September 2005 Notes Biol. Pharm. Bull.

28(9) 1805—1808 (2005) 1805

Effect of Antifungal Drugs on Cytochrome P450 (CYP) 2C9, CYP2C19,

and CYP3A4 Activities in Human Liver Microsomes
Toshiro NIWA,*,a,1) Toshifumi SHIRAGA,b,2) and Akira TAKAGIa,1)
a
Post-marketing Development Research Center, Fujisawa Pharmaceutical Co., Ltd.; 3–4–7 Doshomachi, Chuo-ku, Osaka
541–8514, Japan: and b Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co.,
Ltd.; 2–1–6 Kashima, Yodogawa-ku, Osaka 532–8514, Japan. Received March 22, 2005; accepted May 31, 2005

The effects of five antifungal drugs, fluconazole, itraconazole, micafungin, miconazole, and voriconazole, on
cytochrome P450 (CYP) 2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4
-hy-
droxylation, and CYP3A4-mediated nifedipine oxidation activities in human liver microsomes were compared. In
addition, the effects of preincubation were estimated to investigate the mechanism-based inhibition. The IC50
value against tolbutamide hydroxylation was the lowest for miconazole (2.0 m M), followed by voriconazole (8.4 m M)
and fluconazole (30.3 m M). Similarly, the IC50 value against S-mephenytoin 4
-hydroxylation was the lowest for
miconazole (0.33 m M), followed by voriconazole (8.7 m M) and fluconazole (12.3 m M). On the other hand, micafun-
gin at a concentration of 10 or 25 m M neither inhibited nor stimulated tolbutamide hydroxylation and S-
mephenytoin 4
-hydroxylation, and the IC50 values for itraconazole against these were greater than 10 m M. These
results suggest that miconazole is the strongest inhibitor of CYP2C9 and CYP2C19, followed by voriconazole and
fluconazole, whereas micafungin would not cause clinically significant interactions with other drugs that are me-
tabolized by CYP2C9 or CYP2C19 via the inhibition of metabolism. The IC50 value of voriconazole against
nifedipine oxidation was comparable with that of fluconazole and micafungin and higher than that of itracona-
zole and miconazole. The stimulation of the inhibition of CYP2C9-, CYP2C19-, or CYP3A4-mediated reactions
by 15-min preincubation was not observed for any of the antifungal drugs, suggesting that these drugs are not
mechanism-based inhibitors.
Key words antifungal drug; fluconazole; itraconazole; micafungin; miconazole; voriconazole

Cytochrome P450s (CYP) comprise a superfamily of en- there are few studies comparing the effects of antifungal
zymes that catalyzes the oxidation of a wide variety of xeno- drugs on human hepatic CYP-mediated drug-metabolizing
biotic chemicals, including drugs and carcinogens.3—5) Multi- activity under the same experimental conditions.
ple-drug therapy is a common therapeutic practice, particu- Many inhibitors are known to be activated metabolically
larly in patients with several diseases or conditions, and to a reactive intermediate(s) that, in turn, is irreversibly
many drug–drug interactions involving metabolic inhibition or quasi-irreversibly bound to the enzyme(s).19) Some
are reported.6,7) acetylenes, including those synthetic steroids such as gesto-
Antifungal drugs, including fluconazole, itraconazole, mi-
cafungin, miconazole, and voriconazole (Fig. 1), are widely
used in the treatment of systemic candidal infections and my-
coses. The mechanism of action of these antifungal drugs is
the inhibition of fungal CYP (14a -sterol demethylase), an
enzyme responsible for the conversion of lanosterol to 14a -
demethyllanosterol in the ergosterol biosynthetic pathway,8,9)
except that micafungin inhibits 1,3-b -D-glucan synthase,
leading to disruption of the growing fungal cell wall and
death of the fungal cell.10,11) Because antibiotics, including
antifungal drugs, are coadministered in most cases, the possi-
bility of interactions between them and other drugs exists.
Recently, we have investigated the effects of antifungal drugs
excluding voriconazole on CYP3A4 activity and multidrug
resistance protein 1 (MDR1), and found that itraconazole and
miconazole, as well as ketoconazole, had greater inhibitory
effects on both CYP3A4 metabolic and MDR1 transport ac-
tivities than fluconazole and micafungin.12) In addition, it has
been demonstrated that the Ki values of fluconazole and mi-
cafungin against nifedipine oxidation activity, a marker en-
zyme activity of CYP3A4, are 10.7 m M and 17.3 m M, respec-
tively.13) Zhang et al.14) reported that miconazole competi-
tively inhibits several CYPs, including CYP2C9, CYP2C19,
and CYP3A4, with Ki values ranging from 0.01 to 7.3 m M.
Furthermore, it is likely that fluconazole and voriconazole in-
hibit CYP2C9, CYP2C19, and CYP3A4.9,15—18) However, Fig. 1. Chemical Structures of Antifungal Drugs

∗ To whom correspondence should be addressed. e-mail: toshiro.niwa@jp.astellas.com © 2005 Pharmaceutical Society of Japan
1806 Vol. 28, No. 9

dene and ethynyl estradiol, have been demonstrated to cause hibition against CYP activities was conducted for 1—30 min,
mechanism-based inactivation of CYP3A4.20) Sorivudine is and obvious preincubation-dependent increases in the inhibi-
converted by gut flora to (E)-5-(2-bromovinyl)uracil (BVU), tion rates were observed in more than 5—10 min preincuba-
which is metabolized to dihydro-BVU by dihydropyrimidine tion.20,22—30) In addition, Draper et al. examined the in-
dehydrogenase (DPD), and the dihydro-BVU binds to DPD hibitory effects of 5-min preincubation on coumarine 7-hy-
itself.21) It has been reported that these mechanism-based droxylase (CYP2A6) activity for 47 chemicals, including flu-
inhibitors exhibit preincubation time dependence of in- conazole and itraconazole.31) Therefore the incubation mix-
hibition.18—31) ture without substrate was preincubated at 37 °C for 15 min.
In the present study, we compared the effects of five anti- After preincubation, the substrate was added and then incu-
fungal drugs on specific activities of CYP2C9, CYP2C19, bated at 37 °C as mentioned above.
and CYP3A4 in human liver microsomes under the same ex- Data Analysis All data were analyzed using the average
perimental conditions. In addition, the effects of preincuba- of duplicate, triplicate, or quadruplicate determinations. In
tion were estimated to investigate whether these antifungal preliminary experiments, the linearity of reaction with incu-
drugs are mechanism-based inhibitors. bation time and protein concentration was confirmed for each
assay condition. IC50 values were calculated using the Win-
MATERIALS AND METHODS Nonlin Standard computer program (Version 4.1, Pharsight,
CA, U.S.A.). Because the substrate concentrations are around
Materials Pooled human liver microsomes from 46 or the Km values, Ki values are expected to be equal to IC50/2 or
50 individuals (lot no. 0210171 or lot no. 0310241) were ob- IC50 in competitive or noncompetitive inhibition, respec-
tained from XenoTech (Lenexa, KS, U.S.A.). Micafungin tively.31) Statistical differences for the determination of the
and voriconazole were synthesized in the Medicinal Chem- effect of preincubation were determined with Student’s t-test
istry Research Laboratories, Fujisawa Pharmaceutical Co., using the SAS program (SAS Institute Inc, Cary, NC,
Ltd. Miconazole and fluconazole were purchased from ICN U.S.A.), and the significance level was set at p0.05.
Biomedicals, Inc. (Irvine, CA, U.S.A.) and itraconazole from
Janssen-Kyowa (Tokyo, Japan). Tolbutamide and nifedipine RESULTS
were obtained from Sigma Chemical Co. (St. Louis, MO,
U.S.A.). S-Mephenytoin, 4
-hydroxymephenytoin, hydroxy- Inhibition of CYP Activities by Antifungal Drugs We
tolbutamide, and oxidized nifedipine were purchased from have reported the inhibitory effects of antifungal drugs ex-
Ultrafine Chemicals (Manchester, U.K.). Phenobarbital cluding voriconazole on CYP3A4 activity.12) Therefore the
sodium and p-hydroxybenzoic acid isopropyl were obtained effects of voriconazole on CYP3A4 activity and the effects of
from Wako Pure Chemicals (Osaka, Japan) and Tokyo five antifungal drugs on CYP2C9 and CYP2C19 activities
Chemical Industry (Tokyo, Japan), respectively. All other were investigated in this paper (Fig. 2, Table 1). The IC50
reagents were of the highest purity commercially available. value against tolbutamide hydroxylation (CYP2C9) was the
Determination of Human CYP Activities Tolbutamide lowest for miconazole (2.0 m M), followed by voriconazole
hydroxylation activity (CYP2C9), S-mephenytoin 4
-hydrox- (8.4 m M) and fluconazole (30.3 m M). Similarly, the IC50 value
ylation activity (CYP2C19), and nifedipine oxidation activity
(CYP3A4) in human liver microsomes in the presence or ab-
sence of antifungal drugs were determined as described pre-
viously.32,33) The incubation mixture consisted of human mi-
crosomes, 2 mM NADP, 10 mM glucose-6-phosphate, 5 mM
magnesium chloride, 1 unit/ml of glucose-6-phosphate dehy-
drogenase, 100 mM phosphate buffer (pH 7.4), and 5 m l of
methanol or 0.001—5 mM antifungal drugs dissolved in
methanol in a final volume of 500 m l.21,22) The maximum
concentration of itraconazole in the incubation mixture was
10 m M, because itraconazole at a concentration greater than
1 mM was not dissolved in methanol. The microsomal protein
concentration in the mixture was 0.05 (nifedipine oxidation)
or 0.5 mg/ml (for tolbutamide hydroxylation and S-mepheny-
toin 4
-hydroxylation). Because the Km values for tolbu-
tamide hydroxylation, S-mephenytoin 4
-hydroxylation, and
nifedipine oxidation by human liver microsomes were 150.8,
27.3, and 12.2 m M, respectively (data not shown),21,22) the
concentrations of tolbutamide, S-mephenytoin, and nifedip-
ine were 200, 30, and 10 m M, respectively, which are around
the expected Km values. Incubation was carried out at 37 °C
for 10 min (for the determination of IC50 against nifedipine
oxidation), 15 min (for the determination of the effects of
preincubation on nifedipine oxidation), or 30 min (for tolbu- Fig. 2. Effect of Antifungal Drugs on CYP2C9 and CYP2C19 Activities
tamide hydroxylation and S-mephenytoin 4
-hydroxylation). in Human Liver Microsomes
Preincubation in many studies of the mechanism-based in- The results are shown as means of duplicate determinations.
September 2005 1807

Table 1. IC50 Values of Antifungal Drugs against CYP2C9, CYP2C19,

and CYP3A4 Activities

IC50 (m M)
Antifungal drug
CYP2C9 CYP2C19 CYP3A4

Fluconazole 30.3 12.3 13.1a)

Itraconazole 10 10 0.0326a)
Micafungin No inhibitionb) No inhibitionb) 13.5a)
Miconazole 2.0 0.33 0.0742a)
Voriconazole 8.4 8.7 10.5

a) Sakaeda et al.12) b) No inhibition was observed at 10 and 25 m M concentrations.

against S-mephenytoin 4
-hydroxylation (CYP2C19) was the

lowest for miconazole (0.33 m M), followed by voriconazole
(8.7 m M) and fluconazole (12.3 m M). IC50 values of itracona-
zole against these metabolic activities were greater than 10
m M. The IC50 value of voriconazole against nifedipine oxida-
tion (CYP3A4) was comparable with that of fluconazole and
micafungin and higher than that of itraconazole and micona-
zole. On the other hand, micafungin at a concentration of 10
or 25 m M neither inhibited nor stimulated CYP2C9 and
CYP2C19 activities (Fig. 3).
Effects of Preincubation on Inhibitory Effects of Anti-
fungal Drugs The effects of 15-min preincubation on the
inhibitory effects of antifungal drugs are shown in Fig. 3.
The stimulation of the inhibition of CYP2C9-, CYP2C19-, or
CYP3A4-mediated reactions by 15-min preincubation was
not observed for any of the antifungal drugs, suggesting that
these drugs are not the mechanism-based inhibitors. On the
other hand, a slightly decrease in the inhibition of the
CYP2C19-mediated reaction after preincubation was ob-
served for miconazole and voriconazole.

DISCUSSION Fig. 3. Effect of Preincubation on the Inhibition of Antifungal Drugs

The IC50 value of voriconazole against CYP3A4 activity
(10.5 m M) was higher than that of itraconazole and micona-
zole, and comparable with that of fluconazole and micafun-
gin (Table 1). Itraconazole and miconazole are potent clini-
cally important inhibitors of the clearance of CYP3A4 sub-
strates.15) In addition, it has been reported that fluconazole
(200 mg/d) elevates the plasma/blood concentrations of mi-
dazolam and cyclosporine,15,34—38) which are mainly metabo-
lized by CYP3A4,39) and that voriconazole increases the
blood concentrations of cyclosporin.17,40) However, the blood
concentrations of tacrolimus and cyclosporin are not affected
by coadministration of micafungin.41,42) Fluconazole and
voriconazole are orally administered, whereas micafungin is
given only by infusion. It has been reported that the maxi-
mum plasma concentration (Cmax) after an oral dosing of
100 mg of fluconazole is 1.88 m g/ml (6.1 m M),34) that Cmax
after oral dosing with voriconazole (200 mg bid) is
1.89 m g/ml (5.4 m M),9) and that Cmax after 1-h infusion of
150 mg of micafungin is 14.30 m g/ml (11.3 m M).43) In addi-
tion, protein binding of fluconazole,34) voriconazole,9,17) and
micafungin43,44) in human serum or plasma is reported to be
11—12%, 58%, and 99.8%, respectively. Therefore it is
speculated that fluconazole and voriconazole, but not mica-
fungin, might be sufficiently distributed to the liver to cause
drug interactions, although they have similar inhibitory ef-
against CYP2C9, CYP2C19, and CYP3A4 Activities in Human Liver Mi-
crosomes
The results are shown as meansS.D. (n3—4). Values in parentheses show the
concentrations of antifungal drugs (m M). ∗ p0.05, ∗∗ p0.01.
fects in vitro. In addition, because the intestinal first-pass me-
tabolism mediated by CYP3A4 has been shown to be clini-
cally relevant for several drugs,45) drug interactions with oral
antifungal drugs might occur not only in the liver but also
small intestine.
IC50 values against tolbutamide hydroxylation (CYP2C9)
and S-mephenytoin 4
-hydroxylation (CYP2C19) were the
lowest for miconazole (0.33—2.0 m M), followed by voricona-
zole (8.4—8.7 m M) and fluconazole (12.3—30.3 m M). IC50
values for itraconazole against these metabolic activities
were greater than 10 m M (Table 1). On the other hand, mica-
fungin at 10 or 25 m M neither inhibited nor stimulated
CYP2C9 and CYP2C19 activities (Fig. 3). Although the IC50
values of miconazole against CYP2C9 and CYP2C19 were
higher than those against CYP3A4, the IC50 values of flu-
conazole and voriconazole against CYP2C9 and CYP2C19
were comparable with those against CYP3A4. Therefore it is
possible to speculate that fluconazole and voriconazole,
but not micafungin, would clinically cause drug interactions
with other drugs, which are metabolized by CYP2C9 or
CYP2C19 as well as CYP3A4. It has been reported that the
plasma concentrations of phenytoin and S-warfarin, which
1808 Vol. 28, No. 9

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