0090-9556/01/2907-10681072$3.

00
DRUG METABOLISM AND DISPOSITION
Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:10681072, 2001

Vol. 29, No. 7

331/915884
Printed in U.S.A.

DRUG INTERACTION BETWEEN SIMVASTATIN AND ITRACONAZOLE IN MALE AND

FEMALE RATS
MICHI ISHIGAMI, KIYOSHI KAWABATA, WATARU TAKASAKI, TOSHIHIKO IKEDA, TORU KOMAI, KIYOMI ITO,
YUICHI SUGIYAMA

AND

Drug Metabolism and Pharmacokinetics Research Laboratories, New Drug Development Division and Product Strategy Department, Sankyo
Co., Ltd., Shinagawa-ku, Tokyo, Japan (M.I., K.K., W.T., T.I., T.K.); School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo,
Japan (K.I.); and Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan (Y.S.)
(Received January 2, 2001; accepted April 10, 2001)

This paper is available online at http://dmd.aspetjournals.org

ABSTRACT:
Taking into account the species and sex differences in drug
interactions based on the inhibition of cytochrome P450 (P450)mediated drug metabolism, we examined whether the interaction between simvastatin and itraconazole observed in humans
could also occur in rats, the most commonly used animal species for pharmacokinetic studies. Itraconazole inhibited the in
vitro metabolism of simvastatin in female rat liver microsomes,
but not in male rat liver microsomes. Using anti-P450 antisera,
the main P450 isozyme responsible for the metabolism of simvastatin was identified as CYP3A in female rats and CYP2C11 in
male rats. Therefore, the sex difference in the inhibition of simvastatin metabolism by itraconazole seems to be caused by a
difference in the P450 isozymes responsible for the metabolism

of simvastatin in male and female rats and the different ability of

itraconazole to inhibit CYP3A and CYP2C11. In addition, the
effect of itraconazole on the pharmacokinetics of simvastatin in
rats was also investigated. The area under the curve value of
simvastatin was increased approximately 1.6-fold by the concomitant use of itraconazole (50 mg/kg) in female rats, whereas
in male rats, itraconazole had no effect. In conclusion, it was
found that the results obtained in male rats did not reflect the
results in humans as far as the inhibition of simvastatin metabolism by itraconazole was concerned. The P450 isozymes involved in the metabolism of drugs should be taken into consideration when rats are used as a model animal for humans in the
investigation of drug interactions.

Drug interactions can be classified into two types: in one case the
pharmacological effects or side-effects of drugs are altered by concomitantly administered drugs, and in the other case the effects and sideeffects of the concomitantly administered drugs are altered by the original
drugs. In both cases, the drug interactions have been evaluated based on
changes in plasma drug levels in clinical situations. In the case of
HMG-CoA1 reductase inhibitors, it has been reported in clinical situations that plasma levels of simvastatin and lovastatin, which are in their
prodrug lactone forms, were increased more than 10-fold by the concomitantly administered antifungal agent, itraconazole (Neuvonen and Jalava,
1996; Neuvonen et al., 1998). Itraconazole is known as a potent inhibitor
of CYP3A4, one of the major cytochrome P450 (P450) isozymes in
humans (Olkkola et al., 1994; Varhe et al., 1994).
We have investigated the causes of the increase in the plasma levels of
the prodrug-type lactone forms of HMG-CoA reductase inhibitors by
concomitantly administered itraconazole. We used a pharmacokinetic
model (Ito et al., 1998a,b) involving the pharmacokinetic parameters for
itraconazole and the Ki values obtained in in vitro studies using human

liver microsomes. The predicted increase in plasma simvastatin levels by

the concomitant use of itraconazole agreed reasonably well with those
observed in clinical situations (Ishigami et al., 2001).
In the case of drugs at the development stage, in vivo drug interaction studies have been sometimes conducted with experimental
animals because it is difficult to conduct such studies at this stage in
humans (Damanhouri et al., 1988; Ikeda et al., 1988). It has become
possible, to a certain extent, to predict the possibility of in vivo drug
interactions in humans from results obtained in in vitro systems using
human liver microsomes. For the scale-up of human metabolism from
in vitro to in vivo, some information about the drug concentration in
liver is required; however, the measurement is usually impossible. In
experimental animals administered with the drug, the concentrations
in liver can be easily measured, and the information can be referred to
for prediction of in vivo drug interaction in human from in vitro
metabolism. For the prediction to be more accurate, it is necessary to
choose an appropriate animal as a model. However, there is a possibility that the results obtained in experimental animals do not reflect
the drug interactions in humans because of species (Nelson et al.,
1996; Eagling et al., 1998) and sex differences (Kato and Kamataki,
1982; Kamataki et al., 1983) in P450 isozymes.
Accordingly, taking into account the species and sex differences in
the drug interactions based on the inhibition of P450 activities, we
have investigated whether a drug interaction in humans actually
occurs in rats, the most commonly used animal species in pharmacokinetic studies. In vitro and in vivo inhibition studies were conducted

Abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme

A; V0, initial formation rate; HPLC, high-performance liquid chromatography; TLC,
thin-layer chromatography; CMC, carboxymethylcellulose; AUC, area under the
curve.
Address correspondence to: Yuichi Sugiyama, Professor, Graduate School
of Pharmaceutical Sciences, University of Tokyo, 3-1,7-Chome, Hongo, Bunkyoku, Tokyo 113-0033, Japan. E-mail: sugiyama@mol.f.u-tokyo.ac.jp

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SEX-DEPENDENT INHIBITION OF SIMVASTATIN METABOLISM IN RATS

in male and female rats to investigate the effect of itraconazole, a
specific inhibitor of CYP3A4 in humans, on simvastatin metabolism,
and the results obtained were compared with the drug interaction
observed in humans.
Materials and Methods
Chemicals and Reagents. 14C-Labeled simvastatin (lactone form), simvastatin (lactone form), and simvastatin acid (Na salt) used in the present study
were synthesized at Sankyo Co., Ltd. (Tokyo, Japan). Rat liver microsomes
were prepared from male and female Sprague-Dawley rats (Japan SLC,
Hamamatsu, Japan) according to conventional methods. Anti-rat P450 antisera
preparations (anti-rat CYP2C11 prepared from goat and anti-rat CYP3A2
prepared from rabbit) were purchased from Daiichi Pure Chemicals Co., Ltd.
(Tokyo, Japan). All other chemicals and reagents used were commercially
available and of guaranteed purity.
In Vitro Metabolism of Simvastatin. After preincubation of 0.2 ml of rat
liver microsome (0.2 mg of protein/ml) containing an NADPH-generating
system (2.5 mM NADP, 25 mM glucose 6-phosphate, 2 units of glucose-6phosphate dehydrogenase, and 10 mM MgCl2) at 37C for 3 min, an ethanol
solution of [14C]simvastatin was added. After a 10-min incubation at 37C, the
reaction was stopped by adding 0.4 ml of ethanol and vortex mixing. The
mixture was centrifuged at 10,000 rpm for 3 min, and the quantity of metabolites in the supernatant was analyzed by HPLC or TLC. The HPLC conditions
were as follows: column, C8 ET250/4 Nucleosil 100-5; mobile phase (linear
gradient), acetonitrile/0.05% phosphoric acid 35:75 (0 min) 3 75:35 (25
min); and flow rate, 1 ml/min. The HPLC eluate was collected at intervals of
30 s, and a certain volume of scintillation cocktail (Pico-Fluor, Packard
Instrument Co., Meriden, CT) was added to each eluate. The radioactivity was
then counted in a liquid scintillation counter (2250 CA, Packard). The TLC
analysis of simvastatin and its metabolites was performed under the following
TLC conditions: silica-gel plates, 0.25 mm thickness, 60 F254 (Merck KgaA,
Darmstadt, Germany); and development solvent system, toluene/acetone/acetic
acid (50:50:0.5, v/v/v). The amount of unchanged drug and its metabolites was
determined by radioluminography using BAS 2000 equipment (Fuji Photo
Film Co., Tokyo, Japan).
To identify the P450 isozymes responsible for the metabolism of simvastatin, an inhibition study using anti-rat P450 antisera was performed. Anti-rat
P450 antisera or corresponding control sera (0 0.25 mg of IgG) were added to
rat liver microsomes (pooled for five male rats or five female rats, 10 mg of
protein/ml, 10 l), and the resulting mixture was preincubated at room temperature for 30 min. Then, in the same manner as described above, NADPHgenerating system was added and preincubated for 3 min at 37C. Subsequently, an ethanol solution of [14C]simvastatin was added to each microsomal
preparation to give a final concentration of 20 M and then incubated for 5
min at 37C (0.2 mg of protein/ml, total volume of 0.5 ml). The reaction was
stopped by addition of 1 ml of methanol, and the quantity of metabolites in the
supernatant was determined by TLC.
Inhibition of Simvastatin Metabolism by Itraconazole. Itraconazole
(dimethylacetamide solution; final concentrations: 0 20 M) and simvastatin
(ethanol solution; final concentration: 20 M) were added to male or female
rat liver microsomal preparations (0.2 mg of protein/ml). After incubation for
10 min at 37C, the amount of simvastatin metabolites formed was determined
in the same manner as described above.
We calculated Ki values from Dixon plots by concurrent fitting of the data
to the following equation using the WinNonlin program:
1/V 0 1 K m/S/Vmax I Km/Ki S Vmax

1069

14

istration of [ C]simvastatin. Immediately after sampling, the plasma was

separated by centrifuging the blood sample at 10,000 rpm for 3 min under
refrigerated conditions. Then a 50-l aliquot of each plasma sample was
transferred to a glass vial and solubilized by mixing with 0.1 ml of tissue
solubilizer (NCS-II, Amersham Pharmacia Biotech, Inc. (Piscataway, NJ).
Subsequently, a liquid scintillation cocktail (Hionic Fluor, Packard) was added
to each solubilized sample, and the radioactivity was counted in a liquid
scintillation counter (2250 CA, Packard). For quantification of the metabolites,
200 l of acetonitrile was added to 100 l of each plasma sample, and after
centrifugation of the mixture at 10,000 rpm for 3 min, the supernatant was
collected. Furthermore, the metabolites remaining in the precipitate were
re-extracted with a mixture of acetonitrile/water (3:1, v/v), and the supernatant
obtained was combined with the supernatant collected above and evaporated to
dryness under the stream of N2 gas. The residue was dissolved in acetonitrile/
water (3:1, v/v), and the metabolites were separated by TLC and quantified by
liquid scintillation counting. The AUC values (0 6 h or 0 8 h) were calculated by the trapezoidal method.

Results
Characteristics of Simvastatin Metabolism in Rat Liver Microsomes. During the incubation of simvastatin with female rat liver
microsomes, metabolites M-1 (6-hydroxy simvastatin), M-2 (3,5dihydrodiol simvastatin), and simvastatin acid were formed as the
main metabolites, which were identical to those observed in human
liver microsomes, although their relative amounts differed. On the
other hand, in male rat liver microsomes, metabolite M-3, which was
not detected in human and female rat liver microsomes, and simvastatin acid were detected as main metabolites (Fig. 1).
The formation of M-1 and M-2, the main metabolites in female rat
liver microsomes, was inhibited by about 80 and 50%, respectively,
by the addition of 0.25 mg of IgG anti-CYP3A2 antiserum, while the
formation of these metabolites was scarcely affected by the addition
of anti-CYP2C11 antiserum (Fig. 2, a and b). These results suggest
that the P450 isozyme responsible for the metabolism of simvastatin
to M-1 and M-2 in female rats is CYP3A. On the other hand, the
formation of M-3, the main metabolite in male rat liver microsomes,
was inhibited by about 90% by the addition of 0.25 mg of IgG
anti-CYP2C11 antiserum, but not by the addition of anti-CYP3A2
antiserum (Fig. 2c). These findings suggest that the P450 isozyme
responsible for the metabolism of simvastatin to M-3 in male rats is
CYP2C11.
Effect of Itraconazole on the Formation of Simvastatin Metabolites (M-1, M-2, and M-3) in Rat Liver Microsomes. The metabolism of simvastatin in female rat liver microsomes was inhibited

(1)

where V0 is the initial rate of metabolism, Km is the Michaelis-Menten

constant, Vmax is the maximum rate of metabolism, and S and I indicate the
concentration of the substrate (simvastatin) and the inhibitor (itraconazole),
respectively.
In Vivo Interaction Study. [14C]Simvastatin suspended in 0.5% carboxymethylcellulose (CMC) solution was administered orally to fasted male
and female rats at a dose of 10 mg/kg immediately after oral administration of
itraconazole suspended in 0.5% CMC solution at a dose of 50 mg/kg or 0.5%
CMC solution. Blood samples (0.3 ml each) were taken from the jugular vein
of each rat using heparinized syringes at various time points after the admin-

FIG. 1. HPLC chromatograms of [14C]simvastatin metabolites produced by

human liver microsomes and rat liver microsomes.
[14C]simvastatin (20 M) was incubated at 37C for 30 min with human liver
microsomes (0.2 mg/ml) or rat liver microsomes (0.2 mg/ml) in the presence of an
NADPH-generating system.

1070

ISHIGAMI ET AL.

FIG. 2. Effects of anti-P450 sera on the formation of M-1(a), M-2(b), and M-3(c) from simvastatin in female and male rat liver microsomes.
Pooled rat liver microsomes (five female or five male rats, 0.1 mg of protein/10 l) were preincubated for 30 min at room temperature with 0.05 to 0.25 mg of IgG
of anti-rat P450 sera or control sera, preincubated for 3 min at 37C with NADPH-generating system, and then incubated with simvastatin (20 M) at 37C for 10 min.
Results (mean S.E.) were based on triplicate determinations. a, female; b, female; c, male. , anti-CYP3A2; , anti-CYP2C11.

by itraconazole in a concentration-dependent manner (Fig. 3a),

whereas the formation of M-3 in male rat liver microsomes was
scarcely inhibited by itraconazole (Fig. 3b).
The inhibition of simvastatin metabolism by itraconazole was kinetically analyzed with Dixon plots of the data obtained from female
rat liver microsomes. As a result, the inhibition of the formation of
M-1 (Fig. 4a) and M-2 (Fig. 4b) by itraconazole was demonstrated to
be competitive. In addition, the Ki values for the inhibition of the
formation of M-1 and M-2 by itraconazole were calculated to be 0.690
and 1.22 M, respectively.
Effect of Concomitantly Administered Itraconazole on the
Pharmacokinetics of Simvastatin in Male and Female Rats. Figure
6 shows the time courses of the plasma concentration of simvastatin after oral administration of simvastatin (10 mg/kg), with or
without concomitant oral administration of itraconazole (50 mg/
kg) to male and female rats. The AUC and Cmax values and the
degree of increase in these parameters produced by itraconazole
are summarized in Table 2. The AUC and Cmax values of the
unchanged drug after oral administration of simvastatin to female
rats was increased about 1.6- and 2.0-fold, respectively, by the

concomitant administration of itraconazole (Fig. 5a; Table 1).

However, the plasma concentration of the unchanged drug after an
oral administration of simvastatin to male rats was not affected by
the concomitant administration of itraconazole (Fig. 5b, Table 1).
Discussion
Simvastatin is metabolized mainly by CYP3A4 in humans
(Vickers et al., 1990; Prueksaritanont et al., 1997). In rats, a sex
difference in the pharmacokinetics of simvastatin has been reported (Ohtawa and Uchiyama, 1992); however, the P450
isozyme(s) responsible for simvastatin metabolism have not been
identified. In the present study, the main simvastatin metabolites
formed by male and female rat liver microsomes were found to be
different, and the main metabolites in female rat liver microsomes,
M-1 (6-hydroxy simvastatin) and M-2 (3,5-dihydrodiol simvastatin), were found to be identical to the main metabolites in human
liver microsomes following comparison of their HPLC retention
times and TLC Rf (ratio at flow) values (Ishigami et al., 2001) (Fig.
1). In contrast, the main metabolite in male rat liver microsomes,
M-3, was shown not to correspond to any of the metabolites

FIG. 3. Inhibition of the formation of simvastatin metabolites, M-1, M-2, and M-3, by itraconazole in female (a) and male (b) rat liver microsomes.
Simvastatin (20 M) was incubated at 37C for 10 min with pooled rat liver microsomes (five male or five female rats, 0.2 mg of protein/ml) in the absence or presence
of itraconazole (0.0042 8.4 M). Control activities were obtained in the absence of itraconazole. Results (mean S.E.) were based on triplicate determinations. , M-1;
, M-2; F, M-3.

1071

SEX-DEPENDENT INHIBITION OF SIMVASTATIN METABOLISM IN RATS

FIG. 4. Dixon plots for the inhibition of the formation of simvastatin metabolites, M-1 (a) and M-2 (b), by itraconazole in female rat liver microsomes.
Simvastatin (550 M) was incubated at 37C for 10 min with pooled female rat liver microsomes (five female rats, 0.2 mg of protein/ml) in the absence or presence
of itraconazole (0.1 0.5 M). Results (mean S.E.) were based on triplicate determinations. E, 5 M simvastatin; , 10 M; , 20 M; , 50 M. V0, M-1 or M-2
formation rate (nmol/min/mg).

FIG. 5. Influence of itraconazole on plasma concentrations of simvastatin in female (a) and male (b) rats.
Itraconazole was administrated orally at a dose of 50 mg/kg, and then simvastatin was administrated orally at a dose of 10 mg/kg 1 min later. Each point represents the
mean S.D. (n 3). , plasma concentration of simvastatin alone; , plasma concentration of simvastatin in the presence of itraconazole.
TABLE 1
Pharmacokinetic parameters of simvastatin in the absence or presence of itraconazole
AUC

AUC(I)

AUC(I)/AUC

Cmax

ng h/ml

Female
Male

91.7 8.9
58.3 8.4

Cmax(I)

Cmax(I)/Cmax

44.5 1.8
16.1 0.7

1.96
0.944

ng/ml

149 8
60.1 9.4

1.62
1.03

formed in human liver microsomes. The chemical structure of M-3

was proposed to be 3-hydroxy simvastatin based on the structure
of the main metabolite formed in male rat liver microsomes already
identified (Ohtawa and Uchiyama, 1992). From the inhibition
study with anti-rat P450 antisera, the P450 isozyme responsible for
simvastatin metabolism in male rats was demonstrated to be different from that in female rats. In the formation of M-3 by male rat
liver microsomes, CYP2C11 was suggested to play the main role,
while the CYP3A family was mainly responsible in the formation
of M-1 and M-2 in female rat liver microsomes (Fig. 2). Furthermore, itraconazole inhibited the metabolism of simvastatin in
female rats but not in male rats (Fig. 4). Considering the previous
finding that itraconazole inhibited the metabolism mediated by
CYP3A2 in male rats (Yamano et al., 1999), the sex difference in

22.7 4.1
17.0 1.3

the inhibition by itraconazole was suggested to be attributable to a

difference in the ability of itraconazole to inhibit CYP2C11 and
CYP3A activity. In addition, the metabolism of simvastatin in
human liver microsomes was also inhibited by itraconazole, indicating that female rats rather than male rats reflect the in vitro
inhibition in humans. In the investigation of the effect of concomitantly administered itraconazole on the pharmacokinetics of simvastatin in rats, the in vitro metabolism of simvastatin was not
inhibited by itraconazole in male rats. In female rats in which
inhibition of the in vitro metabolism of simvastatin was observed,
the AUC of simvastatin was increased, although the degree of
increase was significantly lower than that observed in clinical
situations (more than 10-fold) (Neuvonen et al., 1998). As seen in
Fig. 1, the amount of M-1 and M-2 formed relative to the acid form

1072

ISHIGAMI ET AL.

was less in female rat liver microsomes than in human liver

microsomes. Because the formation of M-1 and M-2 is more
susceptible to inhibition by itraconazole than that of the acid form,
the difference in the formation ratio of metabolites in female rats
and humans might be one of the causes for the smaller inhibitory
effect of the concomitantly administered itraconazole in female
rats, compared with that observed in humans.
The drug interaction based on the inhibition of simvastatin
metabolism mediated by CYP3A4 in humans could not be reproduced in the study with male rats. On the other hand, in the study
with female rats, a drug interaction was observed, although the
degree of increase in the AUC of simvastatin was smaller in rats
than in humans. These results suggest that female rats are a more
appropriate animal model than their male counterparts for the
investigation of the drug interaction based on the inhibition of
simvastatin metabolism mediated by CYP3A4. Since species and
sex differences are observed in P450 isozymes, the establishment
of appropriate experimental conditions, taking into account the
P450 isozymes responsible for drug metabolism, should be confirmed as far as drug interaction studies using rats as a model
animal for humans are concerned.
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