Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil

Article abstract of DOI:10.1124/dmd.111.041947

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other gluc

Full text of DOI:10.1124/dmd.111.041947

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2011/09/12/dmd.111.041947.DC1.html
0090-9556/11/3912-2421–2430$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:2421–2430, 2011
Vol. 39, No. 12
41947/3731444
Printed in U.S.A.
Studies to Further Investigate the Inhibition of Human Liver
Microsomal CYP2C8 by the Acyl-␤-Glucuronide of Gemfibrozil
□
S
S. M. Jenkins, T. Zvyaga, S. R. Johnson, J. Hurley, A. Wagner, R. Burrell, W. Turley, J. E. Leet,
T. Philip, and A. D. Rodrigues
Bristol-Myers Squibb Molecular Sciences & Candidate Optimization, Wallingford, Connecticut (S.M.J., T.Z., J.H., A.W.,
R.B., W.T., J.E.L., T.P.); and Bristol-Myers Squibb Molecular Sciences & Candidate Optimization, Princeton, New Jersey
(S.R.J., A.D.R.)
Received July 26, 2011; accepted September 12, 2011
ABSTRACT:
In previous studies, gemfibrozil acyl-␤-glucuronide, but not gem- When the aromatic methyl groups on the gemfibrozil moiety were
fibrozil, was found to be a mechanism-based inhibitor of cyto- substituted with trifluoromethyls, the resulting glucuronide conju-
chrome P450 2C8. To better understand whether this inhibition is gate was a weaker inhibitor of CYP2C8 and mechanism-based
specific for gemfibrozil acyl-␤-glucuronide or whether other gluc- inhibition was abolished. However, the glucuronide conjugates of
uronide conjugates are potential substrates for inhibition of this monomethyl gemfibrozil analogs were mechanism-based inhibi-
enzyme, we evaluated several pharmaceutical compounds (as tors of CYP2C8, although not as potent as gemfibrozil acyl-␤-
their acyl glucuronides) as direct-acting and metabolism-depen- glucuronide itself. The ortho-monomethyl analog was a more po-
dent inhibitors of CYP2C8 in human liver microsomes. Of 11 com- tent inhibitor than the meta-monomethyl analog, indicating that
pounds that were evaluated as their acyl glucuronide conjugates, CYP2C8 favors the ortho position for oxidation and potential inhi-
only gemfibrozil acyl-␤-glucuronide exhibited mechanism-based bition. Molecular modeling of gemfibrozil acyl-␤-glucuronide in the
inhibition, indicating that CYP2C8 mechanism-based inhibition is CYP2C8 active site is consistent with the ortho-methyl position
very specific to certain glucuronide conjugates. Structural analogs being the favored site of covalent attachment to the heme. More-
of gemfibrozil were synthesized, and their glucuronide conjugates over, hydrogen bonding to four residues (Ser100, Ser103, Gln214,
were prepared to further examine the mechanism of inhibition. and Asn217) is implicated.
Introduction
found that gemfibrozil acyl-␤-glucuronide was a direct-acting and
metabolism-dependent inhibitor of CYP2C8 with a KI of 20 to 52 ␮M
and a kinact of 0.21 minϪ1. When gemfibrozil was coadministered with
repaglinide (a substrate for CYP3A4 and CYP2C8) in a clinical study,
an increase in the area under the curve was observed for repaglinide
that persisted for at least 12 h after the dose, indicating that this
mechanism-based inhibition occurs in vivo (Tornio et al., 2008).
The effect is rapid and is evident 1 h after gemfibrozil adminis-
tration (Honkalammi et al., 2011). In a recent study, Baer et al.
(2009) showed that gemfibrozil acyl-␤-glucuronide was covalently
bound to the heme of CYP2C8 and proposed P450-catalyzed
oxidation of either of the benzylic methyl groups to form a benzyl
radical intermediate (Fig. 1A).
Other recent reports of glucuronide metabolites interacting di-
rectly with CYP2C8 have been published, including the CYP2C8-
mediated hydroxylation of the acyl glucuronide of diclofenac and
the 17␤-O-glucuronide of estradiol (Kumar et al., 2002; Delaforge
et al., 2005). Acyl glucuronides have long been known to be
reactive, capable of undergoing reactions such as hydrolysis, rear-
rangement, and covalent binding to proteins, which possibly result
in pharmacological or toxicological effects (Bailey and Dickinson,
2003).
There have been several reports of clinical interactions between
5-(2,5-dimethylphenoxy)-2,2-dimethyl-pentanoic acid (gemfibrozil,
Lopid; Parke-Davis, Ann Arbor, MI) and CYP2C8 substrates such as
cerivastatin, repaglinide, rosiglitazone, and pioglitazone (Backman et
al., 2002; Niemi et al., 2003a,b; Jaakkola et al., 2005). Although in
vitro studies indicate that gemfibrozil is a more potent inhibitor of
CYP2C9 than CYP2C8 (Wen et al., 2001), the results of clinical
studies have shown that gemfibrozil is a more significant inhibitor of
CYP2C8. This discrepancy was explained by demonstrating that the
major metabolite of gemfibrozil, gemfibrozil acyl-␤-glucuronide, is a
potent inhibitor of CYP2C8 based on in vitro studies. These same
authors demonstrated that gemfibrozil acyl-␤-glucuronide inhibits the
CYP2C8-mediated metabolism of cerivastatin in vitro, as well as the
organic anion-transporting peptide mediated uptake of cerivastatin
(Shitara et al., 2004). In subsequent studies, Ogilvie et al. (2006)
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.041947.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: HLM, human liver microsomes; BTFM gemfibrozil, 5-(2,5-bis(trifluoromethyl)phenoxy)-2,2-dimethylpentanoic acid; P450,
cytochrome P450; UDPGA, UDP-glucuronic acid; HPLC, high-performance liquid chromatography; SPE, solid-phase extraction; MS/MS, tandem
mass spectrometry; LC, liquid chromatography; MS, mass spectrometry; ESI, electrospray ionization; Me, methyl.
2421

2422
JENKINS ET AL.
FIG. 1. A, reaction pathway for time-dependent inhibition of
CYP2C8 by gemfibrozil acyl-␤-glucuronide proposed by Baer et al.
(2009). B, reaction pathway for time-dependent inhibition as pro-
posed in this article, indicating that o-Me is the predominant con-
figuration for oxidation to a reactive metabolite.
microsomes (Supersomes) derived from baculovirus-infected insect cells were
obtained from BD Biosciences (Woburn, MA). Liver 9000g supernatant frac-
tion of Aroclor 1254-treated rats (ArRS9) was purchased from Moltox Inc.
(Boone, NC). Alamethicin, bovine serum albumin, NADPH, and UDPGA
were obtained from Sigma-Aldrich. MgCl2 was purchased from Ambion
(Austin, Texas). NH4OAc was purchased from EM Science (Cherry Hill, NJ).
HPLC-grade acetonitrile and water were purchased from Mallinckrodt Baker
(Phillipsburg, NJ). All other reagents were obtained from commercial sources.
Synthesis of Gemfibrozil Analogs and Isolation of Glucuronide Conju-
gates. See supplemental data and Fig. 2.
Time-Dependent Inhibition of CYP2C8 in HLM. Test compounds and
positive controls (60 nl, in dimethyl sulfoxide) at 10 concentrations were
incubated with 15 ␮l of HLM (0.1 mg/ml HLM, 1 mM NADPH, 100 mM
potassium phosphate buffer, pH 7.4, and 2 mM MgCl2) for 30 min (or as
specified) at 37°C. This mixture was then diluted with an equal volume (15 ␮l)
of substrate mixture (4 ␮M amodiaquine, 1 mM NADPH, 100 mM potassium
phosphate buffer, pH 7.4, and 2 mM MgCl2), and the reaction continued for 10
min at 37°C. To determine the IC50 value of the test compounds at the 0-min
The purpose of this study was to determine whether the acyl-␤-
glucuronide of gemfibrozil is a unique mechanism-based inhibitor of
CYP2C8 or whether other glucuronide conjugates are capable of
inhibiting this enzyme. Several compounds were evaluated as their
acyl glucuronide conjugates in this regard. As follow-up to the work
of Baer et al. (2009), gemfibrozil analogs and their glucuronide
conjugates were synthesized and evaluated as inhibitors of CYP2C8
activity in HLM (Fig. 2). Molecular modeling was also conducted to
better understand how the gemfibrozil acyl-␤-glucuronide and its
analogs interact with the CYP2C8 active site.
Materials and Methods
Chemicals and Reagents. Amodiaquine, clotrimazole, diclofenac, gemfi-
brozil, ibuprofen, indomethacin, ketoconazole, ketoprofen, mefenamic acid,
phenelzine, saccharic acid lactone, and simvastatin were purchased from
Sigma-Aldrich (St. Louis, MO). Atorvastatin, dihydroketoprofen, montelukast,
and all glucuronide conjugates (except those prepared below) were purchased
from Toronto Research Chemicals (North York, ON, Canada). (R)- and (S)- time point, test compounds were first mixed with 15 ␮l of the substrate mixture
Naproxen were purchased from VWR (Bridgeport, NJ). HLM and P450
and then were immediately diluted with an equal volume (15 ␮l) of HLM
FIG. 2. Chemical preparation of gemfibrozil
analogs. DMF, dimethylformamide.

INHIBITION OF CYP2C8 BY GEMFIBROZIL GLUCURONIDE ANALOGS
2423
mixture and incubated for 10 min at 37°C. At the end of 10-min incubation,
photodiode-array detector (scanning 200–600 nm at 5 Hz; Agilent) and then
these reactions were terminated by the addition of 30 ␮l of quench buffer (94% to a LCQ Deca Xp Plus ion trap mass spectrometer (Thermo Fisher Scientific).
water-5% acetonitrile-1% formic acid) containing internal standard (0.15 ␮M The electrospray ion source was operated in negative ionization mode with
[2H5]-N-desethylamodiaquine).
data-dependent product ion scanning. The product ion MSn spectra were
obtained by collision-induced dissociation using normalized energy of 35%
and an isolation width of 3 Da.
In one experiment, the test compounds were first preincubated (30 min at
37°C) with alamethicin-treated (25 ␮g/ml) HLM (0.1 mg/ml) with and without
2 mM UDPGA, followed by addition of NADPH (1 mM final) and further
incubation for 0, 15, and 30 min at 37°C. At the end of the incubation time with
NADPH, the reactions were continued by addition of the probe substrate
mixture, containing 4 ␮M amodiaquine, 1 mM NADPH, 100 mM phosphate
buffer (pH 7.4), and 2 mM MgCl2. After 10 min of incubation at 37°C, the
reaction was terminated by addition of the quench buffer containing internal
standard (described above).
Immediately before sample analysis, the denatured protein was precipitated
by centrifugation for 10 min at 2500g, and the supernatant was used to perform
sample analysis. The amount of N-desethylamodiaquine produced in each
reaction was determined by RapidFire mass spectrometry (BIOCIUS Life
Sciences, Wakefield, MA) using on-line solid-phase extraction (SPE) with
tandem mass spectrometry (MS/MS). Four compounds, known CYP2C8 in-
hibitors, were tested as positive controls in each experiment. They included
three reversible CYP2C8 inhibitors (montelukast, clotrimazole, and ketocona-
zole) and one time-dependent inhibitor (phenelzine).
Metabolic Profiling. Gemfibrozil, gemfibrozil glucuronide, 5-(2,5-bis(tri-
fluoromethyl)phenoxy)-2,2-dimethylpentanoic acid (BTFM gemfibrozil), and
BTFM gemfibrozil glucuronide (50 ␮M) were individually incubated at 37°C
with pooled HLM (1 mg/ml) or recombinant CYP2C8 (ϳ50 pmol/ml) in
phosphate buffer (100 mM, pH 7.4). Reactions were started by addition of
NADPH (3 mM) in phosphate buffer. Sample aliquots were taken at various
times: 0, 30, and 60 min. Reactions were quenched by addition of equal
volumes of acetonitrile containing 4% formic acid, and the precipitated pro-
teins were removed by centrifugation (1000g for 5 min). The supernatant was
analyzed by direct injection onto the LC-UV-MSn system for metabolic
profiling.
Quantification of N-desethylamodiaquine (CYP2C8 inhibition). The amount
of substrate probe metabolite (N-desethylamodiaquine) was measured using
SPE-MS/MS analysis on a RapidFire MS/MS system, which consisted of a
RapidFire 200 HT System (BIOCIUS Life Sciences) and a 4000 QTRAP
hybrid triple quadrupole linear ion trap mass spectrometer (AB Sciex, Foster
City, CA) using a Turbo V source with an ESI probe. The samples (20 ␮l) were
loaded onto the BIOCIUS extraction SPE C4 column with mobile phase A
(water with 0.01% trifluoroacetic acid-0.09% formic acid) at 1.5 ml/min for
3.2 s and were eluted with mobile phase B (CH3CN with 0.01% trifluoroacetic
acid-0.09% formic acid) at 1.25 ml/min for 3 s, followed by a reequilibration
of the SPE cartridge with mobile phase A at 1.5 ml/min for 0.5 s. The total
cycle time was ϳ10 s/injection. The SPE eluent was then introduced to a 4000
QTRAP mass spectrometer. Selected reaction monitoring was used for analysis
of metabolite and internal standard. The selected reaction monitoring instru-
ment parameters were as follows: ESI in positive ion mode; m/z 328.2 3 282.8
transition for N-desethylamodiaquine; m/z 331.2 3 282.8 transition for [2H5]-N-
desethylamodiaquine; 55 V declustering potential; 26 eV collision energy; and 80
ms dwell time. The acquired data were processed with RapidFire peak integration
software, and the results were exported as an Excel file for IC50 calculation.
Data Analysis. The signal intensity of the N-desethylamodiaquine (from
MS/MS analysis) was normalized to the signal of internal standard, [2H5]-N-
desethylamodiaquine; thus, signal intensity in each reaction was expressed as
signal ratio. The sample signal ratios were then normalized to the average
signal ratio of the reactions performed in the absence of the test substance
(solvent control, 0%inhibition) and in the absence of enzyme (background,
100% inhibition). Results were expressed as percentage inhibition of the
enzyme activity with solvent control, calculated as
Analytical Procedures. Synthesis of gemfibrozil analogs. Microwave re-
actions were performed using a CEM Discover microwave (ramp, 1 min;
stirring, On; power Max, On). Column chromatography was performed using
a flash chromatography system (Biotage, LLC, Charlotte, NC). HPLC analysis
was performed using a ProStar diode array detector (model 335; Varian, Inc.,
Palo Alto, CA) with ProStar pumps (model 215; Varian) with 25-ml pump
heads and a SunFire C18 column (3.5 ␮m, 4.6 ϫ 150 mm; Waters, Milford,
MA). High-resolution mass spectrometry analysis was performed with an
Exactive Fourier transform Orbitrap mass spectrometer (Thermo Fisher Sci-
entific, Waltham, MA) in negative ionization electrospray mode using Xcali-
bur software (Xcalibur, Inc., Reston, VA). Proton NMR spectra were recorded
on a 400-MHz DPX400A spectrometer (Bruker, Newark, DE).
S Ϫ B
%Inhibition ϭ 1 Ϫ
ꢀ 100
ͩ ͪ
ͫ
ͬ
T Ϫ B
where S is sample, T is average solvent control, and B is average background signals.
The results were then imported into in-house curve fitting software (Curve-
Master), which uses MathIQ (ID Business Solutions, Ltd., Guildford, Surrey,
UK) to determine the IC50 value for each test compound. The IC50 is defined
as the concentration corresponding to 50% inhibition of the enzyme activity
observed with solvent control, derived from the fitted 10-point curve as
determined using a four-parameter logistic regression model:
Isolation of glucuronides. In the course of isolation, chromatographic frac-
tions were screened by LC/MS for glucuronide products with a Thermo
Finnigan Deca LCQ LC/MS system and SunFire C18 column (5 ␮m, 4.6 ϫ
150 mm). The mobile phase was 10 mM NH4OAc with CH3CN (95:5; solvent
A) and 10 mM NH4OAc with CH3CN (5:95; solvent B). Conditions were as
follows: start, 85:15 (A:B) to 0:100 (A:B) over 25 min, held at 100% solvent
B for 5 min, and then back to the initial conditions over 2 min (32 min total);
flow rate, 1.2 ml/min; wavelength, 254 nm; and ESI-MS, negative ion mode.
NMR data were recorded in CD3OD (5-mm tube) using a Bruker Avance III
500 MHz spectrometer.
Metabolite identification. HPLC analyses were performed using an 1100
series separation module (Agilent Technologies, Santa Clara, CA) and a
Synergi Hydro-RP column (2.0 ϫ 150 mm, 4 ␮m; Phenomenex, Torrance,
CA) maintained at room temperature. The mobile phase consisted of a 98:2
mixture of 10 mM NH4OAc and CH3CN (solvent A) and a 70:30 mixture of
CH3CN and CH3OH (solvent B). Separations of components present in the
incubation mixture was achieved under the following gradient conditions:
initially solvents were held isocratically at 0% solvent B for 1 min, followed
by a linear gradient to 100% solvent B over 30 min. Solvent B was then held
B Ϫ A
Y ϭ A ϩ
ͫ ͬ
1 ϩ (C/X)D
where A is minimum inhibition, B is maximum inhibition, X is inhibitor
concentration, C ϭ X, at which Y ϭ Aϩ (B Ϫ A)/2 (i.e., concentration of
inhibitor at which half of the maximal inhibition is observed), and D is the Hill
coefficient (slope). IC50 values (where %inhibition ϭ 50) for each compound
at each time point were determined. IC50 values and percent inhibition at the
highest concentration are reported for each tested time point.
Molecular Modeling. All compounds were sketched using ChemDraw and
converted to three-dimensional structures using Omega (version 2.2; OpenEye
Scientific Software, LLC, Santa Fe, NM). The structures were minimized
using Batchmin (Schro¨dinger, Inc., New York, NY) using the OPLS2005
forcefield with implicit solvation. The CYP2C8 X-ray crystal structure was
retrieved from the Research Collaboratory for Structural Bioinformatics (Pro-
tein Data Bank code 2NNI). The protein was prepared with the Protein Prep
Wizard within Maestro (version 8.5; Schro¨dinger, Inc.) using the standard
defaults for metal treatment and amino acid deletions, among others. Docking
was performed with Glide (Schro¨dinger, Inc.) using flexible conformations for
isocratic at 100% for 3 min. Thereafter, solvents were immediately brought the ligand. Grids were prepared using the Glide Grid generation tool by
back to the initial conditions, 0% solvent B in 0.1 min and reequilibrated
column for 4.9 min with a total chromatographic run time of 38 min.
The eluent from the HPLC column was routed in-line to an 1100 series
selecting montelukast to specify the binding site. No constraints were applied
for docking. Poses were evaluated using the Glide score and by visual inspec-
tion. Poses that included an interaction with the heme group were preferred.

2424
JENKINS ET AL.
point [IC50(30) ϭ 1.4 ␮M]. These results are similar to those reported
Results
by Ogilvie et al. (2006): IC50(0) ϭ 24 Ϯ 5 ␮M and IC50(30) ϭ 1.8 Ϯ
0.5 ␮M. No time/mechanism-dependent effect was observed when
gemfibrozil itself was tested in this assay [IC50(0)/IC50(30) ratio ϳ1.0],
also similar to results reported by Ogilvie et al. Therefore, gemfibrozil
itself is not a metabolism-dependent inhibitor of CYP2C8. Controls
for the CYP2C8 inhibition experiments included three reversible
inhibitors, montelukast (IC50 ϭ 0.03 ␮M), clotrimazole (IC50 ϭ 0.7
␮M), and ketoconazole (IC50 ϭ 2.8 ␮M), and one time-dependent
Effect of Various Glucuronide Conjugates on CYP2C8 Activity.
The acyl glucuronide conjugates of 11 compounds were evaluated as
direct-acting and time/mechanism-dependent inhibitors of CYP2C8
activity in HLM. Activities were assessed with a marker substrate
(amodiaquine) at 2 ␮M, a concentration close to its reported Km of
1.86 ␮M (Walsky and Obach, 2004). The results are summarized in
Table 1. Of the compounds tested, only gemfibrozil acyl-␤-glucuro-
nide elicited a time/mechanism-dependent effect, with a 16-fold shift
in IC50 at the 0-min time point [IC50(0) ϭ 21 ␮M] to the 30-min time inhibitor, phenelzine [IC50(0) ϭ Ͼ200 ␮M, IC50(30) ϭ 60 ␮M].
TABLE 1
IC50 values for inhibition of CYP2C8-catalyzed amodiaquine N-deethylation by various glucuronide conjugates after incubation with NADPH-fortified HLM for 0 and
30 min
a
Average IC50
Structure
Substance
Ratio: IC50(0)/IC50(30)
IC50(0)
IC50(30)
␮M
CH3
O
OH
Gemfibrozil
120 Ϯ 10
110 Ϯ 10
1.1
16
O
CH3
HO
O
CH3
O
OH
O
O
OH
Gemfibrozil acyl-␤-D-glucuronide
21 Ϯ 1.3
1.4 Ϯ 0.03
OH
HO
O
CH3
CH3
O
O
CH3
O
Simvastatin acyl-␤-D-glucuronide
Mefenamic acyl-␤-D-glucuronide
3.8 Ϯ 0.5
8.5 Ϯ 0.9
3.5 Ϯ 0.4
8.4 Ϯ 0.8
1.1
1.0
O
H3C
CH3
OH
O
OH
OH
CH3
OH OH
O
HO
O
O
OH
O
OH
OH
NH
O
CH3
CH3
Cl
HO
NH
O
OH
OH
Diclofenac acyl-␤-D-glucuronide
rac-Ketoprofen acyl-␤-D-glucuronide
Indomethacin acyl-␤-D-glucuronide
14 Ϯ 0.7
26 Ϯ 0.7
26 Ϯ 2.2
9.6 Ϯ 0.5
22 Ϯ 0.9
26 Ϯ 2.3
1.4
1.1
1.0
Cl
O
O
OH
O
O
HO
OH
O
CH3
O
O
OH
OH
O
Cl
HO
OH
CH3
O
O
O
N
O
O
OH
OH
O
OH
HO
O
H3
C
CH3
O
O
OH OH
OH
OH
O
N
N
H
OH
Atorvastatin acyl-␤-D-glucuronide
Ibuprofen acyl-␤-D-glucuronide
(R)-Naproxen acyl-␤-D-glucuronide
45 Ϯ 3.7
35 Ϯ 3.1
1.3
Ͼ1.0
Ͻ1.0
O
F
HO
OH
CH3
O
O
Ͼ100 (39 Ϯ 4)b
Ͼ100 (27 Ϯ 6)b
Ͼ100 (47 Ϯ 4)b
Ͼ100 (37 Ϯ 4)b
OH
O
CH3
OH
O
H3C
HO
HO
OH
CH3
O
O
OH
O
OH
O
O
HO
CH3
O
OH
OH
O
O
(S)-Naproxen acyl-␤-D-glucuronide
Ͼ100 (17 Ϯ 6)b
Ͼ100 (1 Ϯ 5)b
Ͼ100 (16 Ϯ 7)b
Ͼ100 (5 Ϯ 6)b
1.0
1.0
OH
HO
O
O
HO
OH
OH
CH3
O
O
OH
O
Dihydro-ketoprofen acyl-␤-D-glucuronide
OH
O
a Data are reported as the mean Ϯ S.D. of n ϭ 4 determinations. IC50(0) and IC50(30) represent the IC50 determined after 0 and 30 min of incubation with NADPH-fortified HLM.
b %inhibition observed at 100 ␮M Ϯ S.D.

INHIBITION OF CYP2C8 BY GEMFIBROZIL GLUCURONIDE ANALOGS
2425
Control values were in agreement with published results (Polasek et (ibuprofen glucuronide, (R)- and (S)-naproxen glucuronide, and dihy-
al., 2004; Walsky et al., 2005) and were included for all CYP2C8 droketoprofen glucuronide) (Table 1).
inhibition experiments.
The aglycone (parent) compounds were also tested as inhibitors
Although no other compounds were time-dependent inhibitors of of CYP2C8 (Table 2). Simvastatin, mefenamic acid, and atorva-
CYP2C8 as their acyl glucuronides, three could be considered statin were moderate to weak inhibitors of CYP2C8, with IC50
“strong” inhibitors (simvastatin glucuronide Ͼ mefenamic glucuro- values similar to those of their glucuronide conjugates. Ibuprofen,
nide Ͼ diclofenac glucuronide) with IC50 values less than 20 ␮M and (R)- and (S)-naproxen, and dihydroketoprofen did not show
greater than 90% inhibition. Three compounds were “weak” inhibitors CYP2C8 inhibition, also similar to their glucuronide conjugates.
(ketoprofen glucuronide Ͼ indomethacin glucuronide Ͼ atorvastatin However, the glucuronide conjugates of diclofenac and indometh-
glucuronide) with IC50 values between 25 and 50 ␮M and greater than acin were approximately 2-fold more potent as CYP2C8 inhibitors
65% inhibition. Four compounds did not exhibit CYP2C8 inhibition compared with the corresponding aglycones. The glucuronide con-
TABLE 2
IC50 values for inhibition of CYP2C8-catalyzed amodiaquine N-deethylation by various aglycones after incubation with NADPH-fortified HLM for 0 and 30 min
a
Average IC50
Structure
Substance
Ratio: IC50(0)/IC50(30)
IC50(0)
IC50(30)
␮M
CH3
CH3
O
OH
Gemfibrozil
83 Ϯ 7.1
102 Ϯ 4.0
0.8
O
CH3
O
O
CH3
Simvastatin
8.3 Ϯ 1.1
14.9 Ϯ 3.0
21.9 Ϯ 1.3
10.2 Ϯ 1.4
22.0 Ϯ 1.8
22.5 Ϯ 1.8
0.8
0.7
1.0
H3C
CH3
OH
CH3
OH OH
O
H3C
N
H
Mefenamic acid
Atorvastatin
CH3
O
OH
H3C
CH3
N
OH
O
OH
O
OH
N
H
F
HO
O
Cl
H
Diclofenac
54 Ϯ 11
57 Ϯ 8
0.9
N
Cl
Cl
O
N
Indomethacin
Dihydroketoprofen
Ibuprofen
88 Ϯ 16
Ͼ200 (46 Ϯ 6)b
Ͼ200 (44 Ϯ 3)b
Ͼ200 (23 Ϯ 1)b
Ͻ0.45
1.0
CH3
OH
O
OH
O
OH
CH3
OH
Ͼ200 (41 Ϯ 4)b
Ͼ200 (23 Ϯ 3)b
O
CH3
OH
CH3
1.0
O
H3C
O
CH3
OH
Ketoprofen
Ͼ200 (14 Ϯ 3)b
Ͼ200 (17 Ϯ 5)b
Ͼ200 (6 Ϯ 5)b
Ͼ200 (4 Ϯ 4)b
Ͻ1.0
Ͻ1.0
O
CH3
OH
(R)-Naproxen
HO
HO
O
O
O
CH3
OH
(S)-Naproxen
Ͼ200 (9 Ϯ 3)b
Ͼ200 (9 Ϯ 3)b
1.0
O
a Data are reported as the mean Ϯ S.D. of n ϭ 4 determinations. IC50(0) and IC50(30) represent the IC50 determined after 0 and 30 min of incubation with NADPH-fortified HLM.
b %inhibition observed at 200 ␮M Ϯ S.D.

2426
JENKINS ET AL.
jugate of ketoprofen showed the greatest inhibition (versus its absence of NADPH at 0 min, gemfibrozil acyl-␤-glucuronide did
aglycone), with a greater than 5-fold lower IC50. No time- not demonstrate CYP2C8 inhibition, but showed increasing inhi-
dependent CYP2C8 inhibition was observed for the parent agly- bition after a longer preincubation time in the presence of NADPH
cones.
[IC50(30) ϭ 4.0 ␮M], indicating the dependence on metabolism for
Metabolism-Dependent Inhibition of CYP2C8 by Gemfibrozil inhibition. Although the metabolism of gemfibrozil to gemfibrozil
and BTFM Analog. Gemfibrozil, BTFM gemfibrozil, and gemfi- acyl-␤-glucuronide was not monitored in this experiment, it ap-
brozil acyl-␤-glucuronide were preincubated with HLM in the pears that incomplete formation of the glucuronide conjugate oc-
presence and absence of UDPGA and NADPH (0, 15, and 30 min) curred, as indicated by the discrepancy in IC50 in the two experi-
before addition of amodiaquine to monitor CYP2C8 activity (data ments. These results indicate that the glucuronide conjugate has to
not shown). BTFM gemfibrozil was a CYP2C8 inhibitor (IC50 be prepared before its incubation with HLM and that insufficient
15–26 ␮M), but the inhibition was not dependent on UDPGA or glucuronide would be formed in a reaction mixture containing the
NADPH, indicative of direct (reversible) inhibition of CYP2C8 by aglycone and cofactor-fortified HLM. For this reason, glucu-
this analog. In the presence of UDPGA and NADPH, gemfibrozil ronides of gemfibrozil analogs were biosynthesized from their
was a time/mechanism-dependent inhibitor of CYP2C8 after 30 respective aglycones, either by incubation with HLM (BTFM
min of incubation [IC50(30) ϭ 25 ␮M], indicating that glucuroni- gemfibrozil) or rat liver ArRS9 (o-Me gemfibrozil and m-Me
dation of gemfibrozil is necessary for inhibition of CYP2C8. In the gemfibrozil) in the presence of UDPGA.
TABLE 3
IC50 values for inhibition of CYP2C8-catalyzed amodiaquine N-deethylation by gemfibrozil analogs and their glucuronide conjugates after incubation with NADPH-
fortified HLM for 0 and 30 min
a
Average IC50
Structure
Substance
Ratio: IC50(0)/IC50(30)
IC50(0)
IC50(30)
␮M
CH3
O
OH
Gemfibrozil
72 Ϯ 7.2
72 Ϯ 6.2
1.0
O
CH3
CH3
HO
O
O
OH
O
O
O
OH
Gemfibrozil acyl-␤-D-glucuronide
BTFM gemfibrozil
13 Ϯ 2.7
13 Ϯ 2.1
37 Ϯ 3.2
1.0 Ϯ 0.06
17 Ϯ 2.5
43 Ϯ 3.1
13
OH
CH3
CF3
O
OH
0.7
O
CF3
HO
OH
O
OH
OH
CF3
O
O
O
BTFM gemfibrozil acyl-␤-D-glucuronide
Ͻ1.0
O
CF3
CH3
O
OH
o-Me gemfibrozil
Ͼ80 (15 Ϯ 7)b
Ͼ80 (14 Ϯ 7)b
17 Ϯ 0.3
1.0
3.2
O
HO
O
OH
OH
CH3
O
o-Me gemfibrozil acyl-␤-D-glucuronide
55 Ϯ 6
O
O
OH
O
O
OH
m-Me gemfibrozil
Ͼ80 (10 Ϯ 6)b
Ͼ120 (29 Ϯ 6)c
Ͼ80 (9 Ϯ 5)b
1.0
O
CH3
HO
OH
O
O
OH
O
O
Ͼ120 (40 Ϯ 3)c
Ͼ1.0
OH
m-Me gemfibrozil acyl-␤-D-glucuronide
O
CH3
a Data are reported as the mean Ϯ S.D. of n ϭ 4 determinations. IC50(0) and IC50(30) represent the IC50 determined after 0 and 30 min of incubation with NADPH-fortified HLM.
b %inhibition observed at 80 ␮M Ϯ S.D.
c %inhibition observed at 120 ␮M Ϯ S.D.

INHIBITION OF CYP2C8 BY GEMFIBROZIL GLUCURONIDE ANALOGS
2427
Effect of Gemfibrozil and Analog Glucuronide Conjugates on (HLM) are shown (Fig. 4C). The structures of the metabolites were
CYP2C8 Activity. The IC50 values of the gemfibrozil analogs as well assigned on the basis of their MSn product ion mass spectra compared
as their glucuronide conjugates were evaluated for time/mechanism- with that of gemfibrozil acyl-␤-glucuronide (Fig. 4). Gemfibrozil
dependent inhibition as shown in Table 3. As observed previously, acyl-␤-glucuronide gave a protonated molecular ion at m/z 425. Sub-
gemfibrozil acyl-␤-glucuronide behaved as a time- and metabolism- sequent MS2 fragmentation gave a daughter ion at m/z 249 indicating
dependent inhibitor of CYP2C8. In comparison, BTFM gemfibrozil loss of glucuronide moiety (176 Da). Further fragmentation (MS3) of
was an inhibitor of CYP2C8 (IC50 of 13–17 ␮M), but its acyl-␤- the ion at m/z 249 gave rise to an ion at m/z 121, which corresponded
glucuronide was not a time-dependent inhibitor after a 30-min prein- to the dimethyl-phenoxy part of the molecule. The three metabolites,
cubation with NADPH. The o-Me and m-Me gemfibrozil analogs M1, M2, and M3, produced similar full-scan mass spectra with a
exhibited almost no CYP2C8 inhibition and their corresponding acyl- deprotonated molecular ion at m/z 441, a mass 16 Da higher than
␤-glucuronides demonstrated weak inhibition without preincubation gemfibrozil acyl-␤-glucuronide, denoting that a single oxidation had
with NADPH (Fig. 3). However, after a 30-min preincubation, the occurred. The MS2 spectrum of M1, M2, and M3 produced an
o-Me and m-Me gemfibrozil acyl-␤-glucuronides trended toward abundant ion at m/z 265 (loss of glucuronide moiety). MS3 fragmen-
time-dependent inhibition, with the o-Me gemfibrozil glucuronide tation of m/z 265 produced an ion at m/z 137, implying that oxidation
more potent an inhibitor than the m-Me gemfibrozil glucuronide occurred on the dimethyl-phenoxy moiety for all three metabolites.
(Table 3).
An attempt was made to further characterize these metabolites, but
Metabolism of Gemfibrozil Acyl-␤-Glucuronide and BTFM efforts were unsuccessful because of the very low amounts in the
Gemfibrozil Glucuronide in the Presence of HLM and Human incubates. Under conditions in which gemfibrozil acyl-␤-glucuronide
Recombinant CYP2C8. Gemfibrozil acyl-␤-glucuronide was stable metabolites M1 to M3 were detected, no metabolites of BTFM gem-
to metabolism in the presence of both NADPH-fortified HLM and fibrozil glucuronide were detectable (data not shown).
recombinant CYP2C8 (Ն98% remaining after 60 min of incubation).
Computational Docking Studies. Computational docking studies
Only three minor (Ͻ1%) metabolites, M1, M2, and M3, were detected were performed to investigate the orientation of the gemfibrozil
by HPLC-UV-MS in both incubations; representative chromatograms acyl-␤-glucuronide in the active site of CYP2C8 (Fig. 6). Docking
poses with the o-Me position oriented toward the ␥-meso position of
the heme were superior to those with the m-Me oriented toward the
heme. Both orientations include numerous hydrogen bonds between
the glucuronide and the CYP2C8 active site. The carboxylate of the
glucuronide forms hydrogen bonds to side chains of Asn217 and
Ser100 and to the backbone NH of Ser103. Additional hydrogen
bonds are formed to Gln214 and Ser100. The pose orienting the o-Me
toward the heme (Fig. 5A) forms numerous hydrophobic contacts to
the site, including Thr301, Ile113, Val366, Ser114, and Val477. In
addition, the 5-position methyl fits into small hydrophobic recess
between Val477 and Phe205, contributing to the orientation of the
o-Me position toward the heme.
In addition to the glucuronide, gemfibrozil itself was also docked
into CYP2C8. The docking pose (Fig. 5C) for gemfibrozil (shown
with slate blue carbons) is substantially different compared with that
for the glucuronide metabolite. The pose of gemfibrozil acyl-␤-
glucuronide is shown for reference with cyan carbons. The only
contact residue in common for the poses is Ser103. The dimethyl-
phenyl ring of gemfibrozil is placed in a largely hydrophobic pocket
formed by Ile106, Phe201, Val237, and Ala292. Because gemfibrozil
is not a particularly potent inhibitor of CYP2C8 (Tables 2 and 3), it is
likely that multiple weak binding poses are probable.
Inhibition of CYP2C8 (IC50 ϳ10 ␮M) was observed with mefe-
namic acyl-␤-glucuronide and diclofenac acyl-␤-glucuronide, so the
docking studies described herein were expanded to include these two
glucuronides. In both cases, poses were generated forming hydrogen
bonds to the glucuronide similar to those for the gemfibrozil acyl-␤-
glucuronide. In addition, good hydrophobic contacts are formed with
hydrophobic residues proximal to the heme. However, in neither case
were any functional groups oriented toward the heme, which would
imply probable mechanism-based inhibition through covalent attach-
ment. The methyl substituents of mefenamic acyl-␤-glucuronide were
oriented away from the heme in the favored binding pose (Fig. 6). A
similar result was obtained with diclofenac acyl-␤-glucuronide (data
not shown).
Discussion
FIG. 3. IC50 curves for inhibition of CYP2C8-mediated amodiaquine N-deethyla-
tion by gemfibrozil and analog compounds and their respective acyl-␤-D-gluc-
uronides after incubation with NADPH-fortified HLM for 0 min (E) and 30 min (ꢁ).
Although it is known that gemfibrozil acyl-␤-glucuronide is a
mechanism-based inhibitor of CYP2C8 (Ogilvie et al., 2006), it is not

2428
JENKINS ET AL.
FIG. 4. Negative LC-MS/MS analysis of (A)
gemfibrozil acyl-␤-glucuronide (B) oxidative
metabolite after incubation with NADPH-forti-
fied HLM for 60 min (Gluc, glucuronide) and
(C) UV chromatogram (272 nm) of gemfibrozil
acyl-␤-glucuronide oxidative metabolites after
incubation with HLM and NADPH for 60 min.
Metabolites were also observed using recombi-
nant CYP2C8 (results not shown).
known whether gemfibrozil acyl-␤-glucuronide is unique in its ability of UDPGA and NADPH. However, generation of the glucuronide in
to inhibit CYP2C8 or whether other acyl glucuronides are able to the incubation resulted in less potent inhibition of CYP2C8 compared
inhibit this enzyme, either reversibly or mechanistically. Of the acyl with inhibition observed upon incubation with the gemfibrozil acyl-
glucuronide conjugates tested in this study, 7 of 11 showed some ␤-glucuronide (IC50 after 30 min of incubation is 25 versus 4.0 ␮M).
degree of reversible CYP2C8 inhibition. Although some of these It appears that although generation of the glucuronide conjugate in the
compounds show significant inhibition, the impact on CYP2C8 in incubation mixture indicates the presence of time-dependent inhibi-
vivo is not known. For example, Hatorp et al. (2003) have assessed the tion, incubation with the glucuronide conjugate itself is necessary to
impact of simvastatin on the pharmacokinetics of repaglinide. Unfor- accurately determine the time-dependent shift in IC50. Therefore,
tunately, given the role of CYP2C8 and CYP3A4 in the clearance of further studies with gemfibrozil analogs were also conducted with
repaglinide, the results are difficult to interpret. Additional clinical their respective glucuronide conjugates.
data are needed to place the results described herein in context. It is
NADPH was also required to observe time-dependent inhibition of
worth noting that when the aglycone (parent) compounds were tested CYP2C8 in the presence of gemfibrozil acyl-␤-glucuronide, indicat-
for CYP2C8 inhibition, the IC50 values were similar to those of the ing that the gemfibrozil glucuronide is probably oxidized by P450.
corresponding glucuronide conjugates, indicating that glucuronide Such a hypothesis is supported by the fact that incubation of gemfi-
conjugates may fit into the CYP2C8 pocket just as well as the parent brozil acyl-␤-glucuronide with NADPH-fortified HLM produced
molecule. Also noteworthy is the fact that ketoprofen acyl glucuronide three minor hydroxylation products (Fig. 4). It is unfortunate that
was even more potent than ketoprofen itself. Ketoprofen acyl gluc- turnover was low and the oxidative metabolites could not be further
uronide is present in human plasma and excreted in the urine (Upton characterized (metabolism on the benzylic moiety of gemfibrozil is
et al., 1980), although CYP2C8 inhibition in a clinical setting has not implicated). CYP2C8-catalyzed oxidation of glucuronide is not a
been reported. Only gemfibrozil acyl-␤-glucuronide was a time-de- novel finding, because it has been known for some time that the
pendent inhibitor of CYP2C8, indicating that there is a particular enzyme is capable of hydroxylating estradiol-17␤-glucuronide (Dela-
metabolic liability for this glucuronide conjugate; gemfibrozil itself forge et al., 2005). After a thorough docking of the molecule within
was not a time-dependent inhibitor of CYP2C8 (Table 1).
the CYP2C8 crystal structure, the same authors concluded that the
This study confirms the previous finding that gemfibrozil causes active site is large enough to accommodate a glucuronide conjugate.
time-dependent inhibition of CYP2C8 when incubated in the presence Likewise, Kumar et al. (2002) have described CYP2C8-mediated

INHIBITION OF CYP2C8 BY GEMFIBROZIL GLUCURONIDE ANALOGS
2429
In contrast to what Baer et al. (2009) had observed in their com-
putational docking studies, the orientation of the o-Me position of the
gemfibrozil acyl-␤-glucuronide appears to fit better in the CYP2C8-
binding pocket compared with the m-Me position, based on our
modeling studies (Fig. 6). The pose of orientation of the o-Me position
with the methyl group oriented toward the heme appears to fit better
in the CYP2C8-binding pocket compared with the m-Me being ori-
ented toward the heme position. It appears that more hydrophobic
interactions are present in the o-Me analog-oriented pose than in the
m-Me-oriented pose. The loss of potency of the o-Me glucuronide
analog compared with gemfibrozil acyl-␤-glucuronide may be due
to additional rotational freedom of the ortho-phenyl position in
the active site. It appears that the additional methyl in the meta
position allows the molecule to fit into a shallow pocket of the
CYP2C8 molecule and appears to limit the rotation of the phenyl ring.
Thus, the proposed reaction pathway for gemfibrozil acyl-␤-glucuro-
nide more likely involves oxidation of the o-Me position leading to
covalent binding and inactivation of the CYP2C8 (Fig. 1B), rather
than a lack of a favored oxidation position as indicated by Baer et al.
(2009) (Fig. 1A). It is also possible that a dimethide analog could be
formed as a reactive intermediate, which would help explain the loss
of potency in the mono-methyl analogs, but there is no current
evidence to indicate that this is occurring.
Although most glucuronide conjugates are eliminated without in-
hibiting major metabolic pathways, some have the potential to bring
about drug-drug interactions (Faed 1984). Therefore, the 2008 U.S.
Food and Drug Administration safety guidelines state that if a drug
conjugate is reactive (e.g., acyl glucuronide), then additional safety
assessment may be needed (U.S. Food and Drug Administration,
2008). Although P450 inhibition screening could be part of such an
assessment, such studies are not conducted routinely (Bode 2010). It
has been suggested that glucuronides may be generated by incubation
of a test compound with alamethicin-treated (UDPGA-fortified) HLM
to form the glucuronide, followed by incubation with NADPH and a
probe substrate(s) to assess inhibition of one or more P450s (Ogilvie
et al., 2006; Bode, 2010). However, a more accurate determination of
FIG. 5. Gemfibrozil acyl-␤-glucuronide orientation in CYP2C8 orientation of o-Me
toward heme (A) and orientation of m-Me toward heme (B). Key contact residues
are highlighted in the figure. C, pose of gemfibrozil (slate blue carbons) in CYP2C8.
The pose of gemfibrozil acyl-␤-glucuronide from A is shown for spatial reference
(cyan carbons). Note that the only contact residue in common is a hydrogen bond
to Ser103.
glucuronide metabolism: oxidation of diclofenac acyl glucuronide to
4Ј-hydroxy diclofenac acyl glucuronide. On the basis of the results
presented herein, such metabolism does not render pronounced time-
dependent inhibition of CYP2C8 in HLM (Table 1), in contrast to
what is observed for gemfibrozil acyl-␤-glucuronide.
In a recent study, Baer et al. (2009) have shown that after incuba-
tion with recombinant CYP2C8, the gemfibrozil acyl-␤-glucuronide is
covalently bound to the heme moiety, on the basis of mass spectrom-
etry and deuterium isotope effects. Oxidation of the aromatic o-Me or
p-Me group to form a benzyl radical was proposed as the reactive
intermediate that binds to the ␥-meso position of the heme. The
authors concluded that because of the rotameric flexibility of the alkyl
chain of gemfibrozil acyl-␤-glucuronide, there was no indication that
either the o-Me or the m-Me would have a favored orientation. To
further probe this hypothesis, three gemfibrozil analogs and their
corresponding glucuronides were evaluated in the CYP2C8 inhibition
assay. These three analogs were BTFM gemfibrozil, o-Me gemfibro-
zil, and m-Me gemfibrozil.
When the gemfibrozil aromatic methyl groups were substituted
with trifluoromethyls to give BTFM gemfibrozil, this compound was
a competitive CYP2C8 inhibitor; however, time-dependent inhibition
of CYP2C8 was not observed either for the analog or the correspond-
ing glucuronide conjugate of this analog. This is to be expected, on the
basis of the hypothesis that oxidation of the methyl groups is neces-
sary for time-dependent inhibition of CYP2C8 and that such metab-
olism can be blocked by substitution with trifluoromethyls. In agree-
ment, turnover of BTFM gemfibrozil and its acyl-␤-glucuronide was
not detectable after incubation with NADPH-fortified HLM (Fig. 5).
Time-dependent inhibition of CYP2C8 was not observed with the
mono-methyl gemfibrozil analogs. However, the glucuronide conju-
gate of o-Me gemfibrozil was a time-dependent inhibitor of CYP2C8.
The o-Me gemfibrozil glucuronide analog appears to be a more potent
CYP2C8 inhibitor than the m-Me gemfibrozil glucuronide analog but
less potent than gemfibrozil acyl-␤-glucuronide itself (Table 3).
FIG. 6. Mefenamic acyl-␤-glucuronide docked into the CYP2C8 active site. The
molecule has binding between the protein and the glucuronide similar to that for
gemfibrozil acyl-␤-glucuronide. The methyl substituents are oriented away from the
heme, making covalent attachment unlikely. No poses were identified that would
orient the methyl groups toward the heme.

2430
JENKINS ET AL.
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time-dependent inhibition would need to be conducted with the iso-
lated glucuronide once mechanism-based inhibition was suspected. In
any case, it appears from this study that if the parent compound is an
inhibitor of CYP2C8, the corresponding acyl glucuronide may also
contribute to the inhibition. Although gemfibrozil acyl-␤-glucuronide
has a relatively unique configuration that renders mechanism-based
inhibition and this mechanism is not applicable to all acyl gluc-
uronides, P450 inhibition (including time-dependent inhibition)
should be evaluated in the presence (and absence) of UDPGA for all
compounds that produce acyl glucuronide conjugates. If inhibition is
observed in the presence of UDPGA, the corresponding acyl glucuro-
nide should be generated and tested for P450 inhibition. Although
mechanism-based inhibition of additional P450s (beyond CYP2C8)
by glucuronides has not been reported to date, in vitro testing of
multiple human P450s is advisable.
Acknowledgments
We gratefully acknowledge helpful discussions and suggestions by Drs.
Michael Sinz and Kenneth Santone (Bristol-Myers Squibb).
Authorship Contributions
Participated in research design: Jenkins, Zvyaga, Burrell, Turley, and
Rodrigues.
Conducted experiments: Zvyaga, Hurley, Turley, Wagner, Leet, and Philip.
Contributed new reagents or analytic tools: Burrell, Turley, and Leet.
Performed data analysis: Jenkins, Zvyaga, Johnson, Hurley, Wagner, and
Philip.
Tornio A, Niemi M, Neuvonen M, Laitila J, Kalliokoski A, Neuvonen PJ, and Backman JT
(2008) The effect of gemfibrozil on repaglinide pharmacokinetics persists for at least 12 h after
the dose: evidence for mechanism-based inhibition of CYP2C8 in vivo. Clin Pharmacol Ther
84:403–411.
Upton RA, Buskin JN, Williams RL, Holford NH, and Riegelman S (1980) Negligible excretion
of unchanged ketoprofen, naproxen, and probenecid in urine. J Pharm Sci 69:1254–1257.
U.S. Food and Drug Administration (2008) Guidance for Industry Safety Testing of Drug
Metabolites. U.S. Department of Health and Human Services, Washington, DC.
Walsky RL, Gaman EA, and Obach RS (2005) Examination of 209 drugs for inhibition of
cytochrome P450 2C8. J Clin Pharmacol 45:68–78.
Wrote or contributed to the writing of the manuscript: Jenkins, Zvyaga,
Johnson, Turley, Leet, Philip, and Rodrigues.
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