CYP2C9 genotype-dependent effects on in vitro drug-drug interactions: Switching of benzbromarone effect from inhibition to activation in the CYP2C9.3 variant

Article abstract of DOI:10.1124/mol.105.013763

The CYP2C9.3 variant exhibits marked decreases in substrate turnover compared with the wild-type enzyme, but little is known regarding the effect this variant form may have on the occurrence of drug-drug interactions. To examine this possibility, the effect of the potent CYP2C9 inhibitor, benzbromarone, was studied with regard to CYP2C9.1- and CYP2C9.3-mediated flurbiprofen metabolism to evaluate whether the variant enzyme exhibits differential inhibition kinetics. Although benzbromarone inhibited CYP2C9.1 activity as expected, CYP2C9.3-mediated flurbiprofen 4′-hydroxylation was activated in the presence of benzbromarone. T₁ relaxation studies revealed little change in distances of flurbiprofen protons from the heme iron of either CYP2C9.1 or CYP2C9.3 in the presence of benzbromarone compared with flurbiprofen alone. Spectral binding studies were also performed to investigate whether benzbromarone affected substrate binding, with the addition of benzbromarone having little effect on flurbiprofen-binding affinity in both CYP2C9.1 and CYP2C9.3. Docking studies with the 2C9.1 structure crystallized with a closed active site identified multiple but overlapping subsites with sufficient space for benzbromarone binding in the enzyme when flurbiprofen was positioned closest to the heme. If the closed conformation of 2C9.3 is structurally similar to 2C9.1, as expected for the conservative I359L mutation, then the dynamics of benzbromarone binding may account for the switching of drug interaction effects. In conclusion, the I359L amino acid substitution found in CYP2C9.3 not only reduces metabolism compared with CYP2C9.1 but can also dramatically alter inhibitor effects, suggesting that differential degrees of drug inhibition interactions may occur in individuals with this variant form of CYP2C9. Copyright

Full text of DOI:10.1124/mol.105.013763

0026-895X/05/6803-644–651$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 68:644–651, 2005
Vol. 68, No. 3
13763/3048546
Printed in U.S.A.
CYP2C9 Genotype-Dependent Effects on in Vitro Drug-Drug
Interactions: Switching of Benzbromarone Effect from Inhibition
to Activation in the CYP2C9.3 Variant
Matthew A. Hummel, Charles W. Locuson, Peter M. Gannett, Dan A. Rock,
Carrie M. Mosher, Allan E. Rettie, and Timothy S. Tracy
Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, Minnesota (M.A.H., C.W.L.,
T.S.T.); Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia (P.M.G.); Global
Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Inc., St. Louis, Missouri (D.A.R.); and Department of Medicinal
Chemistry, University of Washington, Seattle, Washington (C.M.M., A.E.R.)
Received April 13, 2005; accepted June 13, 2005
ABSTRACT
The CYP2C9.3 variant exhibits marked decreases in substrate benzbromarone affected substrate binding, with the addition of
turnover compared with the wild-type enzyme, but little is benzbromarone having little effect on flurbiprofen-binding af-
known regarding the effect this variant form may have on the finity in both CYP2C9.1 and CYP2C9.3. Docking studies with
occurrence of drug-drug interactions. To examine this possi- the 2C9.1 structure crystallized with a closed active site iden-
bility, the effect of the potent CYP2C9 inhibitor, benzbromar- tified multiple but overlapping subsites with sufficient space for
one, was studied with regard to CYP2C9.1- and CYP2C9.3- benzbromarone binding in the enzyme when flurbiprofen was
mediated flurbiprofen metabolism to evaluate whether the positioned closest to the heme. If the closed conformation of
variant enzyme exhibits differential inhibition kinetics. Although 2C9.3 is structurally similar to 2C9.1, as expected for the con-
benzbromarone inhibited CYP2C9.1 activity as expected, servative I359L mutation, then the dynamics of benzbromarone
CYP2C9.3-mediated flurbiprofen 4Ј-hydroxylation was acti- binding may account for the switching of drug interaction ef-
vated in the presence of benzbromarone. T1 relaxation studies fects. In conclusion, the I359L amino acid substitution found in
revealed little change in distances of flurbiprofen protons from CYP2C9.3 not only reduces metabolism compared with
the heme iron of either CYP2C9.1 or CYP2C9.3 in the presence CYP2C9.1 but can also dramatically alter inhibitor effects, sug-
of benzbromarone compared with flurbiprofen alone. Spectral gesting that differential degrees of drug inhibition interactions
binding studies were also performed to investigate whether may occur in individuals with this variant form of CYP2C9.
Prediction of drug interactions involving the cytochrome its active site (Shou et al., 1994, 1999, 2001; Hutzler et al.,
P450 (P450) enzyme system requires an accurate estimation
of the inhibition kinetics. New compounds are routinely
tested in an in vitro system using liver microsomes or puri-
fied enzymes against P450 isoform-specific probe substrates
to gauge which and to what extent a particular P450 isoform
is inhibited. One probe substrate is often selected for each
P450 isoform. However, it is known that the CYP2C9 and
CYP3A4 isoforms exhibit atypical kinetic profiles, presum-
ably because of the binding of more than one molecule within
2001, 2003; Galetin et al., 2002; Nakamura et al., 2002). The
crystal structure of CYP2C9 has been solved and has facili-
tated visualization of how multiple substrates may bind
within the active site (Williams et al., 2003; Wester et al.,
2004). This unusual occurrence leads to atypical kinetic phe-
nomena, such as heterotropic or homotropic cooperation
(a.k.a., activation), substrate inhibition, and biphasic kinet-
ics, making correlation to the in vivo situation more difficult
(Hutzler et al., 2001; Tracy et al., 2002). The ability of
CYP2C9 to accept multiple molecules into its active site,
because of its active site volume, may make it possible for a
potential inhibitor of one substrate to have no effect on me-
tabolism of the target substrate but still inhibit the metabo-
lism of a different target substrate. This can result in sub-
strate-dependent inhibition for a given inhibitor.
This study was supported in part by National Institutes of Health grants
GM069753 and GM063215 (T.S.T) and GM032165 (A.E.R.), the University of
Minnesota School of Medicine National Science Foundation grant BIR-961477,
and the Minnesota Medical Foundation.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.105.013763.
ABBREVIATIONS: P450, cytochrome P450; HPLC, high pressure liquid chromatography; PCR, polymerase chain reaction; NMR, nuclear
magnetic resonance.
644

Genotype-Dependent Drug Interactions
645
Allelic variants, such as CYP2C9.3, exhibit reduced sub- of Tracy et al. (2002). Incubation mixtures contained 5 to 20 pmol of
purified P450, NADPH reductase, and cytochrome b5 in a 1:2:1 ratio
reconstituted with dilauroylphosphatidylcholine vesicles extruded
through a 200-nm pore-sized membrane. To study the effects of
benzbromarone on CYP2C9.1-mediated flurbiprofen 4Ј-hydroxyla-
tion, six concentrations of 2 to 300 ␮M (S)-flurbiprofen were incu-
bated with six concentrations of 0 to 300 nM benzbromarone. In the
case of benzbromarone effects on CYP2C9.3-mediated flurbiprofen
4Ј-hydroxylation, six concentrations of 2 to 300 ␮M (S)-flurbiprofen
were incubated with six concentrations of 0 to 300 nM benzbromar-
one. All of the incubations were conducted for 20 min at 37°C in 50
strate turnover that can lead to alterations in in vivo phar-
macokinetics (Haining et al., 1996; Steward et al., 1997;
Yamazaki et al., 1998; Takanashi et al., 2000; Higashi et al.,
2002). Furthermore, the altered pharmacokinetics caused by
this polymorphism can result in profound effects on the ther-
apeutic outcome of clinically important drugs, such as war-
farin (Higashi et al., 2002). In addition, we have previously
reported that the CYP2C9 allelic variants exhibit differential
degrees of dapsone-mediated activation of flurbiprofen me-
tabolism, with CYP2C9.3 showing the greatest percentage mM potassium phosphate buffer, pH 7.4, in a final volume of 200 ␮l.
After a 3-min preincubation, reactions were initiated by the addition
of NADPH (final concentration of 1 mM). Reactions were quenched
by the addition of 200 ␮l of acetonitrile containing internal standard
and 180 ng/ml 2-fluoro-4-biphenyl acetic acid as internal standard.
After quenching, 40 ␮l of half-strength H3PO4 was added to the
reaction mixtures. Samples were then centrifuged at 10,000 rpm for
4 min and placed into autosampler vials, and 50 ␮l was injected onto
the HPLC system.
increase in flurbiprofen 4Ј-hydroxylation activity compared
with CYP2C9.1, CYP2C9.2, and CYP2C9.5 (Hummel et al.,
2004a). However, little is known regarding whether enzyme
inhibition also exhibits differential effects among the
CYP2C9 variants, particularly the clinically relevant
CYP2C9.3 enzyme.
To test the hypothesis that differential inhibition of
CYP2C9 variants may occur, metabolism of the model
CYP2C9 substrate flurbiprofen (Tracy et al., 1995, 1996) was
Metabolite Quantitation. HPLC analysis of 4Ј-hydroxyflurbi-
profen production was conducted as described previously (Tracy et
studied in the presence of the potent CYP2C9 inhibitor ben- al., 2002). The HPLC system consisted of a Waters Alliance 2695XE
chromatographic system and a Waters model 2475 fluorescence de-
tector (Waters, Milford, MA). The mobile phase was pumped through
a Brownlee Spheri-5 C18 4.6 ϫ 100-mm column (PerkinElmer Life
and Analytical Sciences, Boston, MA) at 1 ml/min. For quantification
of 4Ј-hydroxyflurbiprofen, the detector was set at an excitation wave-
length of 260 nm and an emission wavelength of 320 nm and the
mobile phase consisted of 45:55 acetonitrile/20 mM potassium phos-
phate, pH 3.0. The retention times for 4Ј-hydroxyflurbiprofen and
the internal standard were approximately 2.6 and 5.6 min, respec-
tively.
zbromarone (Locuson et al., 2003, 2004) in both CYP2C9.1
and CYP2C9.3 enzyme. To evaluate possible reasons for ob-
served differences, potential changes in substrate-enzyme-
inhibitor interactions were also studied by measuring bind-
ing affinities (Ks) and proton distances from the P450 heme
and by conducting preliminary docking studies.
Materials and Methods
Kinetic Data Analysis. Kinetic parameters for the substrates
were estimated by nonlinear regression analysis using Sigma Plot
8.0 (Systat Software, Richmond, CA). To simplify comparisons, ki-
netic data for activation of CYP2C9.3 as well as inhibition of
CYP2C9.1-mediated flurbiprofen 4Ј-hydroxylation in the presence of
benzbromarone were fit to a two-site model (Korzekwa et al., 1998;
Hutzler et al., 2001).
Acetonitrile, potassium phosphate, glycerol, and EDTA were pur-
chased from Fisher Scientific Co. (Pittsburgh, PA). NADPH, dilau-
roylphosphatidylcholine, poly(vinylpyrrolidone), benzbromarone,
and sodium dithionite were obtained from Sigma-Aldrich (St. Louis,
MO). D2O was obtained from Cambridge Isotopes Laboratory (An-
dover, MA). (S)-Flurbiprofen, 4Ј-hydroxyflurbiprofen, and 2-fluoro
4-biphenyl acetic acid were gifts from Pfizer, Inc. (New York, NY).
Centricon MW cut-off filters were obtained from Millipore Corpora-
tion (Billerica, MA). Human P450 oxidoreductase and human cyto-
chrome b5 were purchased from Invitrogen (Carlsbad, CA).
Enzyme Expression and Incubation Conditions. The 2C9.1
gene was subcloned into the pCWoriϩ vector from the original bac-
ulovirus transfer vector pUC19 (Haining et al., 1999) using a single
PCR with the following primers: forward, 5Ј-CCA TCG ATC ATA
TGG CTC TGT TAT TAG CAG TTT TTC TCT GTC TCT CAT GTT
V
[B]
m ϫ [S]
v ϭ
(1)
[B]
␣KB
␤[B]
␣KB
1 ϩ
1 ϩ
ͩ ͪ ͩ ͪ
KB
Km
ϩ [S]
␤[B]
1 ϩ
1 ϩ
ͩ ͪ ͩ ͪ
␣KB
Appropriateness of the fits was determined by examination and
TGC TTC TCC TTT C-3Ј; and reverse, 5Ј-TCT GTC GAC ACA GGA comparison of the residuals, residual sum of squares, coefficients of
ATG AAG CAC AGC TGG TAG AAG-3Ј. The forward primer encoded determination, and F values.
the MALLLAVFL N-terminal sequence of recombinant bovine P450
Enzyme Sample Preparation for NMR. Enzyme samples for
17␣ to enhance expression in Escherichia coli (Barnes et al., 1991). use in T1 relaxation measurements were prepared as reported pre-
NdeI and SalI restriction sites were also engineered into the forward viously (Hummel et al., 2004b). Concentrated enzyme samples were
and reverse primers, respectively, to facilitate downstream subclon- diluted 50-fold into 50 mM potassium phosphate, pH 7.4, in D2O to
ing. The PCR product and pCWoriϩ vector were digested with NdeI remove the majority of the glycerol and H2O. CYP2C9.1 was then
and SalI, gel-purified, ligated together for 10 min at 25°C, and finally added to the sample tube at a concentration of 0.014 ␮M in a final
transformed into DH5␣ FЈIQ cells. The 2C9.3 construct was gener- volume of 750 ␮l containing either 145 ␮M flurbiprofen alone or 145
ated using 2C9.1 as a template. A single PCR was performed using ␮M flurbiprofen plus 300 nM benzbromarone. When CYP2C9.3 was
the following primers: forward, 5Ј-GTC CAG AGA TAC CTT GAC studied, the enzyme was added to the sample tube at a concentration
CTT CTC CCC ACC AGC CTG-3Ј; and reverse, 5Ј-CAG GCT GGT
of 0.030 ␮M in a final volume of 750 ␮l containing either 300 ␮M
GGG GAG AAG GTC AAG GTA TCT CTG GAC-3Ј. These primers flurbiprofen alone or 300 ␮M flurbiprofen plus 300 nM benzbromar-
incorporate the A3C point mutation at base position 1061 that is one. Determination of distances of the benzbromarone protons either
responsible for the Ile3Leu substitution. The final PCR product was alone or in the presence of flurbiprofen was not possible because of
digested with DpnI for 1 h at 37°C and then transformed into DH5␣ the insufficient sensitivity and resolution to monitor the low concen-
FЈIQ cells. All of the constructs were verified by DNA sequencing. trations of benzbromarone (in nanomolar) employed in the study.
Protein expression in E. coli was carried out as described previously
T1 Relaxation Time Measurements. Chemical shift assign-
(Cheesman et al., 2003).
ments for flurbiprofen protons (Fig. 1) were made as described pre-
Metabolic incubations were carried out according to the methods viously (Hummel et al., 2004b). T1 times of substrate protons were

646
Hummel et al.
determined with the NMR (Varian Inova 600 MHz Spectrometer) for 3 min before spectral analysis. Spectra were recorded on an
operating at 600.5 MHz, internally locked on the deuterium signal of Aminco DW-2000 UV-visible spectrophotometer with Olis modifica-
the solvent. The probe was maintained at 298 K for all of the tions (Olis, Inc., Bogart, GA). The spectrophotometer was set to
experiments except when testing for fast exchange conditions (see record spectra between 350 and 500-nm wavelengths with a slit-
below). The Varian T1 inversion-recovery sequence (d1-180-d2-90) width of 6.0 nm and scan rate of 100 nm/min. The temperature was
was used along with presaturation of the residual HOD signal. The held at a constant 28°C. The difference in absorbance between the
90° pulse-width was calibrated on each sample. Spectra were ac- peak (ϳ390) and trough (ϳ420) of the observed type I-binding spec-
quired for 12 ␶ values (d2) ranging from 0.0125 to 25.6 s, and a period trum was calculated and plotted against flurbiprofen concentration.
of 10 ϫ T1 was used between pulses (d1). The Varian software A binding constant (Ks) was determined by fitting the resulting data
routines were used to determine T1 times. Once the paramagnetic to the eq. 2.
effect of the heme iron on substrate protons was measured, carbon
monoxide (CO) was bubbled through the sample for 15 min and
sodium dithionite was then added and allowed to equilibrate for 30
min to determine the diamagnetic contribution of the protein to the
͑Bmax ϫ S͒
Ks ϩ S
⌬A ϭ
(2)
Spectral binding experiments were also performed with flurbipro-
fen in the presence of 300 nM benzbromarone for each enzyme.
Molecular Modeling. MoViT, version 8.0 (Pfizer, Inc.) was used
to dock and minimize benzbromarone in the CYP2C9 crystal struc-
ture (Protein Data Bank 1R9O) (Wester et al., 2004) with flurbipro-
fen after importation of coordinates. The chemical structure of ben-
zbromarone was created in ChemDraw and imported into MoViT
followed by energy minimization of benzbromarone. NMR distances
obtained from the T1 NMR data were used to guide manipulations of
flurbiprofen and benzbromarone. During docking, consideration was
given to possible interactions between key active site residues, in-
cluding Arg108, Phe114, Phe100, and Phe476. The final representa-
tion is a product of multiple energy-minimized docking iterations.
T
1 relaxation times. To ensure adequate diffusion of CO and mixing
of dithionite, samples were removed from the NMR tube and treated
with CO and sodium dithionite and then the sample was placed back
into the NMR tube for the measurements. Stability of the enzyme as
well as the CO-reduced complex was tested, and both were found to
be stable for the duration of the NMR acquisition time.
T1 measurements and the resulting calculated distances are de-
pendent on the substrate being in fast exchange with the enzyme.
The validity of this assumption can be demonstrated by conducting
T1 measurements over a range of temperatures (Regal and Nelson,
2000). Thus, T1 measurements were performed as described above at
three different temperatures (283, 298, and 310 K). Data were col-
lected both in the absence (1/T1,2C9) and presence (1/T1,2C9ϩCO) of
CO/sodium dithionite.
Proton Heme Iron Distance Calculation. Estimates for dis-
tances of protons from the heme iron of CYP2C9 were calculated
using the equation r ϭ C[T1p␣mf(␶c)]1/6, where r is the distance and C
is a constant that is a function of the metal and the oxidation state
and whether it is low or high spin. In this case, Fe3ϩ should be in the
Results
The effects of benzbromarone on the 4Ј-hydroxylation of
flurbiprofen by both CYP2C9.1 and CYP2C9.3 are presented
as three-dimensional contour plots and depicted in Fig. 2, A
low-spin state; thus, the appropriate value for C is 539 (Mildvan and and B. Data for both enzymes were fitted to a two-site kinetic
Gupta, 1978). T1p is the portion of T1obs because of paramagnetic
model (eq. 1) to allow direct comparison of the kinetic param-
eter estimates, and the results of these estimates are pre-
sented in Table 1. The concave nature of the plot in Fig. 2A
resulting from CYP2C9.1-dependent metabolism demon-
strates the inhibitory effect that benzbromarone has upon
the metabolism of flurbiprofen. It is surprising that an oppo-
site effect (activation) was noted (Fig. 2B) when benzbrom-
arone was studied as an effector of CYP2C9.3-mediated
flurbiprofen hydroxylation, resulting in a convex three-di-
Ϫ1
effects alone and is given by T1p ϭ T1obs(Fe3ϩ Ϫ1
assuming that all of the diamagnetic contribution is represented by
1obs(Fe2ϩ) (Regal and Nelson, 2000). This assumption has been used
)
Ϫ T1obs(Fe2ϩ Ϫ1
) ,
T
in many similar studies and seems to be generally valid (Modi et al.,
1996; Poli-Scaife et al., 1997; Shafirovich et al., 2002). The parameter
␣
is equal to [P450]/(Ks ϩ [substrate]) under conditions of fast
m
exchange (Regal and Nelson, 2000). The flurbiprofen Ks values of 7
␮M CYP2C9.1 and 14 ␮M CYP2C9.3 determined from visible spec-
troscopy were used for estimation of the parameter ␣m. The correla-
tion time (␶c) for CYP2C9 has been reported previously (2 ϫ 10Ϫ10 mensional contour plot. Evaluation of the kinetic parameter
s
Ϫ1) (Poli-Scaife et al., 1997) and was used here.
estimates (Table 1) presents interesting contrasts between
the effects of benzbromarone in the two enzymes. The KB for
benzbromarone as an activator of flurbiprofen metabolism in
CYP2C9.3 is roughly 15-fold higher than noted when ben-
zbromarone acts as an inhibitor in the CYP2C9.1 enzyme. In
the CYP2C9.1-mediated process, Km is greatly increased (␣ ϭ
5.0) by benzbromarone, whereas Vm is reduced by 50%. This
type of kinetic change is typically indicative of a mixed com-
petitive and noncompetitive type of inhibition. In the case of
the activation of CYP2C9.3 by benzbromarone, Km is greatly
reduced (␣ ϭ 0.14) but the Vm of flurbiprofen 4Ј-hydroxyla-
tion is slightly changed (␤ ϭ 1.14).
Spectral Binding. Spectral binding studies to measure enzyme-
substrate affinity were performed as reported previously (Hutzler et
al., 2003). In brief, 300 pmol of enzyme along with 0.2 ␮g/pmol
dilauroylphosphatidylcholine was placed into the sample and refer-
ence cuvettes. For determination of spectral changes at increasing
concentrations of flurbiprofen, 5-␮l aliquots of flurbiprofen were
added to the sample cuvette, whereas 5 ␮l of 50 mM potassium
phosphate buffer, pH 7.4, was added to the reference cuvette. After
mixing, the sample and reference cuvette were allowed to equilibrate
To ascertain whether the changes noted in Km for flurbi-
profen hydroxylation by each of the two enzymes are accom-
panied by alterations in substrate affinity, spectral binding
studies were conducted. The spectral binding constant (Ks)
for flurbiprofen in the presence of CYP2C9.1 was 7 ␮M (Fig.
3A), whereas, in the presence of CYP2C9.3, the Ks for flur-
biprofen was 14 ␮M (Fig. 4A). The addition of 300 nM benz-
bromarone reduced the Ks by 40% (Fig. 3B), which is roughly
comparable with the magnitude of the change noted in Km
Fig. 1. Structure of flurbiprofen with protons numbered as they are
referenced within the text. The 4Ј-H is the site of oxidation.

Genotype-Dependent Drug Interactions
647
(ϳ20% decrease) under these same conditions. It is interest- whether benzbromarone altered the distance of the substrate
ing that, despite the substantial activation of CYP2C9.3- (flurbiprofen) protons from the heme iron, potentially con-
mediated metabolism of flurbiprofen and concomitant reduc- tributing to the alterations in metabolism. The estimated
tion in Km caused by the addition of benzbromarone, no distances of flurbiprofen protons from the heme iron of
changes were noted in binding affinity (Ks) of flurbiprofen CYP2C9.1 and CYP2C9.3 are listed in Tables 2 and 3, re-
with CYP2C9.3 in the presence of benzbromarone (Fig. 4, A spectively. In the presence of CYP2C9.1, the distances of the
and B). Furthermore, with the CYP2C9.3 enzyme, in the flurbiprofen protons from the heme iron were relatively un-
presence of flurbiprofen alone, no demonstrable peak was affected by benzbromarone. Likewise, with CYP2C9.3, the
noted at ϳ390 nm, whereas, when 300 nM benzbromarone presence of 300 nM benzbromarone had little effect on dis-
was added, a substantial ϳ390-nm peak was noted similar to tances of flurbiprofen protons to the heme iron. When studied
that observed with wild-type enzyme (either in the absence in the presence of only 100 nM benzbromarone, similar re-
or presence of benzbromarone).
sults were noted. It is also interesting to note that the flur-
T1 NMR experiments were next conducted to determine biprofen protons are essentially the same distance to the
Fig. 2. Two-site model fit of the benzbromarone inhibition of CYP2C9.1 (A) and CYP2C9.3 (B). Data points represent mean of duplicate determina-
tions.
TABLE 1
Kinetic parameter estimates for flurbiprofen 4Ј-hydroxylation in the presence of varying concentrations of benzbromarone
Data were fit to a two-site model (see eq. 1). Parameter estimates are reported as the estimate (mean Ϯ S.E. of the estimate) resulting from nonlinear regression of the data.
KB, the binding constant for the effector; ␣, the change in Km resulting from effector binding; and ␤, the change in Vm resulting from effector binding. An ␣ value Ͻ1 indicates
a decrease in Km. A ␤ value Ͼ1 indicates an increase in Vm, whereas a ␤ value Ͻ1 indicates a decrease in Vm
.
Enzyme
Vm
Km
KB
␣
␤
R2
pmol/min/pmol P450
␮M
nM
CYP2C9.1
CYP2C9.3
3.98 (0.13)
1.43 (0.09)
16.4 (1.98)
147 (20.6)
39.9 (14.2)
627 (516)
5.02 (1.94)
0.14 (0.08)
0.50 (0.13)
1.14 (0.11)
0.977
0.991
Fig. 3. Binding spectra of flurbiprofen to CYP2C9.1 alone (A) and in the presence of 300 nM benzbromarone (B). Inset graph is the fit of the spectral
difference data used to determine the Ks.

648
Hummel et al.
heme iron in CYP2C9.3 (I359L) as in CYP2C9.1, despite flurbiprofen oriented near the heme iron, are presented in
reduced substrate turnover by the variant enzyme. The plots Fig. 5, A and B. Although the space directly above the heme
of 1/T1p versus 1/temperature for the T1 relaxation studies is occupied by flurbiprofen and further constricted by resi-
for CYP2C9.1 and CYP2C9.3 exhibited a positive linear slope dues Leu362 and Leu366, suitable space for docking of ben-
for all of the protons, indicating temperature dependence and zbromarone was found above flurbiprofen (Fig. 5, A and B).
thus fast exchange (data not shown).
Maintaining flurbiprofen closest to the heme, benzbromarone
Docking of flurbiprofen and benzbromarone was conducted populated two overlapping positions that together covered
using the 1R9O crystal structure of CYP2C9 (Wester et al., most of the major substrate recognition sites (SRS regions),
2004). Depictions of lowest energy conformations of benzbro- including the B–C loop, the F, G, and I helices, and the
marone docked within the active site, while maintaining C-terminal loop containing Phe476. Both positions of benz-
Fig. 4. Binding spectra of flurbiprofen to CYP2C9.3 alone (A) in the presence of 100 nM benzbromarone (B) and in the presence of 300 nM
benzbromarone (C). Inset graph is the fit of the spectral difference data used to determine the Ks.
TABLE 2
T1 relaxation rate-estimated distances of flurbiprofen protons from the heme iron of CYP2C9.1 in the absence and presence of 300 nM
benzbromarone
Errors for measurements are shown within parentheses. Errors in the T1 values were those reported by the fitting routine. Errors in the reported distances (r) were
determined by propagation of error from the T1 calculation. Flurbiprofen concentration was 145 ␮M; benzbromarone concentration was 300 nM when studied together. See
Fig. 1 for the numbering scheme of the flurbiprofen protons. T1 calculations for the HC-CO2H proton could not be accurately determined because of interference from the
residual glycerol resonances. T1 values are in seconds. ͓P450 2C9͔ ϭ 0.014 ␮M; ␣M ϭ ͓P450͔/(Ks ϩ ͓substrate͔), KS (flurbiprofen) ϭ 7.8 ␮M, ␣M (flurbiprofen) ϭ 9.16 ϫ 10Ϫ5
.
Distance values are in angstroms (Å), r ϭ C͓T1P ϫ ␣M ϫ f(␶c)͔Ϫ1/6, C ϭ 539 (Mildvan and Gupta, 1978), 1/T1P ϭ 1/T1, 2C9 Ϫ 1/T1, 2C9 ϩ CO (Mildvan and Gupta, 1978), f(␶c) ϭ
2 ϫ 10Ϫ10 sϪ1
.
CYP2C9.1
CYP2C9.1 with Benzbromarone
Proton
2C9
2C9 ϩ CO
r
2C9
2C9 ϩ CO
r
2Ј,6Ј
3Ј,5Ј
5
2.46 (0.03)
2.49 (0.03)
2.22 (0.05)
3.86 (0.16)
1.95 (0.03)
2.67 (0.06)
0.71 (0.01)
2.83 (0.03)
2.85 (0.03)
2.43 (0.05)
4.67 (0.19)
2.20 (0.04)
3.15 (0.08)
0.73 (0.01)
4.52 (0.06)
4.53 (0.06)
4.77 (0.11)
4.65 (0.19)
4.46 (0.07)
4.45 (0.11)
4.76 (0.04)
2.41 (0.03)
2.49 (0.03)
2.12 (0.06)
3.32 (0.10)
1.92 (0.05)
2.54 (0.08)
0.69 (0.01)
2.83 (0.04)
2.85 (0.03)
2.64 (0.06)
4.43 (0.21)
2.16 (0.04)
3.13 (0.10)
0.72 (0.01)
4.41 (0.07)
4.53 (0.06)
4.12 (0.12)
4.27 (0.21)
4.46 (0.13)
4.28 (0.14)
4.44 (0.05)
4Ј
6
2
CH3
TABLE 3
T1 relaxation rate-estimated distances of flurbiprofen protons from the heme iron of CYP2C9.3 in the absence and presence of 300 nM
benzbromarone
Errors for measurements are shown in parentheses. Errors in the T1 values were those reported by the fitting routine. Errors in the reported distances (r) were determined
by propagation of error from the T1 calculation. Flurbiprofen concentration was 300 ␮M; benzbromarone concentration was 300 nM when studied together. See Fig. 1 for the
numbering scheme of the flurbiprofen protons. T1 calculations for the HC-CO2H proton could not be accurately determined because of interference from the residual glycerol
resonances. T1 values are in seconds. ͓P450 2C9͔ ϭ 0.014 ␮M; ␣M ϭ ͓P450͔/(Ks ϩ ͓substrate͔), KS (flurbiprofen) ϭ 13.8 ␮M, ␣M (Flurbiprofen) ϭ 9.16 ϫ 10Ϫ5. Distance values
are in angstroms (Å), r ϭ C͓T1P ϫ ␣M ϫ f(␶c)͔Ϫ1/6, C ϭ 539 (Mildvan and Gupta, 1978), 1/T1P ϭ 1/T1, 2C9 Ϫ 1/T1, 2C9 ϩ CO (Mildvan and Gupta, 1978), f(␶c) ϭ 2 ϫ 10Ϫ10 sϪ1
.
CYP2C9.3
CYP2C9.3 with Benzbromarone
Proton
2C9
2C9 ϩ CO
r
2C9
2C9 ϩ CO
r
2Ј,6Ј
3Ј,5Ј
5
2.40 (0.02)
2.43 (0.02)
2.13 (0.04)
3.44 (0.07)
1.96 (0.03)
2.66 (0.04)
0.68 (0.01)
2.78 (0.03)
2.84 (0.03)
2.44 (0.05)
4.18 (0.14)
2.14 (0.04)
3.06 (0.07)
0.70 (0.01)
4.50 (0.04)
4.46 (0.04)
4.46 (0.10)
4.57 (0.15)
4.74 (0.08)
4.61 (0.11)
4.89 (0.06)
2.41 (0.02)
2.44 (0.03)
2.16 (0.03)
3.82 (0.12)
1.95 (0.03)
2.64 (0.05)
0.69 (0.01)
2.79 (0.02)
2.85 (0.03)
2.46 (0.04)
4.88 (0.18)
2.16 (0.03)
3.11 (0.06)
0.71 (0.01)
4.50 (0.39)
4.47 (0.05)
4.49 (0.08)
4.50 (0.16)
4.62 (0.06)
4.50 (0.09)
4.79 (0.06)
4Ј
6
2
CH3

Genotype-Dependent Drug Interactions
649
Fig. 5. A and B, potential docking locations and energy
minimized flurbiprofen and benzbromarone within the ac-
tive site of CYP2C9 based on the 1R90 crystal structure of
CYP2C9. Flurbiprofen is colored white, and benzbromar-
one is colored blue.
bromarone also overlap the docked position of dapsone
(Wester et al., 2004).
To examine the reasons underlying the benzbromarone-
induced switching to activation kinetics in the variant
CYP2C9.3 enzyme, several additional studies were con-
ducted. T1 relaxation NMR studies can be used to estimate
substrate proton to heme iron distances of cytochrome P450
enzymes (Regal and Nelson, 2000; Hummel et al., 2004b).
Thus, we studied the distances of flurbiprofen protons from
the heme iron in both the CYP2C9.1 and CYP2C9.3 enzymes
in the absence and presence of benzbromarone. In the pres-
ence of benzbromarone, the time-averaged distances of flur-
biprofen protons from the heme of CYP2C9.1 were relatively
unaffected by the presence of benzbromarone. This is pre-
sumed to be due to the mutual exclusivity of flurbiprofen or
to benzbromarone being near enough to the heme iron for
measurement of distances. Likewise, benzbromarone had no
substantial effect on distances of the flurbiprofen protons
relative to the heme iron in CYP2C9.3. This finding differs
from that observed in the activation of flurbiprofen metabo-
lism by dapsone with the CYP2C9.1 enzyme (Hummel et al.,
2004b). Thus, changes in flurbiprofen proton to heme iron
distances do not seem to account for the activation of flurbi-
profen metabolism by benzbromarone in CYP2C9.3. Unfor-
tunately, we were unable to measure the T1 relaxation of the
benzbromarone protons because of the large abundance of
flurbiprofen (300 ␮M flurbiprofen versus 300 nM benzbrom-
arone) in the sample, which prevented resolution of the res-
onances for benzbromarone protons. This prevented us from
definitively determining whether benzbromarone was simul-
taneously binding within the active site along with flurbipro-
fen. It is interesting to note that the distances of flurbiprofen
alone in CYP2C9.3 are similar to the distances of flurbipro-
fen observed in CYP2C9.1. This result suggests that, even
though the flurbiprofen protons are at a similar distance
from the heme iron when bound to CYP2C9.3 compared with
CYP2C9.1, it either assumes a less productive orientation in
the active site or that the CYP2C9.3 variant results in re-
duced substrate turnover through some mechanism other
than change in substrate orientation.
Discussion
Prediction of drug interactions resulting from P450 inhibi-
tion may be confounded by many factors. The ability of P450
enzymes, such as CYP2C9, to accommodate multiple sub-
strate molecules within their active sites can affect interac-
tions between substrates (Korzekwa et al., 1998; Hutzler et
al., 2001; Tracy et al., 2002; Wienkers, 2002), resulting in
substrate-dependent inhibition. CYP2C9 also exhibits ge-
netic polymorphisms that contribute to interindividual vari-
ability in metabolism rates (Haining et al., 1996; Takanashi
et al., 2000; Dickmann et al., 2001; Tracy et al., 2002), yet
little is known regarding whether CYP2C9 polymorphisms
might also affect the degree of inhibition-based drug-drug
interactions. To this end, the inhibition of flurbiprofen by
benzbromarone, the most potent inhibitor of CYP2C9 re-
ported so far, was tested in CYP2C9.1 and CYP2C9.3 to
examine whether variant-dependent inhibition exists for
these enzymes. In contrast to its inhibitory effect in
CYP2C9.1, in the CYP2C9.3 enzyme, benzbromarone was a
potent activator of flurbiprofen metabolism. This unexpected
finding suggests that the extent and type of drug interaction
observed may be dependent on the genotype of a person,
further complicating the potential for predicting drug-drug
interactions.
As predicted, the turnover of flurbiprofen by CYP2C9.3 is
greatly reduced compared with wild-type enzyme. Thus, it
was surprising that benzbromarone, a potent inhibitor of this
same process in wild-type enzyme, activated flurbiprofen
hydroxylation in the CYP2C9.3 variant; a complete reversal
of effect. Other activators of CYP2C9-mediated flurbiprofen
metabolism, such as dapsone and selected analogs, have been
identified, but these compounds exhibit activation kinetics,
regardless of the CYP2C9 variant studied (Hummel et al.,
2004a). Marks et al. (2004) have reported a similar change
from inhibition to activation for ibuprofen inhibition of Vivid
Red metabolism by CYP2C9.1 and CYP2C9.3, respectively.
However, this occurred at micromolar ibuprofen concentra-
tions as opposed to the nanomolar concentrations observed
with benzbromarone. Studying amiodarone, a compound
structurally related to benzbromarone, Egnell et al. (2003)
observed that amiodarone, which is typically a weak inhibi-
tor of CYP2C9 metabolism, activated 7-methoxyfluorocouma-
rin metabolism at nanomolar concentrations. However, this
phenomenon was only studied with the wild-type CYP2C9
enzyme.
To assess other possible reasons for the observed switching
of benzbromarone effect, the binding affinity (Ks) of flurbi-
profen for each of the two enzymes (CYP2C9.1 and
CYP2C9.3) was determined in the absence and presence of
benzbromarone. Benzbromarone coincubation caused a mod-
est change in flurbiprofen-binding affinity in CYP2C9.1 but
no change in CYP2C9.3, despite the observed changes in
turnover. This phenomenon of activation without a corre-
sponding increase in binding affinity is analogous to that
observed with N-hydroxydapsone activation of flurbiprofen

650
Hummel et al.
hydroxylation in CYP2C9.1 (Hutzler et al., 2003) and con- reducing the competition for this binding orientation/site and
enabling activation to occur.
trasts with the increase in flurbiprofen-binding affinity with
dapsone. These results reinforce previous observations that
the activation of cytochrome P450-mediated metabolism may
occur through multiple mechanisms and that the mecha-
nisms involved can be dependent on effector or enzyme.
It is interesting that the measurement of difference spectra
in the case of CYP2C9.3 gave somewhat unusual results (Fig.
4, A–C). It is possible that the Ks of flurbiprofen does change
in the presence of benzbromarone but that the abnormal
difference spectra obscured this change. Upon titration of
flurbiprofen, 2C9.3 failed to give a measurable increase in
the 390-nm peak of the difference spectra associated with
conversion of the heme iron from low spin to high spin (Fig.
4A). When benzbromarone was added during the titrations
(Fig. 4, B and C), the 390-nm peak appeared, whereas the
negative low-spin peak at 420 nm reached its minimum at
the same concentration of flurbiprofen in every experiment.
This suggests that the Ks determination was not affected by
the anomalous nature of the 390-nm peak of the difference
spectra. All of the difference spectra of P450s taken after the
addition of ligand result from perturbation of the solvent
network near the distal face of the heme or coordination to
the iron. Unfortunately, the precise reason for the variable
difference spectra in the 390-nm region is currently unknown
and perhaps is better explored using other methods (e.g.,
EPR). Whether this phenomenon is associated with altered
enzyme activity (e.g., that of CYP2C9.3) is unclear, because
the CYP2C9 substrate diclofenac produces only modest
changes in the high-spin peak relative to the decrease in the
low-spin peak (M. A. Hummel, C. W. Locuson, P. M. Gannett,
D. A. Rock, C. Mosher, A. E. Rettie, and T. S. Tracy, unpub-
lished observations) yet exhibits a high kcat value similar to
that of flurbiprofen (Dickmann et al., 2001).
To gain additional insight into the binding orientation for
benzbromarone, docking studies were performed with the
wild-type enzyme. Although the docking carried out does not
allow the movement of the enzyme in any manner, it dem-
onstrates that there is sufficient space for heteroactivator
binding within the same active site as the substrate. In
addition, the Ile359 residue, which is conservatively changed
to leucine in CYP2C9.3, has no way of directly contacting
either substrate or effector, despite switching of benzbrom-
arone effect to activation in the CYP2C9.3 enzyme. The
leucine at position 359 may alter adjacent residue side chain
packing so that the enzyme gains the volume or flexibility to
accommodate benzbromarone in a different orientation than
in wild-type enzyme, causing an enhancement of flurbiprofen
metabolism. Examining a series of benzbromarone analogs to
probe the CYP2C9-active site, Locuson et al. (2003, 2004)
proposed that benzbromarone binds in CYP2C9 by interact-
ing edge-to-face with Phe114 and ion pairing with Arg108.
Arg108 was also found to be important for obtaining a type
I-binding spectrum for both flurbiprofen and benzbromarone
(Dickmann et al., 2004). This Arg108 residue binding of
benzbromarone probably results in benzbromarone inhibi-
tion of flurbiprofen metabolism, because both molecules
would compete for this Arg residue in CYP2C9.1. In the case
of CYP2C9.3, perhaps the phenol of the benzbromarone or
the carboxylic acid of flurbiprofen is able to bind more readily
to another positively charged residue in CYP2C9.3 (Fig. 5B),
Although this change in type of interaction is substrate-,
effector-, and variant-dependent, it is an important finding,
especially for low therapeutic window drugs, because roughly
10% of the white population carries at least one allele ex-
pressing CYP2C9.3 (Lee et al., 2002). For example, a patient
homozygous for CYP2C9.3 may empirically have their dose
lowered because of the coadministration of a CYP2C9 inhib-
itor. However, if the CYP2C9.1 inhibitor activates CYP2C9.3,
the amount of parent drug in the body would be reduced,
thereby decreasing efficacy in contrast to what would be
expected with inhibition. In addition, intentional activating
drug-drug interactions could conceivably prove useful in al-
tering drug plasma levels to treat overdoses or increase ac-
tive metabolite production depending on one’s genetics.
In summary, CYP2C9.1-mediated flurbiprofen metabolism
was inhibited by the presence of benzbromarone, whereas
CYP2C9.3-mediated flurbiprofen metabolism was activated
by coincubation with this same effector. This seemingly mod-
est amino acid substitution (I359L) may alter active site
conformation and/or substrate/effector binding in such a way
that it results in the switching of effect from inhibition to
activation. Results, such as these demonstrating alterations
in drug interaction effects in variant P450 enzymes, will
undoubtedly further complicate prediction of drug interac-
tions in the early stages of drug development.
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perimental and Clinical Pharmacology, College of Pharmacy, University
of Minnesota, 308 Harvard St., S.E., Minneapolis, MN 55455. E-mail:
tracy017@umn.edu