Characterization of cytochrome P450s mediating ipriflavone metabolism in human liver microsomes

Article abstract of DOI:10.1080/00498250601146962

Ipriflavone, a synthetic flavonoid for the prevention and treatment of osteoporosis, has been reported to be extensively metabolized in man to seven metabolites (M1-M7). This study was performed to characterize the human liver cytochrome P450s (CYP) responsible for the metabolism of ipriflavone. Hydroxylation at the β-ring to M3, O-dealkylation to M1 and oxidation at isopropyl group to M4 and M5 are major pathways for ipriflavone metabolism in three different human liver microsome preparations. The specific CYPs responsible for ipriflavone oxidation to the active metabolites, M1, M3, M4 and M5 were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP enzymes. The inhibitory potencies of ipriflavone and its five metabolites, M1-M5 on seven clinically important CYPs were investigated in human liver microsomes. Our results demonstrate that CYP3A4 plays the major role in O-dealkylation of ipriflavone to M1 and CYP1A2 plays a dominant role in the formation of M3, M4 and M5. Ipriflavone and/or its five metabolites were found to inhibit potently the metabolism of CYPs 1A2, 2C8, 2C9 and 2C19 substrates.

Full text of DOI:10.1080/00498250601146962

Xenobiotica, March 2007; 37(3): 246–259
Characterization of cytochrome P450s mediating
ipriflavone metabolism in human liver microsomes
Y. MOON1, S. Y. KIM1, H. Y. JI1, Y. K. KIM1, H.-J. CHAE2,3
S.-W. CHAE2,3, & H. S. LEE1,3
,
2
1College of Pharmacy, Wonkwang University, Iksan, Korea, Department of Pharmacology,
3
School of Medicine, Chonbuk National University, Jeonju, Korea, and Clinical Trial Center,
Chonbuk National University, Jeonju, Korea
(Received 25 October 2006; revised 30 November 2006; accepted 30 November 2006)
Abstract
Ipriflavone, a synthetic flavonoid for the prevention and treatment of osteoporosis, has been reported
to be extensively metabolized in man to seven metabolites (M1–M7). This study was performed to
characterize the human liver cytochrome P450s (CYP) responsible for the metabolism of ipriflavone.
Hydroxylation at the ꢀ-ring to M3, O-dealkylation to M1 and oxidation at isopropyl group to M4 and
M5 are major pathways for ipriflavone metabolism in three different human liver microsome
preparations. The specific CYPs responsible for ipriflavone oxidation to the active metabolites, M1,
M3, M4 and M5 were identified using a combination of correlation analysis, immuno-inhibition,
chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP
enzymes. The inhibitory potencies of ipriflavone and its five metabolites, M1–M5 on seven clinically
important CYPs were investigated in human liver microsomes. Our results demonstrate that CYP3A4
plays the major role in O-dealkylation of ipriflavone to M1 and CYP1A2 plays a dominant role in the
formation of M3, M4 and M5. Ipriflavone and/or its five metabolites were found to inhibit potently the
metabolism of CYPs 1A2, 2C8, 2C9 and 2C19 substrates.
Keywords: Ipriflavone, CYP3A4, CYP1A2, inhibition of P450s, inhibition of CYPs 1A2, 2C8, 2C9, 2C19
Introduction
Ipriflavone (7-isopropoxy-3-phenyl-chromen-4-one) (Figure 1) is a synthetic flavonoid
which is used in the prevention and treatment of osteoporosis (Reginster 1993; Head 1999).
Ipriflavone is extensively metabolized in rats, dogs, rabbits and humans and undergoes
an extensive first-pass metabolism (Ferenc and Istvan 1995; Kim et al. 2000; Kim
and Lee 2002). Seven metabolites (M1–M7) were identified (Figure 1) in animals and
humans (Yoshida et al. 1985; Ronelli et al. 1991; Ferenc and Istvan 1995; Rohatagi and
Correspondence: H. S. Lee, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea. E-mail: hslee@wonkwang.ac.kr
ISSN 0049-8254 print/ISSN 1366-5928 online ß 2007 Informa UK Ltd.
DOI: 10.1080/00498250601146962

Ipriflavone metabolism
247
CH
O
O
3
HO
O
CH
O
O
O
3
CH
3
CH OH
2
CYP3A4
CYP1A2
O
O
M1
Ipriflavone
M4
CYP1A2
CYP1A2
HO
O
O
CH
3
O
O
CH
O
O
3
CH
COOH
3
O
O
OH
OH
M2
M5
M3
CH
3
O
O
CH
O
O
O
3
OH
COOH
COOH
+
O
OH
M6
M7
Figure 1. Metabolic pathways of ipriflavone.
Barrett 1997; Rohatagi et al. 1997). Ipriflavone is metabolized mainly in the liver by
oxidation of the isopropyl group and/or hydroxylation of the ꢀ-ring followed by phase II
glucuronidation or sulfation. Ipriflavone is biotransformed to M1, M3 and M4.
Subsequently, M4 is biotransformed to M5, which is further metabolized to M6 and M7.
Also, M1 and M3 are converted to M2 (Figure 1). Ipriflavone metabolites, M1, M2, M3 and
M5 have been shown to possess pharmacological activity related to bone formation and bone
resorption and may contribute to the overall efficacy of ipriflavone (Benvenuti et al. 1991;
Cheng et al. 1994; Bassleer et al. 1996; Giossi et al. 1996).
The reduction of theophylline clearance by concomitant ipriflavone administration
observed by Takahashi et al. (1992) is primarily due to the inhibition of CYPs responsible
for theophylline N-demethylation by ipriflavone and/or its metabolites M1 and M5
(Monostory and Vereczkey 1995, 1996). Ipriflavone and M1 have been shown to be
extensive inhibitors of phaenacetin O-deethylase (CYP1A2) and tolbutamide hydroxylase
(CYP2C9) in human liver microsomes, whereas M5 had no effect on CYPs 1A2, 2A6, 2C9,
2C19, 2D6, 2E1 and 3A4 activities (Monostory et al. 1998). The total body clearance values
of ipriflavone obtained after infusion of ipriflavone in rats pretreated with CYP inducers and
inhibitors were compared with that in control rats, suggesting that CYP1A1/2, 2B1/2 and
2C11 might be responsible for ipriflavone metabolism in rats (Chung et al. 2006).
The information of specific CYP enzyme(s) involved in the biotransformation of a large
number of therapeutic agents can be of considerable clinical impact in regard to potential
drug interactions and inter-individual variation in drug metabolism. Consequently, the
evaluation of human CYP enzyme(s) responsible for the metabolism of ipriflavone can be
useful in understanding the disposition and the in vivo behavior of its active metabolites as
well as ipriflavone and may have clinical implications.

248
Y. Moon et al.
The present study describes an in vitro investigation into the metabolism of ipriflavone to
identify the human CYP enzymes responsible for the biotransformation of ipriflavone to the
active metabolites and to investigate the inhibitory effects of ipriflavone and its five
metabolites, M1–M5 on major human CYP enzymes in human liver microsomes.
Materials and methods
Chemicals
Ipriflavone, M1, M3, M4 and M5 (purity of 99.5%) were supplied by Samchundang
Pharm. Co. (Seoul, Korea). 2-(3,4-Dimethoxy-phenyl)-5,7-dihydroxy-chromen-4-one
(DPDC, internal standard) was synthesized by Dong-A Pharm. Co. (Yongin, Korea).
Daidzein (M2), NADPþ, glucose-6-phosphate, glucose-6-phosphate dehydrogenase,
furafylline, ketoconazole, sulfaphenazole and triethylenethiophosphoramide (thioTEPA)
were obtained from Sigma Chemical Co. (St Louis, MO, USA). Acetonitrile and
dichloromethane (HPLC grade) were obtained from Burdick & Jackson Inc. (Muskegon,
MI, USA) and the other chemicals were of the highest quality available.
Human liver microsomes (coded H161, H003, H023, H042, H043, H056, H066, H070,
H089, H093, H112, HK23 and HK34) and microsomes derived from baculovirus-infected
insect cells transfected with human P450 cDNA over-expressing CYPs1A2, 2B6, 2C8, 2C9,
2C19, 2D6, 2E1 and 3A4 were purchased from the Gentest Corporation (Woburn, MA,
USA). Human selective antibodies for the immunoinhibition of human CYPs, i.e. anti-
CYP1A2 (Cat. No. 458312, CYP1A2-specific), anti-CYP2D6, anti-CYP2E1 and anti-
CYP3A4 (Cat. No. 458334, reactive for CYPs3A4 and 3A5), and antiserum of CYP2C
were obtained from Gentest Co.
Metabolism of ipriflavone with human liver microsomes or cDNA-expressed CYP enzymes
Incubation mixtures contained 50 mM potassium phosphate buffer (pH 7.4), human liver
microsomes (0.15 mg protein mlꢀ1) or eight cDNA expressing CYP enzymes (4 pmol),
NADPH generating system (1.3 mM NADP, 3.3 mM glucose-6-phosphate and 0.8 U mlꢀ1
glucose-6-phosphate dehydrogenase), 3 mM magnesium chloride and ipriflavone or
metabolite M4 (2 mM, added in methanol, final solvent concentration not exceeding
0.5%, v/v) in a final volume of 200 ml. Preliminary experiments showed that the formation
of the three major metabolites, i.e. M1, M3 and M4, from ipriflavone was linear with
respect to both incubation time over 45 min and liver microsomal protein concentration
(0.1–0.3 mg mlꢀ1) at 37ꢁC. Thus, a 20-min incubation time and a 0.15 mg mlꢀ1 microsomal
protein concentration were selected. The reactions were terminated by the addition of 50 ml
of 500 mM hydrochloric acid, 10 ml of DPDC (internal standard, 500 ng mlꢀ1) and 1000 ml
of dichloromethane. Following mixing with a vortex mixer and centrifugation, an aliquot
(800 ml) of the organic layer was evaporated under a nitrogen stream. The residue was
dissolved in 50 ml of 50% acetonitrile and an aliquot (10 ml) was analyzed by an LC-MS/MS
method.
In kinetic experiments, seven concentrations of ipriflavone (1.0–100 mM) were incubated
in duplicate with three different human liver microsome preparations (H043, H056
and H093) or cDNA-expressed CYP1A2, 2C9, 2C19 and 3A4 enzymes. Several enzyme
kinetic equations were fitted to the untransformed data using enzyme kinetics software

Ipriflavone metabolism
249
(version 1.1, SPSS Science Inc., Richmond, CA, USA). The equation, V ¼ Vmax
ꢂ
S/(Km þ S) best described the kinetics of biotransformation of ipriflavone to M1, M3 and
M4 in human liver microsomes and cDNA-expressed CYPs 1A2, 2C9, 2C19 and CYP3A4
enzymes. V is the velocity of the reaction at substrate concentration [S]; Vmax, the maximum
velocity; and Km, the substrate concentration at which the reaction velocity is 50% of Vmax
.
The intrinsic clearance (Clint) of in vitro incubation was calculated as Vmax/Km.
Contributions of each cytochrome to ipriflavone oxidation were normalized for mean
values of the relative abundance of individual cytochromes in the liver. The relative
abundances of CYP1A2 (12.7%), CYP3A4 (28.8%), CYP2C9 (14.7%) and CYP2C19
(3.5%) and wide inter-individual variability in the expression of CYP3A4 and CYP1A2 have
been reported (Shimada et al. 1994; Venkatakrishnan et al. 1998). The Clint attributable to
CYPs 1A2, 2C9, 2C19 and 3A4 was multiplied by the relative abundance of each CYP in
the liver. Percentages of the Clint of an individual CYP relative to the overall contribution of
four CYPs (CYP1A2, -2C9, -2C19 and -3A4) were calculated.
For correlation analysis, comparative metabolic rates of ipriflavone in 12 different human
liver microsomes were investigated by incubating 2 mM of ipriflavone with 0.15 mg mlꢀ1
microsomal protein for 20 min. The formation rates of three metabolites, M1, M3 and
M4 were correlated with specific CYP activities in human liver microsomes reported by
Gentest using Pearson product moment correlation (SigmaStat software version 2.0, SPSS
Science). For pairs with p values below 0.05, there is significant relationship between the two
variables.
Inhibition experiments
Immunoinhibition studies were performed by incubating human liver microsomes with
various amounts of human selective antibodies, anti-CYP1A2, anti-CYP2C, anti-CYP2D6,
anti-CYP2E1 or anti-CYP3A4 for 15 min on ice before the addition of buffer, ipriflavone or
M4 (2 mM) and an NADPH generating system. As a control, comparable incubations were
done with microsomes and 25 mM Tris buffer.
To explore which CYP enzymes are involved in the metabolism of ipriflavone, the
inhibitory effects were determined using selective CYP enzyme inhibitors. The concentra-
tions of specific CYP enzyme inhibitors were chosen based on the published IC50 or Ki
values for CYP enzyme-specific reactions: 10 mM furafylline for CYP1A2 (Sesardic et al.
1990), 100 mM coumarin for CYP2A6 (Yun et al. 1991), 5 mM thioTEPA for CYP2B6 (Rae
et al. 2002), 0.1 mM montelukast for CYP2C8 (Walsky et al. 2005), 10 mM sulfaphenazole
for CYP2C9 (Baldwin et al. 1995), 1 mM S-benzylnirvanol for CYP2C19 (Suzuki et al.
2002; Walsky and Obach 2003), 10 mM quinidine for CYP2D6 (Newton et al. 1995), 10 mM
diethyldithiocarbamate for CYP2E1 (Newton et al. 1995) and 1 mM ketoconazole for
CYP3A4 (Baldwin et al. 1995). Incubations were performed with CYP-selective inhibitor,
pooled human liver microsomes (H161, 0.15 mg mlꢀ1) and ipriflavone or M4 (2 mM).
Furafylline and diethyldithiocarbamate were pre-incubated for 10 min with microsomes and
an NADPH generating system before addition of ipriflavone or M4 to initiate the reaction.
Activities of all the inhibitors were compared with that of inhibitor-free controls.
LC-MS/MS analysis
The concentrations of ipriflavone and its five metabolites, M1, M2, M3, M4 and M5, in the
microsomal incubates were analyzed by the modification of the previous LC-MS/MS

250
Y. Moon et al.
method (Ji et al. 2005). The chromatographic system consisted of a Nanospace SI-2 HPLC
system (Shiseido, Tokyo, Japan). The separation was performed on an XTerra C18 column
(5 mm, 3.0 mm i.d. ꢃ 50 mm, Waters, USA) using a mixture of acetonitrile-ammonium
formate (10 mM, pH 3.0) (50:50, v/v) at a flow rate of 0.5 ml minꢀ1. The column and
autosampler tray temperature was 30ꢁC and 4ꢁC, respectively. The analytical run time was
11 min. The eluent was introduced directly into the positive ionization electrospray source of
a tandem quadrupole mass spectrometer (Quattro LC, Micromass UK Ltd, UK). The ion
source and desolvation temperature were held at 120ꢁC and 350ꢁC, respectively.
The optimum cone voltages were 32, 35, 25, 35 and 55 V for M1, M3, M4, M5 and
DPDC (internal standard), respectively. Multiple reaction monitoring (MRM) mode
was employed for the quantitation: m/z 238.9 ! 165.3 for M1, m/z 297.6 ! 254.9 for M3,
m/z 297.3 ! 239.1 for M4, m/z 311.1 ! 238.8 for M5 and m/z 315 ! 299 for DPDC.
Peak areas for all components were automatically integrated using MassLynx version 3.5
software (Micromass UK Ltd.). Calibration curves were linear over the concentration
range of 0.5–500 pmol of M1–M5. The inter-batch relative error values for M1–M5 ranged
from ꢀ3.7% to 2.3% with coefficient of variation values of 2.2–5.7% at three QC levels,
i.e. 1.0, 60 and 400 pmol.
Inhibitory potency of ipriflavone and its metabolites on CYP activities
in human liver microsomes
The inhibitory potency of ipriflavone and its five metabolites, M1–M5, was determined with
cytochrome P450 assays in the absence and presence of ipriflavone, M1, M2, M3, M4 or
M5 (final concentrations of 1–100 mM with methanol concentration less than 0.5% v/v)
using pooled human liver microsomes (H161). All experiments were performed in
duplicate. Phenacetin O-deethylase, coumarin 7-hydroxylase, paclitaxel 6ꢁ-hydroxylase,
diclofenac 4-hydroxylase, (S)-mephenytoin 4-hydroxylase, bufuralol 10-hydroxylase and
midazolam 10-hydroxylase were determined as probe activities for CYP1A2, CYP2A6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively, using cocktail
incubation and tandem mass spectrometry with the modification of the method of Dierks
et al. (2001). In brief, the incubation mixtures containing pooled human liver microsomes
(0.25 mg mlꢀ1), CYP-selective substrates and ipriflavone or its metabolites (0.1–100 mM)
were pre-incubated for 5 min at 37ꢁC. The reaction was initiated by adding an NADPH
generating system, and the reaction mixtures were incubated for 20 min at 37ꢁC in a shaking
water bath. The substrates were used at concentrations approximately equal to their
respective Km values: 50 mM phenacetin, 2.5 mM coumarin, 10 mM paclitaxel, 10 mM
diclofenac, 100 mM [S]-mephenytoin, 5 mM bufuralol and 2.5 mM midazolam.
Results
Kinetics of ipriflavone oxidation in human liver microsomes
From the incubation of ipriflavone with human liver microsomes in the presence of
NADPH-generating system, its four metabolites, i.e. M1, M3, M4 and M5 were identified
but M2, M6 and M7 were not detected by LC-MS analysis. The incubation of M4 with
human liver microsomes in the presence of NADPH-generating system resulted in the
formation of M5.

Ipriflavone metabolism
251
The oxidation of ipriflavone to M1, M3 and M4 in three different human liver
microsomes followed single-enzyme Michaelis–Menten kinetics (Figure 2). The formation
rates of M5 were very low, and therefore kinetic parameters for M5 could not be
determined. The apparent kinetic parameters for O-dealkylation of ipriflavone to M1 in
three human liver microsomes were Km of 3.0 ꢄ 0.8 mM and Vmax of 56.5 ꢄ 37.0 pmol
minꢀ1 mg proteinꢀ1 (Table I). The apparent kinetic parameters for isopropyl hydroxylation
of ipriflavone to M4 in three human liver microsomes were Km of 2.3 ꢄ 0.5 mM and Vmax
of 105.2 ꢄ 111.7 pmol minꢀ1 mg proteinꢀ1 (Table I). The apparent kinetic parameters for
ꢀ-ring hydroxylation of ipriflavone to M3 in three human liver microsomes were Km of
2.3 ꢄ 0.6 mM and Vmax of 51.0 ꢄ 48.4 pmol minꢀ1 mg proteinꢀ1 (Table I). The metabolism
of authentic standard M4 to M5 in three different human liver microsomes followed
M1
M3
50
40
30
20
10
0
30
25
20
15
10
5
30
25
20
15
10
5
40
30
20
10
0
0
0
5
10
15
20
25
0
2
4
6
8
10 12 14
V/[ipriflavone]
V/[ipriflavone]
0
0
20
40
60
80
100
0
20
40
60
80
100
Concentration of ipriflavone (µM)
Concentration of ipriflavone (µM)
M4
M5
40
30
20
10
0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
50
40
30
20
10
0
0
5
10
15
20
0.0 0.5 1.0 1.5 2.0 2.5
V/[ipriflavone]
V/[M4]
0
20
40
60
80
100
0
20
40
60
80
100
Concentration of ipriflavone (µM)
Concentration of M4 (µM)
Figure 2. Representative Michaelis–Menten plots for the biotransformation of ipriflavone to M1, M3
and M4 and the formation of M5 from M4 in human liver microsome H093 with inset Eadie–Hostee
plots demonstrating single enzyme kinetics. Each point represents the average of two determinations.

252
Y. Moon et al.
Table I. Kinetics of the formation of M1, M3 and M4 from ipriflavone and M4 oxidation to M5 in three human
liver microsome preparations.
Metabolite formation
Human liver microsomes
M1
M3
M4
M5
H043
Km (mM)
3.2
28.2
8.8
2.0
21.5
10.8
1.8
33.3
18.5
14
Vmax (pmol mg proteinꢀ1 minꢀ1
)
)
)
)
89.8
6.4
Clint (ml mg proteinꢀ1 minꢀ1
)
H056
Km (mM)
Vmax (pmol mg proteinꢀ1 minꢀ1
Clint (ml mg proteinꢀ1 minꢀ1
H093
Km (mM)
3.7
98.4
26.6
3.0
106.9
35.6
2.7
233.9
86.6
20.9
200.5
9.6
)
2.1
42.9
20.4
2.0
24.7
12.4
2.5
48.5
19.4
19.7
41.3
2.1
Vmax (pmol mg proteinꢀ1 minꢀ1
Clint (ml mg proteinꢀ1 minꢀ1
)
Mean ꢄ SD
Km (mM)
3.0 ꢄ 0.8
56.5 ꢄ 37.0
18.6 ꢄ 9.0
2.3 ꢄ 0.6
51.0 ꢄ 48.4
19.6 ꢄ 13.9
2.3 ꢄ 0.5
105.2 ꢄ 111.7
41.5 ꢄ 39.1
18.2 ꢄ 3.7
110.5 ꢄ 81.6
6.0 ꢄ 3.8
Vmax (pmol mg proteinꢀ1 minꢀ1
Clint (ml mg proteinꢀ1 minꢀ1
)
single-enzyme Michaelis–Menten kinetics (Figure 2). The apparent kinetic parameters for
oxidation of M4 to M5 in three human liver microsomes were Km of 18.2 ꢄ 3.7 mM and
Vmax of 110.5 ꢄ 81.6 pmol minꢀ1 mg proteinꢀ1 (Table I). The order of the metabolic
pathways for ipriflavone in human liver microsomes were hydroxylation to M4 (Clint
;
41.5 ꢄ 39.1 ml mg proteinꢀ1 minꢀ1) > hydroxylation to M3 (Clint
;
19.6 ꢄ 13.9 ml mg
proteinꢀ1 minꢀ1) ffi O-dealkylation to M1 (Clint; 18.6 ꢄ 9.0 ml mg proteinꢀ1 minꢀ1) > oxida-
tion of M4 to M5 (Clint; 6.0 ꢄ 3.8 ml mg proteinꢀ1 minꢀ1).
Ipriflavone metabolism in human cDNA-expressed CYPs
A screen of ipriflavone oxidation at 2 mM concentration using microsomes containing
human cDNA-expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4
identified the possible roles of CYPs 1A2, 2C9, 2C19, 2D6 and 3A4 for the oxidation of
ipriflavone to M1, M3 and M4. CYP2A6, CYP2B6, CYP2C8 and CYP2E1 did not catalyze
the biotransformation of ipriflavone to M1, M3 and M4 at a detectable rate
(<0.006 pmol minꢀ1 pmol CYPꢀ1). Screening of M4 oxidation to M5 in nine human
cDNA-expressed CYP microsomes showed that only CYP 1A2 formed M5 from M4.
Table II shows the kinetic parameters for the formation of M1, M3 and M4 from
ipriflavone and M4 oxidation to M5 in human cDNA-expressed CYPs 1A2, 2C9, 2C19 and
3A4. The formation of M1, M3, M4 and M5 was consistent with single-enzyme Michaelis–
Menten kinetics. Contributions of these latter CYPs were determined after normalization for
the predicted relative abundance of each CYP to ipriflavone oxidation. Normalization
for the relative abundance of CYPs confirmed that CYP3A4 played a predominant role
in O-dealkylation of M1 from ipriflavone. CYP1A2 played a major role in hydroxylation of
ipriflavone to M3 and M4 as well as M4 oxidation to M5. Also, CYP2C9 partly contributed
to ipriflavone oxidation to M1, M3 and M4.

Ipriflavone metabolism
253
Table II. Kinetic parameters for the formation of M1, M3 and M4 from ipriflavone and M4 oxidation to M5 in
expressed human CYP enzymes. Since the role of each CYP has been determined relative to others, their Clint
(Vmax/Km) values have been normalized for the relative hepatic abundance of each CYP.
Expressed human CYPs
M1
M3
M4
M5
CYP 3A4
Km (mM)
5.9
0.94
0.16
62.3
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
N.C.
Vmax (pmol pmol CYPꢀ1 minꢀ1
)
)
)
)
Clint (ml pmol CYPꢀ1 minꢀ1
Clint (% of total)
)
CYP 1A2
Km (mM)
3.8
4.0
0.86
0.22
77.9
3.9
0.33
0.085
59.7
23.8
0.58
Vmax (pmol pmol CYPꢀ1 minꢀ1
0.12
0.03
5.4
Clint (ml pmol CYPꢀ1 minꢀ1
Clint (% of total)
)
0.024
100.0
CYP 2C9
Km (mM)
6.2
0.78
0.13
25.1
4.1
0.2
3.9
0.17
0.044
35.6
N.C.
N.C.
N.C.
N.C.
Vmax (pmol pmol CYPꢀ1 minꢀ1
Clint (ml pmol CYPꢀ1 minꢀ1
Clint (% of total)
)
0.049
20.1
CYP 2C19
Km (mM)
2.7
3.3
2.3
N.C.
N.C.
N.C.
N.C.
Vmax (pmol pmol CYPꢀ1 minꢀ1
0.40
0.15
7.0
0.067
0.02
2.0
0.055
0.024
4.6
Clint (ml pmol CYPꢀ1 minꢀ1
Clint (% of total)
)
N.C.: Not calculable.
Table III. Correlation of ipriflavone oxidation rates with other CYP activities in 12 human liver microsome
preparations.
Correlation coefficient (r)
Enzyme activities
CYP
M1
M3
M4
Phenacetin O-deethylation
Coumarin 7-hydroxylation
Paclitaxel 6ꢁ-hydroxylation
Diclofenac 40-hydroxylation
[S]-Mephenytoin 40-hydroxylation
Bufuralol 10-hydroxylation
Chlorzoxazone 6-hydroxylation
Testosterone 6ꢀ-hydroxylation
1A2
2A6
2C8
2C9
2C19
2D6
2E1
0.980*
ꢀ0.115
ꢀ0.0578
0.385
0.984*
ꢀ0.161
ꢀ0.0983
0.332
0.981*
ꢀ0.112
ꢀ0.0879
0.314
0.307
0.322
0.333
ꢀ0.0264
0.0987
ꢀ0.0749
0.00275
0.144
0.0286
0.109
3A4
ꢀ0.0130
ꢀ0.0537
*p < 0.05.
Correlation of rates of ipriflavone oxidation with other enzyme activities
in human liver microsomes
The rates of ipriflavone oxidation at 2 mM concentration to M1, M3 and M4 in the
12 different human liver microsomes were 11.8–83.2, 4.0–49.0 and
8.2–132.7 pmol minꢀ1 mg proteinꢀ1, respectively. Table III shows correlations between the
biotransformation rates of ipriflavone to M1, M3 or M4 in 12 human liver microsome
preparations and their standard CYP isoform-specific activities. Significant correlations
were observed between M1, M3 or M4 formation and CYP1A2-catalyzed phenacetin
O-deethylation.

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Y. Moon et al.
Immuno-inhibition of ipriflavone oxidation in human liver microsomes
To determine which CYP is primarily responsible for ipriflavone metabolism to M1, M3 and
M4, immuno-inhibition studies with anti-CYP1A2, anti-CYP2C, anti-CYP2D6, anti-
CYP2E1 or anti-CYP3A4 were performed in pooled human liver microsomes (H161)
(Figure 3). The formation of M1 was inhibited by anti-CYP3A4, anti-CYP1A2 and anti-
CYP2C, suggesting the involvement of CYPs 3A4, 2C and 1A2 in O-dealkylation of
ipriflavone to M1 (Figure 3a). Anti-CYP1A2 potently inhibited the formation of M3 and
M4 from ipriflavone as well as M4 oxidation to M5 (Figure 3b–d). Anti-CYP3A4, anti-
CYP2C, anti-CYP2E1 and anti-CYP2D6 did not inhibit the formation of M3, M4 and M5.
These results indicate that CYP1A2 may play a major role in the formation of M3, M4 and
M5 from ipriflavone or M4.
(a)
(b)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
2
4
6
8
10
0
2
4
6
8
10
Anti-CYPs (µL)
Anti-CYPs (µL)
(c)
(d)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
2
4
6
8
10
0
2
4
6
8
10
Anti-CYPs (µL)
Anti-CYPs (µL)
Figure 3. Effects of CYP antibodies on the biotransformation of ipriflavone to (a) M1, (b) M3 and
(c) M4, and (d) the oxidation of M4 to M5. Human pooled microsomes (H161, 0.15 mg protein mlꢀ1
)
were pre-incubated with anti-CYP3A4 (f), anti-CYP1A2 (e), anti-CYP2D6 (g), anti-CYP2C (n)
or anti-CYP2E1 (j). Data are derived from the average of two determinations. Rates of control
reactions for the formation of M1, M3, M4 and M5 were 43.1, 26.2, 27.0 and
10.1 pmol minꢀ1 mg proteinꢀ1, respectively.

Ipriflavone metabolism
255
M1 formation
M3 formation
M4 formation
M5 formation
140
120
100
80
60
40
20
0
Figure 4. Effects of CYP isoform-selective inhibitors on the formation of M1, M3 and M4 from
ipriflavone and M4 metabolism to M5. Pooled human liver microsomes (H161, 0.15 mg protein mlꢀ1
)
were incubated at 37ꢁC for 20 min with 2 mM ipriflavone and NADPH generating system in the
presence and absence of inhibitors. Each bar represents the average of two determinations. FURA,
furafylline (10 mM); COUM, coumarin (100 mM); TEPA, triethylenethiophosphoramide (5 mM);
MONT, montelukast (0.1 mM); SULF, sulfaphenazole (10 mM); S-BEN, S-benylnirvanol (1 mM);
QUIN, quinidine (10 mM); DETC, diethyldithiocarbamate (10 mM); KETO, ketoconazole (1 mM).
Rates of control reactions for the formation of M1, M3, M4 and M5 were 46.3, 24.8, 25.4 and
9.4 pmol minꢀ1 mg proteinꢀ1, respectively.
Chemical inhibition of ipriflavone metabolism in human liver microsomes
Figure 4 shows the effects of selective inhibitors of CYP450s on the metabolism of
ipriflavone to M1, M3 and M4 as well as M5 formation from M4 in pooled human liver
microsomes (H161). The rate of M1 formation was potently inhibited up to 70% and 40%
by ketoconazole (1 mM, a CYP3A4 inhibitor) and furafylline (10 mM, a CYP1A2 inhibitor),
respectively, whereas the effect of the other inhibitors tested on the formation of M1 was less
than 15%. Furafylline (10 mM) extensively inhibited the formation of M3 (85%) and M4
(40%) from ipriflavone and M5 formation (60%) from M4. These results suggest that
CYP3A4 might play a major role in O-dealkylation to M1 and CYP1A2 might play the
major role in the formation of M1, M3, M4 and M5.
In vitro inhibition of CYP enzymes by ipriflavone and its five metabolites
Inhibition of CYP activity was evaluated at concentrations up to 100 mM of ipriflavone and
its five metabolites, i.e. M1–M5, to investigate the effect of ipriflavone and its five
metabolites on CYP-mediated drug interactions (Table IV). Ipriflavone was a potent
inhibitor, with IC50 value of 7.3 mM against phenacetin O-deethylase (CYP1A2) activity
and IC50 value of 10.8 mM against [S]-mephenytoin 40-hydroxylase (CYP2C19)
activity. Ipriflavone at a concentration of 100 mM did not affect coumarin 7-hydroxylation
(CYP2A6), paclitaxel 6ꢁ-hydroxylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9),
bufuralol 10-hydroxylation (CYP2D6) and midazolam 1-hydroxylation (CYP3A4).

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Y. Moon et al.
Table IV. Inhibitory potentials of ipriflavone and its metabolites M1–M5 on specific CYP activities in
human liver microsomes.
IC50 (mM)
Enzyme activities
CYP
M1
M2
M3
M4
M5
Ipriflavone
Phenacetin O-deethylase
Coumarin 7-hydroxylase
Paclitaxel 6ꢁ-hydroxylase
Diclofenac 4-hydroxylase
[S]-Mephenytoin 40-hydroxylase
Bufuralol 10-hydroxylase
Midazolam 1-hydroxylase
1A2
2A6
2C8
2C9
2C19
2D6
3A4
33.6
13.4
9.9
–
–
–
65.2
–
6.8
–
–
–
7.3
–
–
10.2
19.6
–
31.8
21.9
1.5
2.5
6.0
–
11.2
4.5
–
–
1.7
56.2
–
10.8
–
21.1
86.6
71.9
–
19.4
63.7
0.1
–
–
-: unaffected at 100 mM.
M1 potently inhibited [S ]-mephenytoin 40-hydroxylation (CYP2C19), paclitaxel 6ꢁ-
hydroxylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9) and coumarin 7-hydro-
xylation (CYP2A6) with IC50 values of 4.5, 9.9, 11.2 and 13.4 mM, respectively. M1 also
inhibited bufuralol 10-hydroxylation (CYP2D6), phenacetin O-deethylation (CYP1A2) and
midazolam 1-hydroxylation (CYP3A4) with IC50 values of 21.1, 33.6 and 86.6 mM,
respectively. M2, daidzein inhibited paclitaxel 6ꢁ-hydroxylation (CYP2C8), diclofenac
4-hydroxylation (CYP2C9) and bufuralol 10-hydroxylation (CYP2D6) with IC50 values of
10.2, 19.6 and 71.9 mM, respectively. M3 potently inhibited [S]-mephenytoin
40-hydroxylase (CYP2C19) activity with an IC50 value of 1.7 mM and weakly inhibited
bufuralol 10-hydroxylation (CYP2D6) and coumarin 7-hydroxylation (CYP2A6) with IC50
values of 56.2 and 65.2 mM, respectively. M4 was a potent inhibitor of [S]-mephenytoin
40-hydroxylation (CYP2C19) and phenacetin O-deethylation (CYP1A2) with IC50 values of
1.5 and 6.8 mM, respectively. M4 inhibited bufuralol 10-hydroxylation (CYP2D6),
diclofenac 4-hydroxylation (CYP2C9), paclitaxel 6ꢁ-hydroxylation (CYP2C8) and mid-
azolam 1-hydroxylation (CYP3A4) with an IC50 value of 19.4, 21.9, 31.8 and 63.7 mM,
respectively. M5 potently inhibited paclitaxel 6ꢁ-hydroxylation (CYP2C8) and diclofenac
4-hydroxylation (CYP2C9) with an IC50 value of 2.5 and 6.0 mM, respectively.
Discussion
Ipriflavone has been reported to be metabolized to seven metabolites, i.e. M1–M7 in animals
and human (Figure 1) but only M1, M3, M4 and M5 were identified from the incubation of
ipriflavone with 12 different human liver microsome preparations in the current study. The
formation rates of M5 from ipriflavone were very low and inconsistent in human liver
microsomes, and therefore, M5 formation was determined from the incubation of M4 with
human liver microsomes. Clint values for M1, M3, M4 and M5 formation in three human
liver microsomes were 18.6 (ꢄ9.0), 19.6 (ꢄ13.9), 41.5 (ꢄ39.1) and 6.0
(ꢄ3.8) ml mg proteinꢀ1 minꢀ1, respectively (Table I), suggesting that isopropyl hydroxylation
to M4, O-dealkylation to M1, ꢀ-ring hydroxylation to M3 and M4 oxidation to M5 are the
major metabolic pathways for ipriflavone metabolism in human liver microsomes in vitro.
The specific CYP isoforms responsible for ipriflavone oxidation to M1, M3, M4 and M5
were identified using a combination of correlation analysis, immuno-inhibition, chemical

Ipriflavone metabolism
257
inhibition in human liver microsomes and metabolism by expressed recombinant CYP
enzymes. From a screen of ipriflavone or M4 metabolism using microsomes containing
cDNA-expressed CYPs, it was shown that the ipriflavone oxidations might be mediated by
CYPs 3A4, 1A2, 2C9 and/or 2C19. The predominant role of CYP3A4 was evident in
ipriflavone oxidation to M1 when the contributions of each CYP were normalized for their
relative abundance in the liver (Table II). CYP1A2 played the major role in the formation of
M3, M4 and M5. CYP2C9 played the role in the formation of M1, M3 and M4 from
ipriflavone. Chemical inhibitor and CYP antibody studies also support the roles for CYPs
3A4, 1A2 and 2C9 in O-dealkylation of ipriflavone to M1 (Figures 3 and 4). Furafylline, a
specific CYP1A2 inhibitor and anti-CYP1A2 antibody potently inhibited the formation of
M3 and M4 from ipriflavone and M4 oxidation to M5. These results collectively suggest that
CYP3A plays the major role in ipriflavone O-dealkylation to M1 and CYP1A2 plays the
predominant role in the formation of M3, M4 and M5. CYP2C9 may partly contribute to
the formation of M1, M3 and M4 from ipriflavone.
Expression of CYP3A4 has been observed to be highly variable among human liver
samples (Wrighton et al. 1990; Shimada et al. 1994; Gibson et al. 2002). Wide inter-
individual variability has also been reported in the expression of CYP1A2 in humans
(Schweikl et al. 1993). Genetic polymorphisms in CYP2C9 have been shown to affect the
metabolism, efficacy and toxicity of clinically important drugs (Goldstein 2001). CYPs 3A4,
1A2 and 2C9 are responsible for the metabolism of ipriflavone, and, therefore, inter-
individual variability in plasma concentrations of ipriflavone may be expected owing to
variable rates of metabolism. CYPs 3A4, 1A2 and 2C9 have been shown to be induced or
inhibited by a variety of xenobiotics (Miners and Birkett 1998; Miners and McKinnon 2000;
Wrighton and Thummel 2000; Luo et al. 2004). Therefore, any co-administered drugs that
can inhibit or induce CYP3A4, CYP1A2 or CYP2C9 may alter the metabolism of
ipriflavone and potentially lead to changes in the pharmacokinetics of ipriflavone in patients.
The effect of ipriflavone and its major metabolites, i.e. M1, M2, M3, M4 and M5, on the
activities of clinically important human CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4
was also investigated in human liver microsomes (Table IV). Phenacetin O-deethylase
(CYP1A2) activity was potently inhibited by ipriflavone and M4 with IC50 values of 7.3 and
6.8 mM, respectively. Diclofenac 4-hydroxylase, a marker of CYP2C9, activity was
extensively inhibited by M5 and M1 with IC50 values of 6.0 and 11.2 mM, respectively.
[S]-mephenytoin 40-hydroxylase (CYP2C19) activity was strongly inhibited by M4, M3, M1
and ipriflavone with IC50 values of 1.5, 1.7, 4.5 and 10.8 mM, respectively. Paclitaxel
6ꢁ-hydroxylation (CYP2C8) was potently inhibited by M5, M1 and M2 with IC50 values of
2.5, 9.9 and 10.2 mM, respectively. These in vitro data suggest that ipriflavone should be
used carefully with drugs metabolized by CYPs 1A2, 2C8, 2C9 and 2C19 to avoid possible
drug–drug interactions. There is evidence that ipriflavone co-administration resulted in a
decrease in theophylline clearance in patients as a result of CYP inhibition (Takahashi et al.
1992; Monostory and Vereczkey 1995, 1996). Coumarin 7-hydroxylation (CYP2A6),
bufuralol 10-hydroxylation (CYP2D6) and midazolam 1-hydroxylation (CYP3A4) activities
were moderately or weakly inhibited, and, therefore, it is very unlikely that ipriflavone
treatment will significantly alter the clearance of other compounds metabolized by CYPs
2A6, 2D6 and/or 3A4.
In conclusion, this study demonstrates that hydroxylation of the isopropyl moiety of
ipriflavone to M4 and M5, ꢀ-ring hydroxylation to M3 and O-dealkylation to M1 are the
major metabolic pathways for ipriflavone in human liver microsomes. CYP3A4 plays the
major role in O-dealkylation to M1 and CYP1A2 plays the dominant role in the formation
of M3, M4 and M5. CYP2C9 are responsible for ipriflavone oxidations. The possibility

258
Y. Moon et al.
of drug–drug interactions would therefore be predicted when prescribing ipriflavone
concomitantly with known inducers or inhibitors of CYPs 3A4, 1A2 and 2C9. Ipriflavone
and its five metabolites were observed to inhibit potently the metabolism of CYPs 1A2, 2C8,
2C9 and 2C19 probes, indicating that ipriflavone may have the significant effects on the
pharmacokinetics of other drugs metabolized by CYPs 1A2, 2C8, 2C9 and/or 2C19.
Acknowledgements
This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health
and Welfare, Republic of Korea (A060520).
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