Eur J Clin Pharmacol (1997) 52: 293±298
PHARMACOKINETICS AND DISPOSITION
T. S. Tracy á C. Marra á S. A. Wrighton á F. J. Gonzalez
K. R. Korzekwa
Involvement of multiple cytochrome P450 isoforms
in naproxen O-demethylation
Received: 16 September 1996 / Accepted in revised form: 20 December 1996
Abstract Objective: A series of studies was undertaken
to determine the cytochrome P450 isoform(s) involved in
naproxen demethylation and whether this included the
same isoforms reported to be involved in the metabolism
of other NSAIDs.
Methods: (S)-Naproxen was incubated with human liver
microsomes in the presence of a NADPH-generating
system and the formation of desmethylnaproxen was
measured by high-performance liquid chromatography
(HPLC). To further clarify the speci®c isoforms in-
volved, experiments were conducted with preparations
expressing only a single P450 isoform (vaccinia virus-
expressed cells and microsomes derived from a lymph-
oblastoid cell line, each transfected with speci®c P450
cDNAs) as well as inhibition studies using human liver
microsomes and putative speci®c P450 inhibitors.
Results: In human liver microsomes (n ˆ 7), desmethyl-
naproxen formation was observed with a mean kM of 92
(21) lmol á l)1, Vmax of 538 pmol á min)1 á mg)1 protein
and Cint2 (re¯ective of a second binding site) of
0.36 ll á min)1 á mg)1 protein. This Cint2 term was added
since Eadie-Scatchard analysis suggested the involve-
ment of more than one enzyme. Studies using putative
speci®c P450 inhibitors demonstrated inhibition of
this reaction by sulfaphenazole, (apparent Ki ˆ
T.S. Tracy (&) á C. Marra
Department of Basic Pharmaceutical Sciences,
School of Pharmacy, West Virginia University,
HSN P.O. Box 9530, Morgantown, WV 26506, USA
Tel. +1-304-293-1474; fax +1-304-293-5483;
e-mail tstracy@wvnvm.wvnet.edu
S.A. Wrighton
Department of Drug Disposition, Eli Lilly and Co.,
Indianapolis, Indiana, USA
F.J. Gonzalez
National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA
K.R. Korzekwa
Center for Clinical Pharmacology, University of Pittsburgh,
Pittsburgh, Pennsylvania, USA
Ó Springer-Verlag 1997
1:6 lmol á l)1), warfarin (apparent Ki ˆ 27 lmol á l)1),
piroxicam (apparent Ki ˆ 23 lmol á l)1) and tolbut-
amide (apparent Ki ˆ 128 lmol á l)1). No e€ect was
observed when a-naphtho¯avone and troleandomycin
were employed as inhibitors, but reaction with furafyl-
line produced, on average, a maximum inhibition of
23%. At a naproxen concentration of 150 lmol á l)1,
formation of desmethylnaproxen was observed in cells
expressing P450 1A2, 2C8, 2C9 and its allelic variant
2C9R144C. To further characterize these reactions,
saturation kinetics experiments were conducted for the
P450s 1A2, 2C8 and 2C9. The kM and Vmax for P450
1A2 were 189.5 lmol á l)1 and 7.3 pmol á min)1 á pmol)1
P450, respectively. Likewise, estimates of kM and Vmax
for P450 2C9 were 340.5 lmol á l)1 and 41.4 pmol á
min)1 á pmol)1 P450, respectively. Reliable estimates of
kM and Vmax could not be made for P450 2C8 due to the
nonsaturable nature of the process over the concentra-
tion range studied.
Conclusion: Multiple cytochrome P450 isoforms (P450
1A2, 2C8 and 2C9) appear to be involved in naproxen
demethylation, although 2C9 appears to be the pre-
dominant form.
Key words Naproxen, Cytochrome P450; human liver
microsomes, vaccinia virus, cDNA expression
Introduction
Naproxen [(S)-6-methoxy-a-methyl-2-naphthalene-ace-
tic acid] has been widely prescribed for the treatment of
pain or in¯ammation for several years and recently was
approved for over-the-counter use in the United States.
Naproxen is somewhat unique among the nonsteroidal
antiin¯ammatory drugs (NSAIDs) in that, although it
possesses a chiral center, it is only available commercially
as the (S)-enantiomer. Furthermore, other than conju-
gative processes, naproxen is thought to undergo only a
single oxidative biotransformation to form the demeth-
ylated metabolite, desmethylnaproxen [1]. Therefore,
294
assessment of naproxen in vivo pharmacokinetics and
metabolic pro®le is much simpler than for NSAIDs ad-
ministered as racemic mixtures.
Following administration of naproxen to humans,
approximately 10% is recovered in the urine as napr-
oxen itself, 40% as the glucuronide conjugate, 5% as
desmethylnaproxen, 12% as the glucuronide of desm-
ethylnaproxen, and approximately 30% as unknown
conjugates of naproxen and desmethylnaproxen [1].
More recently, Kiang et al. [2] have reported the id-
enti®cation of a sulfate conjugate of desmethylnaproxen
in plasma from both normal and uremic volunteers re-
ceiving usual doses of naproxen. Concerning the in-
volvement of speci®c P450 isoforms, Newlands et al. [3]
have reported, in abstract form, the inhibition of
desmethylnaproxen formation by sulfaphenazole, sug-
gesting the involvement of P450 2C9. Furthermore, they
did not observe any correlation between desmethyl-
naproxen formation and other P450 isoform activities
re¯ecting 1A, 3A and 2C19 as measured by correlation
with model substrate activities. More recently, two sep-
arate laboratories have reported that both P450 2C9 and
P450 1A2 were involved in naproxen demethylation but
these reports di€ered on the relative contribution of each
isoform [4, 5].
Since desmethylnaproxen and its conjugates may
comprise between 17 and 47% (vide supra) of recovered
drug following administration of naproxen, it seems
logical to determine the cytochrome P450 isoform(s)
responsible for the oxidation of naproxen to desmeth-
ylnaproxen. We have recently shown cytochrome P450
2C9 to be the primary enzyme responsible for the oxi-
dative metabolism of the NSAID, ¯urbiprofen [6]. Thus,
it is relevant to determine whether NSAIDs from dif-
ferent chemical classes (e.g., ¯urbiprofen and naproxen)
are primarily metabolized by P450 2C9, or, if other
isoforms are involved, the extent of their contribution.
To this end, we undertook a series of studies involving
human liver microsomes, transfected cells expressing
speci®c P450 isoforms, and cytochrome P450 inhibitors
to determine the enzymes involved in the oxidative me-
tabolism of naproxen.
Materials and methods
Chemicals and reagents
Acetonitrile and potassium phosphate were obtained from the
Fisher Co. (Pittsburgh, Pa., USA). Naproxen and desmethylnapr-
oxen were a gift from Syntex Co. (Palo Alto, Calif., USA), 2-
¯uoro-4-biphenyl acetic acid was a gift from the Upjohn Co.
(Kalamazoo, Mich., USA), and furafylline was purchased from
Research Biochemicals Inc. (Natick, Mass., USA). All other
chemicals were obtained from commercial sources and were of the
highest purity available.
Human liver tissue and cDNA -transfected cells
Human liver tissue (n ˆ 7) for preparation of microsomes was
obtained using protocols approved by the Institutional Review
Board at the Medical College of Wisconsin or through the Liver
Tissue Procurement and Distribution System (LTPADS). All
samples of human liver tissue were obtained from liver donor
candidates who were not su€ering from any known hepatic diseases
(e.g., cirrhosis). Based on chart review, none of the patients from
whom tissues were obtained was taking any medications known to
inhibit or induce the P450s. Microsomal samples were prepared
according to established methods [7]. Protein content was measured
by the method of Lowry et al. [8] and P450 content by the method
of Omura and Sato [9].
Microsomal preparations from transfected human B-lymph-
oblastoid cell lines coding for human P450 2A6, 2C19, and 2D6, or
expressing only reductase were obtained from Gentest (Woburn,
Mass., USA). Vaccinia, virus-expressed P450s were obtained by
infecting human HepG2 cells with a recombinant vaccinia virus
containing cDNAs for P450s 1A2, 2C8, 2C9, 2C9R144C, 2E1, and
3A4. The details of construction of the viruses were published
previously [10]. Control cells were infected only with wild-type
vaccinia virus. The cells were harvested 1 day after infection and
stored frozen at )80 °C. Prior to use, the cells were thawed at 4 °C,
spun to remove the supernatant, and resuspended in 50 mM
K2HPO4, pH 7.4. The cells were then sonicated with two 5-s bursts
prior to addition to the incubation mixture.
Liver microsomal incubations
Liver microsomes (0.1 mg á ml)1 ®nal protein concentration) were
incubated in the presence of 1 mmol á l)1 b-NADP, 10 mmol á l)1
glucose-6-phosphate, 0.2 U glucose-6-phosphate dehydrogenase,
and 100 mmol á l)1 K2HPO4, pH 7.4, in a total volume of 200 ll.
Reactions were carried out for 20 min at 37 °C over the naproxen
concentration range of 5±1800 lmol á l)1. Experiments involving
cDNA-expressed P450s were carried out at 150 lmol á l)1 naproxen
concentrations (saturation kinetics experiments were conducted at
the same naproxen concentrations as used with liver microsomes)
under the same conditions, except that the bu€er was 50 mmol á l)1
K2HPO4, pH 7.4, 0.4 U glucose-6-phosphate dehydrogenase was
added, and the total volume was 400 ll. In experiments using
CYP2A6, a Tris bu€er 50 mmol á l)1, pH 7.4, replaced the phos-
phate bu€er, because the phosphate bu€er inhibits the activity of
P450 2A6 in this microsomes preparation (Gentest, product in-
formation).
HPLC assay of desmethylnaproxen.
The measurement of desmethylnaproxen after incubation of
naproxen was carried out as follows. The microsomal incubation
reaction was terminated by the addition of 200 ll of acetonitrile.
To the samples, 480 ng of 2-¯uoro-4-biphenyl acetic acid (internal
standard) and 20 ll of H3PO4 were added. The samples were
centrifuged at 11 000 g for 4 min and 5±22 ll was directly injected
onto the HPLC system. The HPLC system consisted of a Waters
501 HPLC pump, a Waters 717 autosampler, and Waters 470 ¯u-
orescence detector set at an excitation wavelength of 230 nm and
an emission wavelength of 340 nm. The mobile phase consisted of
(30:70) acetonitrile: 20 mmol á l)1 K2HPO4, pH 3, pumped at
1 ml á min)1 through a Brownlee Spheri-5 C18 4.6 ´ 100 mm col-
umn. Inter- and intra-day coecients of variation for this assay
were less than 6%.
Data analysis
Data on desmethylnaproxen formation in human liver microsomes
were analyzed by nonlinear regression (PCNONLIN, Statistical
Consultants, Lexington, Ky., USA) of the substrate concentration
versus velocity plots, according to the following equation,
m ˆ Vkmax
M ‡S
xS
‡ …Cint2 xS†, which is a modi®cation of the Michaelis-
Menten equation to include a second intrinsic clearance (Cint2 ) term
for a nonsaturable component. This intrinsic clearance of the low-
anity site termed Cint2 was included since the observed lack of

saturation and the Eadie-Scatchard plots suggested the possible
involvement of more than one enzyme. The e€ects of sulfa-
phenazole, racemic-warfarin, piroxicam, and tolbutamide on the
formation of desmethylnaproxen were evaluated by estimating the
apparent inhibition constant (Ki) for the inhibitors by Dixon plot
analysis [11].
Results
A representative example of desmethylnaproxen forma-
tion in human liver microsomes is shown in Fig. 1. It is of
note that, similar to those results observed in Fig. 1, none
of the other six liver samples tested showed enzyme sat-
uration at substrate concentrations up to 1800 lmol á l)1.
Furthermore, no metabolite formation was noted in
negative controls, indicating that chemical breakdown of
naproxen was not responsible for the nonsaturable
component of metabolite formation. The apparent kM,
Vmax and Cint2 estimates for each of the seven liver
samples are listed in Table 1.
Chemical inhibition studies, with putative speci®c
inhibitors of various P450 isoforms, were employed to
determine the P450 isoform(s) involved in naproxen
demethylation. Sulfaphenazole, a putative P450 2C9
inhibitor [12], substantially inhibited naproxen demeth-
ylation with an estimated apparent Ki of 1.6 lmol á l)1
(Fig. 2a). Racemic-warfarin, a substrate for both P450
2C9 and P450 1A2 [13], produced inhibition of desm-
ethylnaproxen formation with an estimated apparent Ki
of 27 lmol á l)1 (Fig. 2b). Tolbutamide, which inhibits
both P450 2C8 and P450 2C9 to varying degrees [14],
exhibited an estimated apparent Ki of 128 lmol á l)1
(Fig. 3a). Piroxicam, another NSAID which would be
predicted to be metabolized by similar enzymes [15], was
also studied as an inhibitor. Piroxicam produced sub-
stantial inhibition of naproxen demethylation, similar to
Fig. 1 Formation of desmethylnaproxen in a representative sample of
human liver microsomes (closed squares actual desmethylnaproxen
formation velocities, solid lines nonlinear regression ®ts of desmeth-
ylnaproxen formation data)
295
Table 1 Kinetic parameter estimates for naproxen demethylation
in human liver samples
Sample kM Vmax Cint2 a
(lmol á l)1) (pmol á min)1 á mg)1 protein)
1 111 1411 0.31
2 70 271 0.31
3 97 302 0.29
4 82 285 0.31
5 127 669 0.43
6 71 281 0.4
7 88 546 0.48
Mean (SD) 92 (21) 538 (416) 0.36 (0.07)
a
Intrinsic clearance in ll á min)1 á mg)1 protein
that seen with racemic-warfarin, with an estimated ap-
parent Ki of 23 lmol á l)1 (Fig. 3b). Neither troleando-
mycin [16] (a putative P450 3A subfamily inhibitor) nor
a-naphtho¯avone [17] (an inhibitor of the P450 1A
subfamily) produced inhibition of desmethylnaproxen
formation (data not shown). However, furafylline (a
putative speci®c P450 1A2 inhibitor) [18], when studied
at concentrations of 0.2±100 lmol á l)1 (in incubations of
either 50 or 150 lmol á l)1 naproxen), caused a maximum
inhibition of 23% which was reached at 2 lmol á l)1
furafylline. Increasing concentrations of furafylline
above 2 lmol á l)1 did not result in further inhibition.
Since the formation of desmethylnaproxen was not
saturable at concentrations up to 1800 lmol á l)1, mul-
tiple enzyme involvement was suspected. Thus, an Ea-
die-Scatchard plot was constructed (data not shown),
and this graphical analysis suggested the involvement of
at least two enzymes in naproxen demethylation, be-
cause of the nonlinear nature of the data [19].
To elucidate more conclusively the P450 forms in-
volved in desmethylnaproxen formation, we then un-
dertook studies using P450 cDNA-expressed cells as well
as microsomes derived from transfected lymphoblastoid
cell lines. Naproxen was incubated with cells expressing
either P450 1A2, 2A6, 2C8, 2C9, 2C9R144C, 2C19, 2D6,
2E1, 3A4 or control cells which had not been transfected
with P450 cDNAs. Demethylation of naproxen was
observed by cells expressing P450 1A2, 2C8, 2C9 and
2C9R144C (Fig. 4). To further characterize this oxida-
tive process, saturation kinetics experiments were con-
ducted using cells expressing P450 1A2, 2C8 and 2C9 to
estimate the kM and Vmax for each of these enzymes with
respect to this process (Fig. 5). The estimated kM and
Vmax for desmethylnaproxen formation by P450 1A2
were 189.5 lmol á l)1 and 7.3 pmol á min)1 á pmol)1 P450,
respectively. For P450 2C9, the estimate of kM was
340.5 lmol á l)1 and of Vmax, 41.4 pmol á min)1 á pmol)1
P450. Reliable estimates of kM and Vmax could not be
obtained for P450 2C8 due to the nonsaturable nature of
this reaction over the range of concentrations studied.
However, relative formation rates were substantially less
than those observed for either P450 1A2 or P450 2C9, at
all concentrations studied.
296

Fig. 2 Dixon plot representation of the inhibition of desmethylnapr-
oxen formation by sulfaphenazole (a) and racemic-warfarin (b).
Naproxen was incubated at either 25 lmol á l)1 or 150 lmol á l)1
concentration in the presence of either sulfaphenazole (0±1.6 lmo-
l á l)1) or racemic-warfarin at concentrations ranging from 0 to
100 lmol á l)1. The estimated apparent Ki value for inhibition of
desmethylnaproxen formation by sulfaphenazole was 1.5 lmol á l)1.
The estimated apparent Ki value for inhibition of desmethylnaproxen
formation by warfarin was 27 lmol á l)1
Discussion
Previous studies have suggested that both cytochromes
P450 2C9 and P450 1A2 contribute to naproxen de-
methylation [3±5]. We have extended these studies by
utilizing a larger number of P450 isoforms expressed in
cells transfected with cDNA of speci®c cytochromes
P450, as well as a more sensitive assay to further char-
acterize this process. Similar to the situation with ¯u-
rbiprofen [6], P450 2C9 was the major isoform involved
in naproxen demethylation. The estimated Vmax for this
process by P450 2C9 was approximately equal to that
observed for ¯urbiprofen hydroxylation [6] but the es-
timated kM was roughly 70-fold higher for naproxen
demethylation. Additionally, in contrast to ¯urbiprofen,
cytochrome P450 1A2 was also involved in desmethyl-
naproxen formation resulting in a maximum velocity
approximately one-sixth of that observed with P450
2C9. Conversely, the estimated kM for this process by
P450 1A2 was approximately one-half of that observed
for P450 2C9. Taken together, these data suggest that
the anity of cytochrome P450 2C9 for naproxen is
substantially lower than that for ¯urbiprofen, and that
at naproxen concentrations achieved in vivo [20] the
velocity of this reaction would also be lower.
Due to its lower estimated kM, cytochrome P450 1A2
could play a substantial role in this reaction, though at
concentrations approximately equal to kM (Fig. 5), the
activity of P450 1A2 is approximately one-third that of
P450 2C9. If one uses the estimated kM and Vmax for this
process by P450 2C9 and 1A2 as parameters for the
Michaelis-Menten equation and inserts a therapeu-
tically relevant plasma naproxen concentration (e.g. 50
lmol á l)1), the relative contribution of each isoform can
be estimated. Under these assumed conditions, it would
be expected that the relative contribution of P450 2C9
would be approximately four times higher than that of
1A2. Furthermore, if one assumes that the relative

Fig. 3 Dixon plot representa-

tion of the inhibition of desm-
ethylnaproxen formation by
tolbutamide (a) and piroxicam
(b). Naproxen was incubated at
either 25 lmol á l)1 or 150
lmol á l)1 concentrations in the
presence of either piroxicam or
tolbutamide at concentrations
ranging from 0±100 lmol á l)1.
The estimated apparent Ki value
for inhibition of desmethylnapr-
oxen formation by piroxicam
was 23 lmol á l)1. The estimated
apparent Ki value for inhibition
of desmethylnaproxen forma-
tion by tolbutamide was
128 lmol á l)1

Fig. 4 Formation of desmethylnaproxen by vaccinia virus expressed
P450s or microsomes derived from a P450 cDNA-transfected
lymphoblastoid cell line. Data labeled 2C9v ˆ R144C variant of
P450 2C9. Naproxen substrate concentrations were 150 lmol á l)1 for
all incubations
amounts of P450 2C9 and 1A2 in the liver are roughly
equal [21], and that plasma concentration approximates
the substrate concentration at the enzyme site, then this
prediction of relative contribution should hold true for
the in vivo situation. Thus, it would appear that under
normal in vivo conditions, the role of P450 1A2 in
naproxen demethylation should be relatively minor
compared with P450 2C9. However, due to the ability of
cigarette smoking to increase liver cytochrome P450 1A
activity as much as threefold [22], the participation of
this isoform could be of increasing importance in pa-
tients who smoke, potentially resulting in metabolic ac-
tivities roughly equal to that exhibited by P450 2C9.
Since the expression of both P450 1A2 and 2C9 is het-
erogeneous within the population, one could envision
several scenarios wherein P450 1A2 induction could play
a major, if not dominant role in naproxen demethyla-
Fig. 5 Michaelis-Menten kinetic analysis of desmethylnaproxen
formation from cells or microsomes expressing P450 1A2 (squares),
297
tion. Finally, in contrast to previous reports [4, 5], we
found that cytochrome P450 2C8 also plays a minor role
in the demethylation of naproxen but that the action of
this isoform does not appear to be saturable under the
conditions studied. This minor involvement of P450 2C8
is similar to that seen during the oxidation of ibuprofen
(S.D. Hall, personal communication).
Interestingly, our estimations for kM and Vmax in
human liver microsomes are very similar to those re-
ported previously [4, 5], but when one compares these
kinetic parameter estimates in expressed cells, our results
are only similar to those of Rodrigues et al. [4]. Both our
results and those of Rodrigues et al. estimate the kM and
Vmax for naproxen demethylation in expressed cells to be
higher than that observed in liver microsome prepara-
tions. It is unclear why the apparent anity of the en-
zyme di€ers substantially between liver microsome
preparations (92 lmol á l)1) and expressed cells (341 (32)
lmol á l)1 for P450 2C9 and 190 (37) lmol á l)1 for P450
1A2), though di€erences in reductase levels, b5 levels,
and lipid composition between expressed cells and liver
microsomes may be potential contributing factors to
these di€erences.
Studies using putative speci®c inhibitors of cyto-
chrome P450 isoforms corroborated the involvement of
at least cytochrome P450 2C9 in naproxen demethyla-
tion. Piroxicam, racemic-warfarin, and tolbutamide, all
expected inhibitors of P450 2C9 [13±15], produced in-
hibition of naproxen demethylation. As demonstrated
by their observed apparent Ki values, piroxicam and
racemic-warfarin were more e€ective at inhibiting this
reaction than tolbutamide. This is expected since tol-
butamide has previously been shown to be a relatively
weak inhibitor of NSAID metabolism [15, 22]. Inter-
estingly, P450 1A2 also plays a substantial role in the
metabolism of (R)-warfarin [13] [whereas 2C9 is the
predominantly active form toward (S)-warfarin], and
thus both enantiomers of warfarin would be expected to
inhibit desmethylnaproxen formation. The putative
P450 1A (and 2C9 at higher concentrations) inhibitor,
a-naphtho¯avone [23], produced no inhibition of this
reaction. It is unclear why no inhibition was observed
with a-naphtho¯avone; possibly, higher inhibitor con-
centrations might have had an e€ect. However, the se-
lective P450 1A2 inhibitor, furafylline [24], in
concentrations up to 100 lmol á l)1, did produce a slight
inhibition (23%) of naproxen demethylation, thus con-
®rming the role of P450 1A2 in this process.
This activity of cytochrome P450 2C9 toward napr-
oxen correlates well with the purported role of this iso-
form in the metabolism of other NSAIDs, such as
diclofenac [22], tenoxicam, and piroxicam [15], as well as
¯urbiprofen [6]. However, it does put into question the
utility of correlations of formation with previously de-
termined model reactions in determination of enzyme
involvement since a previous report [3], utilizing only
sulfaphenazole inhibition (a putative 2C9 inhibitor), and
correlation with model reactions suggested the involve-
ment of only 2C9 in naproxen demethylation. However,
298
our studies with cDNA-expressing cells suggest the in-
volvement of other isoforms. Thus, although one
method can be used for preliminary screening, it must be
combined with other con®rmatory procedures, such as
cDNA-expressing cells, antibody inhibition experiments,
or a range of putative speci®c chemical inhibitors, to
conclusively determine isoform involvement. Thus, a
critical application of the techniques available and ap-
propriate interpretation of the results are necessary to
determine isoform involvement.
In conclusion, it appears that cytochrome P450 2C9
plays a substantial role in naproxen demethylation, as
has been noted for other NSAIDs. However, the ob-
served involvement of P450 1A2 and to a smaller extent
2C8, call into question the ability to generalize across
chemical classes of NSAIDs that only P450 2C9 is in-
volved in their oxidative metabolism. Furthermore, the
use of cDNA-expressing cells and subsequent discovery
of multiple enzyme involvement has brought to light the
need for extensive testing of NSAID metabolism, be-
yond the use of putative speci®c inhibitors and correla-
tion with model substrate activities, to conclusively
determine the enzyme(s) involved in the oxidation of a
particular compound.
Acknowledgements Funded in part by NSF grant #OSR-9450578,
a P®zer Undergraduate Research Fellowship and NIH grant
#NO1-DK-6-2274 to the Liver Tissue Procurement and Distribu-
tion System. Presented in abstract form at the American Society for
Clinical Pharmacology and Therapeutics Annual Meeting, Orlan-
do, Florida, 22 March 1996.
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