Influence of CYP2C8 polymorphisms on the hydroxylation metabolism of paclitaxel, repaglinide and ibuprofen enantiomers in vitro

Article abstract of DOI:10.1002/bdd.1842

CYP2C8 plays an important role in the metabolism of various drugs, such as paclitaxel, repaglinide and ibuprofen. Polymorphisms in the CYP2C8 gene were shown to influence interindividual differences in the pharmacokinetics of paclitaxel, repaglinide and i

Full text of DOI:10.1002/bdd.1842

BIOPHARMACEUTICS & DRUG DISPOSITION
Biopharm. Drug Dispos. 34: 278–287 (2013)
Published online 3 June 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/bdd.1842
Influence of CYP2C8 polymorphisms on the hydroxylation
metabolism of paclitaxel, repaglinide and ibuprofen
enantiomers in vitro
Lushan Yua, Da Shia, Liping Maa, Quan Zhoub, and Su Zenga,
*
aLaboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,
310058, China
bDepartment of Pharmacy, the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang, Zhejiang Province, China
ABSTRACT: CYP2C8 plays an important role in the metabolism of various drugs, such as
paclitaxel, repaglinide and ibuprofen. Polymorphisms in the CYP2C8 gene were shown to influence
interindividual differences in the pharmacokinetics of paclitaxel, repaglinide and ibuprofen enantiomers.
In this study, three CYP2C8 allelic variants (CYP2C8.2, CYP2C8.3 and CYP2C8.4) and wild-type CYP2C8
(CYP2C8.1) were co-expressed for the first time with human cytochrome P450 oxidoreductase (POR) and
cytochrome b5 by using a baculovirus-assisted insect cell expression system. Further, the effects of
genotype–phenotype correlations of CYP2C8 alleles on the metabolism of paclitaxel, repaglinide and
ibuprofen enantiomers were evaluated. The CLint values of CYP2C8.2, CYP2C8.3 and CYP2C8.4 for pac-
litaxel were 47.7%, 64.3% and 30.2% of that of CYP2C8.1 (p< 0.01). The CLint values of CYP2C8.2 and
CYP2C8.4 for repaglinide were 77.9% and 80.2% of that of CYP2C8.1 (p< 0.05), respectively, while the
CLint value of CYP2C8.3 was 1.31-fold higher than that of CYP2C8.1 (p< 0.05). The relative CLint values
of CYP2C8.2, CYP2C8.3 and CYP2C8.4 were 110.5%, 72.3% and 49.7% of that of CYP2C8.1 and were
124.6%, 83.4% and 47.4% of that of CYP2C8.1 for R-ibuprofen and S-ibuprofen, respectively. Comparing
hydroxylation by CYP2C8.1 and CYP2C8.3 resulted in higher and lower intrinsic clearance of repaglinide
and ibuprofen enantiomers, respectively. These in vitro findings were consistent with the pharmacokinetics
in volunteers who were heterozygous or homozygous carriers of CYP2C8*3. The results of this study
provide useful information for predicting CYP2C8 phenotypes and may contribute to individualized drug
therapy in the future. Copyright © 2013 John Wiley & Sons, Ltd.
Key words: CYP2C8; genetic polymorphisms; paclitaxel; repaglinde; ibuprofen enantiomers
Introduction
Fourteen CYP2C8 alleles, from CYP2C8*2 to
CYP2C8*14, have been identified to date. CYP2C8*2,
CYP2C8*3 and CYP2C8*4 are the three most
common alleles. CYP2C8 and some of its variants
have been recombinantly expressed using Escherichia
coli (2C8.1 (R139K), 2C8.1 (K399R), 2C8.3 and 2C8.4)
[2] and yeast expression systems (CYP2C8.2, 2C8.3
and 2C8.4) [3]. Baculovirus-assisted insect cell
expression system is also used widely and offers
many advantages, such as eukaryotic post-
translational modifications without complica-
tion [4], proper protein folding and S–S bond
formation, and high expression levels [5].
Cytochrome P450 2C8 (CYP2C8) comprises 7% of
the total hepatic CYP content and plays an impor-
tant role in the metabolism of many exogenous and
endogenous compounds [1]. CYP2C8 is mainly
expressed in the liver but is also present in the gas-
trointestinal tract, kidney, adrenal gland and tonsils.
*Correspondence to: College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058, China.
E-mail: zengsu@zju.edu.cn
Received 4 November 2012
Revised 4 February 2013
Accepted 18 March 2013
Copyright © 2013 John Wiley & Sons, Ltd.

EFFECT OF CYP2C8S ON PACLITAXEL, REPAGLINIDE AND IBUPROFEN ENANTIOMERS
279
Cytochrome P450 2C8 is involved in the metab-
olism of many clinically available drugs, such as
paclitaxel, repaglinide and ibuprofen [6]. Pacli-
taxel is a probe drug for measuring CYP2C8
activity in vitro; however, the fraction of paclitaxel
eliminated by CYP2C8 was estimated to be only
36% [7]. Clinical studies have reported no associa-
tion between CYP2C8 polymorphisms and pacli-
taxel pharmacokinetics in breast cancer patients
[8]. Repaglinide, an insulin secretagogue used in
the treatment of type 2 diabetes, is almost metab-
olized by CYP2C8 and CYP3A4 which have a
comparable in vitro contribution [9]. When a
single dose of repaglinide was administered to
healthy volunteers, the mean area under the
plasma concentration–time curve (AUC) and the
maximum plasma concentration (Cmax) were both
lower in the subjects with the CYP2C8*1/*3 geno-
type than in the wild-type homozygotes [10–12].
However, the effect of genotype–phenotype correla-
tion of CYP2C8 polymorphisms on repaglinide me-
tabolism has not been evaluated in vitro.
research, such as a correlation between CYP2C8*3
and CYP2C9*2 polymorphisms, differences in the ex-
tent of conversion from R-ibuprofen to S-ibuprofen
in vivo, and few CYP2C8*3 homozygotes. Consider-
ing this, the construction of the recombinant CYP2C8
variants in vitro may be useful for estimating the
functional consequence of genetic variations in the
CYP2C8 gene on ibuprofen metabolism.
In the present study, wild-type CYP2C8 and
its three variants, CYP2C8.2, CYP2C8.3 and
CYP2C8.4, were expressed in the baculovirus-
assisted insect cell expression system to evaluate
the impact of CYP2C8 genetic polymorphisms on
the metabolism of repaglinide and ibuprofen.
Paclitaxel 6a-hydroxylation was used as the
marker reaction. Stereoselective hydroxylation
of ibuprofen by CYP2C8 variants was also
investigated.
Materials and Methods
Although it is known that CYP2C8 genetic
polymorphisms are associated with variable
disposition of certain drugs, their influence on the
metabolism of chiral drugs and the possible effects
on the stereoselectivity has not been determined
in vitro. To better understand the stereoselective me-
tabolism by CYP2C8 variants, ibuprofen enantio-
mers were used as substrates in this study.
Ibuprofen, a widely used chiral nonsteroidal anti-
inflammatory drug (NSAID), is clinically adminis-
tered in the racemic form. R-Ibuprofen is converted
unidirectionally to S-ibuprofen in vivo, and mainly
the S-enantiomer contributes to the pharmacological
activity [13]. Ibuprofen is stereoselectively metabo-
lized to hydroxylated metabolites by CYP2Cs,
and CYP2C8 plays a role in R-ibuprofen (< 10%)
and S-ibuprofen (< 13%) clearance [14]. The pres-
ence of CYP2C8 alleles may influence the pharmaco-
kinetics of R-ibuprofen, which can be converted
to S-ibuprofen, resulting in different dispositions of
both enantiomers. To our knowledge, the effect of
CYP2C8 polymorphisms on the stereoselective hy-
droxylation of ibuprofen enantiomers has not been
investigated to date. Meanwhile, data regarding the
association between CYP2C8 polymorphisms and
ibuprofen pharmacokinetics are conflicting and
inconclusive [15]. Potential explanations for the
discrepancies include inherent factors in in vivo
Materials
Polymerase chain reaction (PCR) primers were
synthesized by Sangon (Shanghai, China). The
pMD19-T vector was purchased from TaKaRa
Biotechnology Co. (Dalian, China). The donor
plasmid pFastBacTM1 vector, DH10 BacTM compe-
tent cells and TRIzolW reagent were purchased
from Invitrogen (Carlsbad, CA, USA). The primary
anti-histidine antibody was purchased from Merck
(Darmstadt, Germany). Paclitaxel, nateglinide,
7-hydroxycoumarin (chemical purity > 99.0%)
were purchased from the National Institutes
for Food and Drug Control (Beijing, China).
R-Ibuprofen and S-ibuprofen were obtained
from Biomol Research Laboratories Inc. (Plymouth
Meeting, PA, USA). 4-Androstene-3,17-dione,
repaglinide and 6a-hydroxy paclitaxel were pur-
chased from Sigma Chemical Co. (St Louis, MO,
USA). All other solvents and chemicals were
obtained from commercial sources.
Construction of human CYP2C8 variant cDNAs
Human liver total RNA was obtained from
human liver tissue by TRIzol reagent. This RNA
was utilized as a template to obtain CYP2C8*1
cDNA by reverse transcription-PCR using the for-
ward primer 50-CCCAAGCTTATGGAACCTTTT
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280
L. YU ET AL.
GTGGTCCTG-30
and the reverse primer
(Amersham Biosciences, USA). The relative expres-
sions of CYP2C8s were analysed using Quantity
One analysis software (Bio-Rad).
50-CCGCTCGAGTCAATGATGATGATGATGATG
GACAGGGATGAAGCAGATC-30. cDNA encoding
CYP2C8*1 tagged with histidine (His6) was
subcloned to pMD19-T vector. Using pMD19-T/
CYP2C8*1 vector as the template, CYP2C8
alleles were generated by site-directed mutagenesis
PCR with the following 50-phosphorylated primers:
50-TATCGATTGCTTCCTGtTCAAAATGGAGCA
GGA-30 for CYP2C8*2, 50-TTGGGATGGGGA
AGAaGAGCATTGAGGACCG-30 and 50-GTG
CTACATGATGACAgAGAATTTCCTAATCCAA
A-30 for CYP2C8*3, 50-ATCCTCGGGACTTTA
TgGATTGCTTCCTGATCA-30 for CYP2C8*4. The
sites of the mutated bases are shown in lowercase
bold. To ensure accuracy during the mutagenesis, all
PCR products were verified by DNA sequencing.
Enzymatic assay of CYP2C8 toward paclitaxel,
regaglinide and ibuprofen enantiomers
Kinetic parameters for the hydroxylation of
paclitaxel, repaglinide and ibuprofen enantiomers
were determined in recombinant CYP2C8.1,
CYP2C8.2, CYP2C8.3 and CYP2C8.4. The incuba-
tion mixture contained the S9 expressing CYP2C8s
(0.25–1.0 mg/ml), the substrate as paclitaxel (1–
30 mM) or repaglinide (0.5–30 mM) or ibuprofen enantio-
mers (16–2000 mM), MgCl2 (15 mM) and NADPH gen-
erating system made up to a final volume (100 ml) with
phosphate buffer solution (0.1 M, pH7.4). Each sample
was pre-incubated for 5 min at 37 ꢁC, initiated by the
addition of NADPH/NADP+ and terminated with
400 ml of internal standard in ice-cold methanol.
Internal standards for paclitaxel, repaglinide and
ibuprofen enantiomers were 4-androstene-3,17-dione,
nateglinide and 7-hydroxycoumarin, respectively. The
reaction mixture was then centrifuged at 16000 ꢀ g
for 20 min. 5 ml of the supernatant was directly
analysed by ultra high performance liquid chromatog-
raphy/tandem mass spectrometry (UPLC-MS).
Recombinant expression of CYP2C8 and its vari-
ants in insect cells (Sf9 cells)
Recombinant expression of CYP2C8 and its variants
was performed in the Bac-to-BacW baculovirus ex-
pression system using the modified procedure de-
scribed by Chen et al. [16]. The Sf9 cells infected
with recombinant baculovirus were harvested after
96 h. The cells were lysed in lysis buffer (20% glycerol,
0.1 M K3PO4, 1mM EDTA and 0.1 mM PMSF, pH 7.4)
by ultrasonication for 30 min. The whole-cell lysate
(S9) was prepared by centrifugation at 9000 ꢀ g for
10 min at 4 ꢁC, and then stored at ꢂ80 ꢁC until analy-
sis. The protein concentrations were measured by the
Bradford method. The functional P450 content was
recorded by the reduced CO difference spectral
method described by Gonzalez et al. [17].
Analytical methods
Quantification of 6a-hydroxy paclitaxel was carried
out by an Agilent 1200 series HPLC system (Agilent
Technologies, Palo Alto, CA, USA). Separation was
performed on a DiamonsilTM C18 column (4.6 mm
200 mm, 5 mm) (Dikma Technologies, Beijing,
China) with methanol–water as the mobile phase.
The elution was performed with a linear gradient
from 60% to 70% methanol (v/v) in 20min at a flow
rate of 1.0 ml/min. The detection wavelength was
set at 230 nm.
A Waters UPLC-TQD system (Waters Acquity,
Waters, Milford, MA) was used to establish an
LC-MS/MS method for quantitation of the hydroxy
metabolites of repaglinide and ibuprofen. Chro-
matographic separation of 30-hydroxy repaglinide
and 2-hydroxy ibuprofen was achieved on a
Hypersil C18 column (50 mm ꢀ 2.1 mm, 3 mm) (Elite,
Dalian, China) and a Waters Acquity UPLCTM HSS
T3 column (100 mm ꢀ 2.1 mm, 1.8 mm) (Waters),
respectively. Data were acquired by using Masslynx
software (version 4.1, Waters) in the multiple
Western blot analysis
Total CYP2C8 protein levels in the Sf9 whole-cell
lysate were determined by western blot analysis.
S9 (25 mg/lane) was separated by 8% sodium
dodecyl sulfate–polyacrylamide gels and trans-
ferred onto a polyvinylidene difluoride mem-
brane (Bio-Rad, Mississauga, ON, Canada). The
membranes were incubated overnight at 4 ꢁC with
monoclonal antibody against histidine as the pri-
mary antibody (1:2000 dilution), and then with a
secondary antibody (goat anti-mouse IgG-HRP,
1:5000 dilution) for 1 h at room temperature.
Immunoreactive proteins were visualized with
enhanced chemiluminescence-plus (ECL) reagents
Copyright © 2013 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 278–287 (2013)
DOI: 10.1002/bdd

EFFECT OF CYP2C8S ON PACLITAXEL, REPAGLINIDE AND IBUPROFEN ENANTIOMERS
281
reaction monitoring mode for the following
transitions: 469.2 m/z to 246.3 m/z for 30-hydroxy
repaglinide, 318.1m/z to 166.0 m/z for nateglinide,
221.5 m/z to 177.2 m/z for 2-hydroxy ibuprofen,
and 161 m/z to 133 m/z for 7-hydroxycoumarin.
30-Hydroxy repaglinide and 2-hydroxy ibuprofen
concentrations were given in arbitrary units (AU) rel-
ative to the peak area ratio of each metabolite to that
of the internal standard in the chromatogram [10].
peak at approximately 450 nm (Figure 1). The
average P450 content of recombinant CYP2C8.1,
CYP2C8.2, CYP2C8.3 and CYP2C8.4 was
35.6ꢃ 2.1, 30.82ꢃ 1.5, 31.72ꢃ 1.6 and 25.40ꢃ 1.9
pmol/mg protein, respectively.
Western blot results showed a single band at
approximately 55 kDa, consistent with the previ-
ously reported molecular mass of CYP2C8 [18]
(Figure 2A). Negligible levels of non-specific pro-
teins were detected in the negative control group.
As shown in Figure 2B, no significant difference
was observed between the protein expression
level of CYP2C8.1 and that of the three CYP2C8
variants, indicating that all CYP2C8 proteins were
expressed uniformly.
Data analysis
The kinetic constants, Km and Vmax, except for the
metabolism of ibuprofen by CYP2C8.4 were esti-
mated using nonlinear regression analysis of
Michaelis-Menten model by GraphPad Prism ver-
sion 5.0 software (GraphPad Software Inc., San
Diego, CA). The kinetic constants of ibuprofen
catalysed by CYP2C8.4 were estimated using the
Hill equation as follows:
Enzymatic activities of CYP2C8s
toward paclitaxel
Paclitaxel is the preferred substrate for CYP2C8
in vitro, and its 6a-hydroxylation has been accepted
widely as a marker reaction [18]. Non-linear regres-
sion curves of Michaelis-Menten kinetics are shown
in Figure 3A and the kinetic parameters are sum-
marized in Table 1. All CYP2C8s exhibited typical
Michaelis-Menten behaviour. CYP2C8.3 had a
1.9-fold higher Km value than that of CYP2C8.1
(p < 0.01) and a slightly higher Vmax value than
that of CYP2C8.1. CYP2C8.2 and CYP2C8.4
had slightly higher Km values than that of
CYP2C8.1 and significantly lower Vmax values
than that of CYP2C8.1 (p < 0.01). The CLint
values of CYP2C8.2, CYP2C8.3 and CYP2C8.4
were 47.7%, 64.3% and 30.2% of that of
CYP2C8.1 (p < 0.01), respectively.
Vmax ꢀ Sn
v ¼
S50 þ Sn
n
where the substrate concentration resulting in 50% of
Vmax (S50) is analogous to the Km parameter, and n is
the Hill coefficient. Intrinsic clearance (CLint) values
were determined as the ratio of Vmax/Km. All values
are expressed as the meanꢃ SD of three separate
experiments derived from independent preparations.
The two-sided Student’s t-test was used for statistical
comparison. A probability value of less than 0.05
(p< 0.05) was accepted as a significant difference.
Results
Enzymatic activities of CYP2C8s
toward repaglinide
Expression of recombinant CYP2C8s in Sf9 cells
CYP2C8.1, CYP2C8.2, CYP2C8.3 and CYP2C8.4
were heterologously co-expressed with human
cytochrome P450 oxidoreductase (POR) and cyto-
chrome b5 in a Bac-to-Bac insect expression system.
The co-expression conditions were optimized to
have a ratio of 3:3:1 (CYPs:POR:b5) in the prelimi-
nary experiment. The expression levels of func-
tional P450 content and CYP2C8 holoproteins
in Sf9 cells were examined using the reduced
carbon monoxide (CO) difference spectra and west-
ern blot analysis, respectively. The CO difference
spectrum of recombinant CYP2C8s showed a Soret
30-Hydroxy repaglinide is the major metabolite of
repaglinide formed after metabolism by CYP2C8
in vitro. The Michaelis-Menten plots for repaglinide
hydroxylation by recombinant CYP2C8 variants
are shown in Figure 3B and the Km values and the
Vmax and CLint values in arbitrary units are given
in Table 2. The Km values of CYP2C8.2 and
CYP2C8.4 were comparable to that of CYP2C8.1,
while the Vmax values were significantly lower than
that of CYP2C8.1, resulting in CLint values of
CYP2C8.2 and CYP2C8.4 that were 77.9% and
80.2% of that of CYP2C8.1 (p < 0.05), respectively.
Copyright © 2013 John Wiley & Sons, Ltd.
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Figure 1. Carbon monoxide difference spectra of recombinant proteins. (A) CYP2C8.1; (B) CYP2C8.2; (C) CYP2C8.3; (D) CYP2C8.4
These results indicated that metabolism by
CYP2C8.2 and CYP2C8.4 decreased repaglinide
hydroxylation, whereas metabolism by CYP2C8.3
significantly increased repaglinide hydroxylation.
Enzymatic activities of CYP2C8s toward
ibuprofen enantiomers
The kinetic plots of 2-hydroxylation of R- and
S-ibuprofen are shown in Figure 3C, and the
corresponding enzyme kinetic parameters are
listed in Table 3. The relative CLint values of
Figure 2. Western blot analysis of recombinant protein expres-
sion in sf9 insect cells after infection. (A) Lane 1/2/3/4, Sf9 cell
lysates co-infected with v-CYP2C8*1/*2/*3/*4, v-POR and v-
Cyt b5; lane 5, Sf9 cell lysates co-infected with v-POR and v-
Cyt b5 as control. (B) The results were expressed as a relative per-
centage compared with CYP2C8.1. Each bar represents the scan-
ning analysis result of the expression level of the above
CYP enzymes. The results are indicated as mean ꢃ SD of
three independent experiments
CYP2C8.2, CYP2C8.3 and CYP2C8.4 were 110.5%,
72.3% and 49.7% of that of CYP2C8.1 and were
124.6%, 83.4% and 47.4% of that of CYP2C8.1
for R-ibuprofen and S-ibuprofen, respectively. The
Hill coefficients of R-ibuprofen and S-ibuprofen
catalysed by CYP2C8.4 were 1.32 and 1.02, respec-
tively. For both R-ibuprofen and S-ibuprofen, the
amino acid substitutions present in CYP2C8.2
increased the metabolic ability, whereas CYP2C8.3
and CYP2C8.4 had a significantly reduced metabolic
ability. The four CYP2C8 allozymes were all pre-
dominantly active toward R- ibuprofen. The CLint
values for R-ibuprofen were over 1.5-fold higher
than those for S-ibuprofen.
CYP2C8.3 and CYP2C8.1 had significantly different
Km (p < 0.01), Vmax (p < 0.01) and CLint values
(p < 0.05). The CLint value of CYP2C8.3 was
1.31-fold higher than that of CYP2C8.1 (p < 0.05).
Copyright © 2013 John Wiley & Sons, Ltd.
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DOI: 10.1002/bdd

EFFECT OF CYP2C8S ON PACLITAXEL, REPAGLINIDE AND IBUPROFEN ENANTIOMERS
283
Figure 3. CYP2C8 activity comparisons for paclitaxel, repaglinide and ibuprofen (IBU) enantiomers hydroxylation among
CYP2C8.1/.2/.3/.4 (n = 3). (A) paclitaxel; (B) repaglinide; (C) ibuprofen enantiomers
Discussion
no obvious peak at 420 nm in CYP2C8.1. The pres-
ence of a large peak at 420 nm has been shown to in-
dicate the altered stability of the proteins or the
formation of an incorrectly folded enzyme resulting
in a significantly lower functional P450 content [19].
The amino acid substitution caused by
CYP2C8*4 polymorphism was I264M in helix H
located on the protein surface, away from regions
involved in heme and substrate binding [20]. As a
highly conserved amino acid among the CYP2C
subfamily of enzymes, Ile264 is important for
heme insertion and protein folding. Because of
the weak iron–thiolate interaction between the
heme and coordinated Cys435, the secondary
and tertiary structures of CYP2C8.4 changed
significantly resulting in the loss of stability.
CYP2C8.4 was thermally unstable and more sensi-
tive to proteinase K digestion [21,22], which may
explain the remarkably poor catalytic activity of
In this study, recombinant CYP2C8.1, CYP2C8.2,
CYP2C8.3 and CYP2C8.4 were systematically
co-expressed with POR and cytochrome b5 using
the Bac-to-Bac insect expression system. To ensure
that the four CYP2C8s were uniformly expressed,
the viral titres of baculovirus (CYP2C8*1, CYP2C8*2,
CYP2C8*3, CYP2C8*4, POR and cytochrome b5)
were determined by real-time PCR (data not shown).
The expression level of CYP2C8 holoprotein in
CYP2C8.2, CYP2C8.3 and CYP2C8.4 was compara-
ble to that in CYP2C8.1 (Figure 2), whereas the func-
tional P450 content in CYP2C8.4 was significantly
lower than that in CYP2C8.1. As shown in Figure 1D,
CYP2C8.4 exhibited a large peak at 420 nm in addi-
tion to the Soret peak at 450 nm. However, the peaks
at 420 nm were relatively small compared with that
at 450 nm in CYP2C8.2 and CYP2C8.3. There was
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L. YU ET AL.
Table 1. Metabolism kinetics of CYP2C8s for paclitaxel hydroxylation (n = 3)
Protein
R2
Km(mmol∙l-1)
Vmax (pmol∙min-1∙pmol-1 protein)
CLint (ml∙min -1∙pmol-1protein)/(% of CYP2C8.1)
CYP2C8.1
CYP2C8.2
CYP2C8.3
CYP2C8.4
0.9980
0.9794
0. 9875
0.9745
8.65 ꢃ 0.25
9.65 ꢃ 0.93
11.81 ꢃ 1.35a
69.83 ꢃ 0.84
36.95 ꢃ 1.30b
84.15 ꢃ 3.47
28.90 ꢃ 1.18b
8.09 ꢃ 0.14 (100)
3.86 ꢃ 0.23b (47.7)
5.20 ꢃ 0.25b (64.3)
2.44 ꢃ 0.20b (30.2)
16.25 ꢃ 1.45b
Significant difference between the marked data and the data for CYP2C8.1,
ap < 0.05,
bp < 0.01.
Table 2. Metabolism kinetics of CYP2C8s for repaglinide hydroxylation (n = 3)
Protein
R2
Km (mmol∙l-1)
Vmax (AU)
CLint (AU)/(% of CYP2C8.1)
CYP2C8.1
CYP2C8.2
CYP2C8.3
CYP2C8.4
0.9985
0.9868
0.9833
0.9974
12.01 ꢃ 1.63
11.34 ꢃ 1.61
7.43 ꢃ 0.84b
11.38 ꢃ 1.00
15.69 ꢃ 0.93
11.56 ꢃ 0.58b
12.74 ꢃ 0.44b
11.91 ꢃ 0.35b
1.31 ꢃ 0.09 (100)
1.02 ꢃ 0.10a (77.9)
1.71 ꢃ 0.13a (130.5)
1.05 ꢃ 0.06a (80.2)
Significant difference between the marked data and the data for CYP2C8.1,
ap < 0.05,
bp < 0.01.
Table 3. Metabolism kinetics of CYP2C8s for R- /S- ibuprofen (IBU) hydroxylation (n = 3)
Protein
R2
Km (mmol∙l-1)
Vmax (AU)
CLint (AU)/(% of CYP2C8.1)
R/S of CLint
CYP2C8.1
CYP2C8.2
CYP2C8.3
CYP2C8.4
R-IBU
S-IBU
R-IBU
S-IBU
R-IBU
S-IBU
R-IBU
S-IBU
0.9861
0.9917
0.9782
0.9901
0.9775
0.9750
0.9726
0.9638
341.3 ꢃ 25.2
388.8 ꢃ 26.1
479.2 ꢃ 55.5a
544.9 ꢃ 43.6b
230.1 ꢃ 22.9a
269.5 ꢃ 29.2b
226.1 ꢃ 27.8
342.5 ꢃ 83.3
4.92 ꢃ 0.13
3.02 ꢃ 0.07d
7.63 ꢃ 0.08b
5.27 ꢃ 0.17b,d
2.40 ꢃ 0.07b
1.74 ꢃ 0.06b,c
1.62 ꢃ 0.08b
1.26 ꢃ 0.12b,c
14.42 ꢃ 0.70 (100)
7.76 ꢃ 0.33d (100)
15.93 ꢃ 1.16 (110.5)
9.67 ꢃ 0.51b,d (124.6)
10.43 ꢃ 0.72b (72.3)
6.47 ꢃ 0.48b,d (83.4)
7.16 ꢃ 0.33b (49.7)
3.68 ꢃ 0.34b,d (47.4)
1.86
1.65
1.61
1.95
Significant difference between CYP2C8.1 group and its variants group
ap < 0.05,
bp < 0.01.
Significant difference between R-IBU group and S-IBU group
cp < 0.05,
dp < 0.01.
CYP2C8.4 toward paclitaxel, repaglinide and
ibuprofen in the present study (Tables 1–3).
145% higher activity than that of CYP2C8.1 in the
E. coli expression system [23].
The catalytic activity of CYP2C8.3 in paclitaxel
6a-hydroxylation was 64.3% of that of CYP2C8.1
(p < 0.01), which was roughly similar to the enzyme
activity measured in the microsomes of yeast cells
or HepG2 cells (50%–75%) [10,19]. Furthermore, in
the human liver microsomes homozygous for
CYP2C8*3, the paclitaxel 6a-hydroxylation activi-
ties represented 31%–84% of the corresponding
median activities in the microsomes homozygous
for CYP2C8*1 [22], similar to the values obtained
in the present study. However, CYP2C8.3 showed
To date, no studies have been performed to
investigate the influence of CYP2C8 genetic poly-
morphisms on the hydroxylation metabolism of
repaglinide in vitro. CYP2C8.2 and CYP2C8.4
had significantly lower repaglinide hydroxylation
activities than CYP2C8.1 (p < 0.05), whereas
CYP2C8.3 showed 130.5% activity of that of
CYP2C8.1 (Table 2). The mean AUC and Cmax of
repaglinide were significantly lower in subjects
with the CYP2C8*1/*3 genotype than in those with
the
CYP2C8*1/*1
genotype
after
being
Copyright © 2013 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 278–287 (2013)
DOI: 10.1002/bdd

EFFECT OF CYP2C8S ON PACLITAXEL, REPAGLINIDE AND IBUPROFEN ENANTIOMERS
285
administered
a
single dose (0.25 mg) of
toward repaglinide and ibuprofen enantiomers,
suggesting that CYP2C8.2 and CYP2C8.3 had
substrate-dependent activities in vitro.
repaglinide [11,12]. The result further validated
that in vitro CYP2C8.3 played a major role in low-
ering the AUC and Cmax of repaglinide in subjects
with the CYP2C8*1/*3 genotype.
The substrate-dependent activities of CYP2C8.2
and CYP2C8.3 can be partly interpreted by evaluat-
ing the structure–function relationship. Several sig-
nificant substrate recognition sites (SRSs) and amino
acid residues have been identified within the rela-
tively large substrate-binding cavity of CYP2C8
[21,28]. The large size of the binding site should
permit more substrate mobility and allow more in-
teractions with the related SRSs. The amino acid
substitutions present in the CYP2C8 variants may
result in structural changes in some specific SRSs,
followed by a changed affinity to related sub-
strates, while the affinity to unrelated substrates
may remain unchanged. Therefore, the influence
of CYP2C8 polymorphisms on substrate affinity
should be expected to be substrate-dependent. It
has been reported that SRS-1 and SRS-5 are impor-
tant for binding to paclitaxel [29]. However, further
studies are required to determine the SRSs respon-
sible for binding to repaglinide and ibuprofen. Fur-
thermore, the substrate-dependent activities of
CYP2C8s may be associated with POR and cyto-
chrome b5, as the modulatory effects of POR and
cytochrome b5 on CYPs were both substrate- and
CYP-dependent [30]. For CYP2C8.3, both amino
acid mutations (R139K and K399R) were located
on the proximal side of the heme-binding region
of cytochrome b5 and POR. Recent studies have
shown that drug metabolism by CYP2C8.3 was
directed by substrate-dependent interactions with
POR and cytochrome b5 [23].
For the stereoselective metabolism of ibuprofen,
our results showed that the four CYP2C8 allozymes
mainly formed 2-hydroxy R-ibuprofen, which
was consistent with the in vivo data [24]. The
Km values of the two enantiomers differed as
much as the corresponding Vmax values (Table 3).
Additionally, both the increased rate of R-ibuprofen
hydroxylation and the higher affinity of the
CYP2C8s to R-ibuprofen, confirmed the prefer-
ence for R-ibuprofen metabolism. Interestingly,
Eadie-Hofstee plots in Figure 3C for CYP2C8.4
indicated that atypical kinetics existed. Therefore,
the Hill equation was used to estimate the kinetic
parameters. Furthermore, no significant difference
was found in the R/S-ibuprofen CLint ratio among
the CYP2C8 allozymes, indicating that the CYP2C8
genetic polymorphisms may have minimal
influence on the stereoselective metabolism of
ibuprofen enantiomers.
Some clinical studies have shown lower clear-
ance of paclitaxel in ovarian cancer patients
heterozygous or homozygous for CYP2C8*3 [25]
and lower clearance of ibuprofen in healthy volun-
teers heterozygous or homozygous for CYP2C8*3
[26], while the CYP2C8*3 allele was associated with
an increased clearance of repaglinide [11,12]. In
healthy white subjects, CYP2C8*3 and the two
novel haplotypes significantly influenced repaglinide
pharmacokinetics in SLCO1B1c.521 T/C heterozy-
gous individuals: haplotype B was associated with
a reduced and haplotype C with an increased
repaglinide AUC (0–1) [27]. Thus, the CYP2C8*3
polymorphism influenced the in vivo drug disposition
in a substrate-dependent manner. The results of this
study were consistent with the data in vivo.
The catalytic activities of CYP2C8 allozymes for pacli-
taxel 6a-hydroxylation were in the order CYP2C8.4
CYP2C8.2 < CYP2C8.3 < CYP2C8.1, while the order
for repaglinide metabolism was CYP2C8.2 ≤
CYP2C8.4 < CYP2C8.1 < CYP2C8.3. For the meta-
bolism of ibuprofen enantiomers, the order was
CYP2C8.4 < CYP2C8.3 < CYP2C8.1 < CYP2C8.2. For
CYP2C8.2 and CYP2C8.3, the significant differ-
ences in enzymatic activities toward paclitaxel were
inconsistent with the corresponding differences
In summary, the clearance profiles of paclitaxel,
repaglinide and ibuprofen mediated by CYP2C8.3
in vitro were consistent with the pharmacokinetics
in volunteers who were heterozygous or homozy-
gous for CYP2C8*3. The results of this study pro-
vide useful information for predicting CYP2C8
phenotypes and may contribute to individualized
drug therapy in the future.
Acknowledgements
This project was supported by the National
Science and Technology Major Project of China
(No. 2012ZX09506001-004) and National Natural
Science Foundation of China (30873122).
Copyright © 2013 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 34: 278–287 (2013)
DOI: 10.1002/bdd

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There are no conflicts of interest.
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