsomal protein.
NOVEL SUBSTRATES FOR CYP2J2
clinical significance of CYP2J2 in the liver is minor given the abun- Download full-text
dance of CYP3A4, whereas the CYP2J2 is more abundant in nonhe-
patic tissues such as heart and intestine, warranting further assessment
to determine its clinical significance.
In summary, eight new structurally diverse substrates for CYP2J2
metabolism were discovered among the 139 substrates evaluated. The
substrates belong to different therapeutic classes and vary in shape
from relatively small rigid structures such as amiodarone to larger
more complex structures such as cyclosporine. There is a strong
overlap in substrate recognition by CYP2J2 and CYP3A4, and the
active site of CYP2J2, although similar in size to that of CYP3A4, is
more restrictive in that many of the substrates are metabolized at a
single site. Among the newly identified substrates, albendazole forms
unique CYP2J2-mediated metabolites. Although our findings demon-
strate the ability of CYP2J2 to metabolize structurally diverse com-
pounds, the clinical significance is probably overshadowed by that
of CYP3A4, suggesting a minor role in overall intestine and liver
Acknowledgments. We thank Drs. Ruth Hyland, Alfin Vaz, and R.
Scott Obach for their thoughtful review and suggestions for our work.
Berecz R, de la Rubia A, Dorado P, Ferna ´ndez-Salguero P, Dahl ML, and LLerena A (2003)
Thioridazine steady-state plasma concentrations are influenced by tobacco smoking and
CYP2D6, but not by the CYP2C9 genotype. Eur J Clin Pharmacol 59:45–50.
Bie `che I, Narjoz C, Asselah T, Vacher S, Marcellin P, Lidereau R, Beaune P, and de Waziers I
(2007) Reverse transcriptase-PCR quantification of mRNA levels from cytochrome (CYP)1,
CYP2 and CYP3 families in 22 different human tissues. Pharmacogenet Genomics 17:731–
Dai Y, Iwanaga K, Lin YS, Hebert MF, Davis CL, Huang W, Kharasch ED, and Thummel KE
(2004) In vitro metabolism of cyclosporine A by human kidney CYP3A5. Biochem Pharmacol
Dehal SS and Kupfer D (1997) CYP2D6 catalyzes tamoxifen 4-hydroxylation in human liver.
Cancer Res 57:3402–3406.
Delozier TC, Kissling GE, Coulter SJ, Dai D, Foley JF, Bradbury JA, Murphy E, Steenbergen
C, Zeldin DC, and Goldstein JA (2007) Detection of human CYP2C8, CYP2C9, and CYP2J2
in cardiovascular tissues. Drug Metab Dispos 35:682–688.
Eap CB, Guentert TW, Scha ˜ublin-Loidl M, Stabl M, Koeb L, Powell K, and Baumann P (1996)
Plasma levels of the enantiomers of thioridazine, thioridazine 2-sulfoxide, thioridazine 2-sul-
fone, and thioridazine 5-sulfoxide in poor and extensive metabolizers of dextromethorphan and
mephenytoin. Clin Pharmacol Ther 59:322–331.
Fabre G, Julian B, Saint-Aubert B, Joyeux H, and Berger Y (1993) Evidence for CYP3A-
mediated N-deethylation of amiodarone in human liver microsomal fractions. Drug Metab
Hashizume T, Imaoka S, Mise M, Terauchi Y, Fujii T, Miyazaki H, Kamataki T, and Funae Y
(2002) Involvement of CYP2J2 and CYP4F12 in the metabolism of ebastine in human
intestinal microsomes. J Pharmacol Exp Ther 300:298–304.
Jacolot F, Simon I, Dreano Y, Beaune P, Riche C, and Berthou F (1991) Identification of the
cytochrome P450 IIIA family as the enzymes involved in the N-demethylation of tamoxifen in
human liver microsomes. Biochem Pharmacol 41:1911–1919.
Johnson EF and Stout CD (2005) Structural diversity of human xenobiotic-metabolizing cyto-
chrome P450 monooxygenases. Biochem Biophys Res Commun 338:331–336.
Jones JP, Katayama JH, Jiang Y, Lee CA, and Totah RA (2008) Identification of danazol as a
selective inhibitor of cytochrome P450 2J2 (Abstract). Drug Metab Rev 40:78.
Kronbach T, Fischer V, and Meyer UA (1988) Cyclosporine metabolism in human liver:
identification of a cytochrome P-450III gene family as the major cyclosporine-metabolizing
enzyme explains interactions of cyclosporine with other drugs. Clin Pharmacol Ther 43:630–
Lafite P, Dijols S, Buisson D, Macherey AC, Zeldin DC, Dansette PM, and Mansuy D (2006)
Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2.
Bioorg Med Chem Lett 16:2777–2780.
Lafite P, Francois A, Zeldin DC, Dansette PM, and Mansuy D (2007) Unusual regioselectivity
and active site topology of human cytochrome P450 2J2. Biochemistry 46:10237–10246.
Lee SS, Jeong HE, Liu KH, Ryu JY, Moon T, Yoon CN, Oh SJ, Yun CH, and Shin JG (2005)
Identification and functional characterization of novel CYP2J2 variants: G312R variant causes
loss of enzyme catalytic activity. Pharmacogenet Genomics 15:105–113.
Li W, Tang Y, Liu H, Cheng J, Zhu W, and Jiang H (2008) Probing ligand binding modes of
human cytochrome P450 2J2 by homology modeling, molecular dynamics simulation, and
flexible molecular docking. Proteins 71:938–949.
Lin Y, Lu P, Tang C, Mei Q, Sandig G, Rodrigues AD, Rushmore TH, and Shou M (2001)
Substrate inhibition kinetics for cytochrome P450-catalyzed reactions. Drug Metab Dispos
Liu KH, Kim MG, Lee DJ, Yoon YJ, Kim MJ, Shon JH, Choi CS, Choi YK, Desta Z, and Shin
JG (2006) Characterization of ebastine, hydroxyebastine, and carebastine metabolism by
human liver microsomes and expressed cytochrome P450 enzymes: major roles for CYP2J2
and CYP3A. Drug Metab Dispos 34:1793–1797.
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
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
Ohyama K, Nakajima M, Nakamura S, Shimada N, Yamazaki H, and Yokoi T (2000) A
significant role of human cytochrome P450 2C8 in amiodarone N-deethylation: an approach to
predict the contribution with relative activity factor. Drug Metab Dispos 28:1303–1310.
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.
Rawden HC, Kokwaro GO, Ward SA, and Edwards G (2000) Relative contribution of cyto-
chromes P-450 and flavin-containing monoxygenases to the metabolism of albendazole by
human liver microsomes. Br J Clin Pharmacol 49:313–322.
Rodrigues AD, Mulford DJ, Lee RD, Surber BW, Kukulka MJ, Ferrero JL, Thomas SB, Shet
MS, and Estabrook RW (1995) In vitro metabolism of terfenadine by a purified recombinant
fusion protein containing cytochrome P4503A4 and NADPH-P450 reductase. Comparison to
human liver microsomes and precision-cut liver tissue slices. Drug Metab Dispos 23:765–775.
Rosi D, Neumann HC, Christiansen RG, Schane HP, and Potts GO (1977) Isolation, synthesis,
and biological activity of five metabolites of danazol. J Med Chem 20:349–352.
Sali A and Blundell TL (1993) Comparative protein modelling by satisfaction of spatial
restraints. J Mol Biol 234:779–815.
Salih IS, Thanacoody RH, McKay GA, and Thomas SH (2007) Comparison of the effects of
thioridazine and mesoridazine on the QT interval in healthy adults after single oral doses. Clin
Pharmacol Ther 82:548–554.
Sridar C, Kent UM, Notley LM, Gillam EM, and Hollenberg PF (2002) Effect of tamoxifen on
the enzymatic activity of human cytochrome CYP2B6. J Pharmacol Exp Ther 301:945–952.
Sta ¨ubli M, Bircher J, Galeazzi RL, Remund H, and Studer H (1983) Serum concentrations of
amiodarone during long term therapy. Relation to dose, efficacy and toxicity. Eur J Clin
von Bahr C, Movin G, Nordin C, Lide ´n A, Hammarlund-Udenaes M, Hedberg A, Ring H, and
Sjo ¨qvist F (1991) Plasma levels of thioridazine and metabolites are influenced by the
debrisoquin hydroxylation phenotype. Clin Pharmacol Ther 49:234–240.
Wo ´jcikowski J, Maurel P, and Daniel WA (2006) Characterization of human cytochrome P450
enzymes involved in the metabolism of the piperidine-type phenothiazine neuroleptic thiorid-
azine. Drug Metab Dispos 34:471–476.
Wu S, Moomaw CR, Tomer KB, Falck JR, and Zeldin DC (1996) Molecular cloning and
expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly
expressed in heart. J Biol Chem 271:3460–3468.
Address correspondence to: Dr. Caroline A. Lee, Pfizer Global Research &
Development, 10646 Science Center Dr., San Diego, CA 92121. E-mail:
LEE ET AL.