Download (HMG-CoA) REDUCTASE INHIBITORS ON THE CYP3A4

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DRUG METABOLISM AND DISPOSITION
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:282–288, 2001
Vol. 29, No. 3
223/886405
Printed in U.S.A.
A COMPARISON OF THE EFFECTS OF 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A
(HMG-CoA) REDUCTASE INHIBITORS ON THE CYP3A4-DEPENDENT OXIDATION OF
MEXAZOLAM IN VITRO
MICHI ISHIGAMI, TOMOYO HONDA, WATARU TAKASAKI, TOSHIHIKO IKEDA, TORU KOMAI,
KIYOMI ITO, AND YUICHI SUGIYAMA
Drug Metabolism and Pharmacokinetics Research Laboratories and Product Strategy Department, Sankyo Co., Ltd., Shinagawa-ku, Tokyo,
Japan (M.I., T.H., 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 August 29, 2000; accepted December 5, 2000)
This paper is available online at http://dmd.aspetjournals.org
HMG-CoA reductase inhibitors can be divided into two groups:
those administered as the prodrug, i.e., the lactone form (e.g.,
simvastatin and lovastatin), and those administered in the active
form, i.e., the acid form (e.g., pravastatin, fluvastatin, atorvastatin,
and cerivastatin). In this study, the influence of the lactone and acid
forms of various HMG-CoA reductase inhibitors on metabolism by
CYP3A4, a major cytochrome P450 isoform in human liver, was
investigated by determining the in vitro inhibition constant (Ki
value) using an antianxiety agent, mexazolam, as a probe substrate. In human liver microsomes, all the lactone forms tested
inhibited the oxidative metabolism of mexazolam more strongly
than did the acid forms, which have lower partition coefficient
(logD7.0) values. In addition, the degree of inhibition of mexazolam
metabolism tended to increase with an increasing logD7.0 value of
the HMG-CoA reductase inhibitors among the lactone and acid
forms. In particular, pravastatin (acid form), which has the lowest
logD7.0 value, failed to inhibit CYP3A4 activity. Taking account of
the lipophilicity of the inhibitors, in conjunction with the CYP3A4inhibitory activity, could be very useful in predicting drug interactions between substrates of CYP3A4 and HMG-CoA reductase
inhibitors.
Drug interactions can be classified roughly into two types. In one
case, the pharmacological effects or side effects of particular drugs are
altered by concomitant administration of other drugs. In the other
case, the effects of concomitant drugs are altered by the first drugs. In
either case, the drug interactions have often been evaluated by the
changes in plasma levels of drugs in clinical situations. In the case of
HMG-CoA1 reductase inhibitors, plasma levels of lactone-form prodrugs simvastatin and lovastatin are reported to be increased 10 times
or more by the concomitant administration of an antifungal agent,
itraconazole, a potent inhibitor of CYP3A4 (Varhe et al., 1994), one
of the isoforms of cytochrome P450 (Neuvonen and Jalava, 1996;
Neuvonen et al., 1998). The possibility has also been discussed of
predicting the drug interaction with HMG-CoA reductase inhibitors.
It was considered that the increase in the plasma levels of the
prodrug type, i.e., the lactone forms of HMG-CoA reductase inhibitors, caused by the concomitant administration of itraconazole may be
due to inhibition of the CYP3A4-mediated metabolism of simvastatin
and lovastatin (Vickers et al., 1990; Wang et al., 1991; Prueksaritanont et al., 1997) by itraconazole. We have determined the in vitro Ki
values of itraconazole using human liver microsomes and applied
them to a pharmacokinetic model (Ito et al., 1998), together with
pharmacokinetic parameters for itraconazole. As a result, we have
found that the predicted increase in plasma levels of simvastatin by
concomitant administration of itraconazole agreed fairy well with the
observed increase in clinical situations (Ishigami et al., 2001). However, as far as the water soluble pravastatin was concerned, when
administered in the active acid form, there was no evidence suggesting
any drug interaction with itraconazole either in clinical situations
(Neuvonen and Jalava, 1996; Neuvonen et al., 1998) or in vitro
(Ishigami et al., submitted). These results strongly suggest that pravastatin does not exhibit a drug interaction with itraconazole because
its metabolism is not mediated by CYP3A4.
As described above, most of the studies that investigated the cause
of drug interactions have focused on the identification of the enzymes
responsible for the metabolism of the tested drugs, and only a few
studies have examined the relationship between drug interactions and
the physicochemical properties of drugs. One of the most important
physicochemical properties of a drug is its partition coefficient
(logD7.0), but few studies have examined the relationship between
logD7.0 values and the inhibition of drug-metabolizing enzymes.
Therefore, in the present study, we investigated the correlation between the logD7.0 values of various HMG-CoA reductase inhibitors
and the potential inhibition of P450. We selected CYP3A4 as the CYP
isoform to be examined, since this isoform has been reported to be
involved in the drug interaction with simvastatin. In the present study,
we selected mexazolam, an anxiolytic benzodiazepine, as a substrate
1
Abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme
A; V0, initial formation rate; logD7.0, partition coefficient; HPLC, high-performance
liquid chromatography.
Send reprint requests to: Yuichi Sugiyama, Professor, Graduate School of
Pharmaceutical Sciences, University of Tokyo, 3-1,7-Chome, Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan. E-mail: [email protected]
282
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ABSTRACT:
283
CYP3A4 INHIBITION BY HMG-CoA REDUCTASE INHIBITORS
FIG. 1. Metabolism of mexazolam by CYP3A4.
Metabolites in brackets are the postulated intermediates that have not yet been isolated.
for CYP3A4. Previous studies with various P450 isoforms expressed
in HepG2 cells demonstrated that mexazolam to M-1 via postulated
intermediates (Fig. 1) is metabolized mainly by the CYP3A family
(Ono et al., 1993).
In the present study, we investigated the inhibitory effects of the
lactone and acid forms of various HMG-CoA reductase inhibitors
(Fig. 2) on CYP3A4 using mexazolam as a probe to monitor CYP3A4
activity, and we evaluated the correlation between the potential inhibition of CYP3A4 and the logD7.0 values of the inhibitors. In addition,
we compared and evaluated the HMG-CoA reductase inhibitors in
terms of potential drug interactions.
Materials and Methods
TABLE 1
HPLC condition for logD7.0 measurement of HMG-CoA reductase inhibitors
Columns used were 150 ⫻ 4.6 mm. For mobile phase, 1 mM KH2PO4 (pH 7.0 NaOH):
Acetonitrile was used in varying ratios as shown under Mobile Phase. The flow rate in all
cases was 1 ml/min.
HMG-CoA Reductase
Inhibitor
Pravastatin (acid)
Atorvastatin (acid)
Cerivastatin (acid)
Fluvastatin (acid)
Atorvastatin lactone
Cerivastatin lactone
Fluvastatin lactone
Column
Mobile Phase
a
Deverosil ODS-UG-5
Deverosil ODS-UG-5
Deverosil ODS-UG-5
Deverosil ODS-UG-5
Capcel pak C18b
Capcel pak C18
Inertsil ODS-2c
80:20
65:35
60:40
65:35
50:50
30:70
65:35
Wavelength
238
238
238
238
237
237
254
nm
nm
nm
nm
nm
nm
nm
a
Chemicals and Reagents. Pravastatin (acid form), pravastatin lactone,
simvastatin (lactone form), simvastatin acid (Na⫹ salt), lovastatin (lactone
form), lovastatin acid (Na⫹ salt), fluvastatin (acid form), fluvastatin lactone,
atorvastatin (acid form), atorvastatin lactone, cerivastatin (acid form), cerivastatin lactone, mexazolam, and a metabolite of mexazolam (M-1) were
synthesized at Sankyo Co., Ltd. (Tokyo, Japan). Human liver microsomes were
obtained from the International Institute for the Advancement of Medicine
(Exton, PA). Polyclonal antibodies for human P450 (anti-CYP2C antiserum
from goat, and anti-CYP1A, -CYP2D6, and -CYP3A4 antisera from rabbit)
were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). All
Deverosil: Nomura Chemical Co., Ltd. (Aichi, Japan).
Capcel pak: Shiseido Co., Ltd. (Tokyo, Japan).
Inertsil: GL Science Inc. (Tokyo, Japan).
b
c
other chemicals and reagents used were of analytical grade and were obtained
commercially.
In Vitro Metabolism of Mexazolam. A final volume (0.2 ml) of a typical
enzyme source mixture consisted of 0.04 mg of liver microsomal protein, 2
␮mol of potassium phosphate buffer (pH 7.4), 2 ␮mol of MgCl2, 0.5 ␮mol of
NADP, 5 ␮mol of glucose 6-phosphate, and 0.4 units of glucose-6-phosphate
dehydrogenase. To the enzyme source preincubated at 37°C for 3 min, mex-
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FIG. 2. Structures of HMG-CoA reductase inhibitors.
284
ISHIGAMI ET AL.
FIG. 3. Effects of anti-CYP sera on the formation of M-1 from mexazolam in male (a) and female (b) human liver microsomes.
half-maximal velocity, and n is the Hill coefficient. The intrinsic metabolic
clearance (CLint) was evaluated using the following equation:
CL int ⫽ Vmax/Km
FIG. 4. Eadie-Hofstee plots for the formation of M-1 from mexazolam in male
and female human liver microsomes.
Mexazolam was incubated for 2 min at 37°C with pooled human liver microsomes (10 male subjects or 10 female subjects, 0.2 mg of protein/ml). Each value
represents the mean ⫾ S.E. of triplicate determinations. ⽧, female; 䡺, male. Solid
lines, theoretical curves calculated using the obtained values of Km, Vmax, and n.
azolam dissolved in ethanol (final concentration, 1%) was added, and the
reaction was continued for 2 min at 37°C and then terminated by the addition
of methanol (0.4 ml). Next, the reaction mixture was centrifuged at 19,000g for
3 min, and the resulting supernatant was analyzed for the mexazolam metabolite (M-1, Fig. 2) by an HPLC method. The HPLC conditions were as follows:
column, Deverosil ODS-UG-5 (250 ⫻ 4.6 mm, Nomura Chemical Co., Ltd.,
Aichi, Japan); mobile phase, acetonitrile/50 mM phosphate buffer (pH 8.0) ⫽
55/45; flow rate, 1 ml/min; and UV-detection wavelength, 235 nm.
Kinetic Analysis of Mexazolam Metabolism. Mexazolam was tested at a
final concentration range of 2 to 100 ␮M in human liver microsomes to
determine the kinetic parameters. The production of the mexazolam metabolite
determined was fitted to the Hill equation (eq. 1) using a nonlinear regression
program (WinNonlin, Scientific Consulting, Inc., Apex, NC) to estimate Km,
Vmax, and n:
V 0 ⫽ V max ⫻ Sn /共Kmn ⫹ Sn 兲
(1)
where V0 is the initial formation rate, Vmax is the maximum metabolic rate, S
is the substrate concentration, Km is the substrate concentration showing a
(2)
For immunoinhibition studies, human liver microsomes (pooled from 10
male or 10 female subjects, 10 mg of protein/ml, 10 ␮l) were first incubated
with antiserum or corresponding control serum (total, 25 ␮l) at room temperature for 30 min. The mixture was added with the cofactor solution and
mexazolam solution (final concentration, 20 ␮M), and then a final volume of
0.5 ml was incubated in a similar manner as described under In Vitro Metabolism of Mexazolam. Finally, 1 ml of methanol was added to terminate the
reaction, and after centrifugation the supernatant was subjected to HPLC.
Inhibition of Mexazolam Metabolism by HMG-CoA Reductase Inhibitors. To define the type of inhibition by HMG-CoA reductase inhibitors on
mexazolam metabolism, mexazolam (5–50 ␮M) was coincubated with various
HMG-CoA reductase inhibitors [50 to 400 ␮M pravastatin (acid form); 20 to
200 ␮M simvastatin acid, fluvastatin (acid form), atorvastatin (acid form), and
cerivastatin (acid form); and 2 to 20 ␮M simvastatin (lactone form)] in the
enzyme source mixture.
The 1/V0(⫹I) values obtained for each concentration of HMG-CoA reductase
inhibitor were applied to Dixon plots, and the Ki values were obtained by
simultaneous fitting using WinNonlin (eq. 3):
1/V 0共⫹I兲 ⫽
1 ⫹ K mn /Sn
I 䡠 K mn
⫹
Vmax
Ki 䡠 Sn 䡠 Vmax
(3)
where “(⫹I)” represents the value after alteration by the drug-drug interaction,
and S and I represent the concentration of substrate (mexazolam) and inhibitor
(HMG-CoA reductase inhibitor), respectively.
In addition, to compare the Ki values for lactone and acid forms of HMGCoA reductase inhibitors, HMG-CoA reductase inhibitors (0.1–200 ␮M) and
mexazolam (20 ␮M) were incubated with human liver microsomes (pooled
from 10 female subjects) for 2 min at 37°C, and the amount of the mexazolam
metabolite M-1 formed was determined. The Ki value was calculated by fitting
the data to the following equation using WinNonlin:
V 0共⫹I兲/V 0 ⫽
K mn ⫹ Sn
Kmn 䡠 共1 ⫹ I/Ki兲 ⫹ Sn
(4)
Measurement of the logD7.0 Value of HMG-CoA Reductase Inhibitors.
Partition coefficients between phosphate buffer (pH 7.0, Britton Robinson
Buffer) and 1-octanol for fluvastatin lactone, cerivastatin lactone, atorvastatin
lactone, fluvastatin (acid form), cerivastatin (acid form), atorvastatin (acid
form), and pravastatin (acid form) were determined by the flask-shaking
method (OECD, 1981). Each drug was dissolved in 1-octanol-saturated buffer,
and then the solution was mixed with buffer-saturated 1-octanol solution. After
shaking for 30 min at 25°C, the 1-octanol and buffer layers were separated by
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Pooled human liver microsomes (10 male subjects or 10 female subjects, 0.1 mg of protein/10 ␮l) were preincubated for 30 min at room temperature with 0.05 to 0.25
mg IgG of anti-human CYP sera or control sera, preincubated for 3 min at 37°C with NADPH-generating system, and then incubated with mexazolam (20 ␮M) at 37°C
for 2 min. Results (mean ⫾ S.E.) were based on triplicate determinations. 䡺, anti-CYP1A; Œ, anti-CYP2C; 〫, anti-CYP2D6; F, anti-CYP3A4.
285
CYP3A4 INHIBITION BY HMG-CoA REDUCTASE INHIBITORS
TABLE 2
Kinetics of mexazolam metabolite formation in human liver microsomes
The kinetic parameters Km, Vmax, and n (mean ⫾ calculated S.D.) were calculated from data shown in Fig. 4 according to eq. 1. The CLint was estimated by dividing Vmax by Km.
Male
Female
Km
Vmax
␮M
nmol/min/mg
n
29.2 ⫾ 10.6
26.5 ⫾ 3.8
0.68 ⫾ 0.12
1.64 ⫾ 0.11
CLint
ml/min/mg
1.34 ⫾ 0.26
1.30 ⫾ 0.10
0.023
0.062
CLint, intrinsic clearance.
centrifugation, and the drug concentrations in each layer were determined by
HPLC as described in Table 1.
The distribution coefficients and logD7.0 values were calculated from the
following equation:
Distribution coefficients 共Dow兲 ⫽ Co/Cw, logD7.0 ⫽ log10Dow
(5)
in which CO and CW represent the concentration (mol/liter) of test drug in
octanol and buffer layer, respectively.
Results
The Characteristics of Mexazolam Metabolism by Human Liver
Microsomes. The formation of the mexazolam metabolite (M-1) in male
and female human liver microsomes was inhibited by addition of antiCYP3A4 serum to the reaction mixture in a concentration-dependent
manner, but was not affected by the addition of anti-CYP1A, -CYP2C or
-CYP2D6 sera (Fig. 3, a and b). When 0.25 mg of IgG of anti-CYP3A4
serum was added, the M-1 formation in human liver microsomes was
inhibited by about 80% in both males and females.
The formation of M-1 at various concentrations of mexazolam in
human liver microsomes was investigated to calculate the kinetic
parameters for mexazolam metabolism. As shown in Fig. 4, an EadieHofstee plot showed a convex curve, and kinetic parameters such as
Km, Vmax, and the Hill coefficient (n) are shown in Table 2. The Km
and n values were similar for males and females, but the Vmax value
in females was about 2.4 times higher than that in males. In addition,
the mean intrinsic clearance (Vmax/Km) in females was about 2.6 times
higher than that in males.
Influence of HMG-CoA Reductase Inhibitors on Mexazolam
Metabolism in Human Liver Microsomes. The Ki values for the
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FIG. 5. Dixon plots for the inhibition of M-1 formation from mexazolam in male (a) and female (b) human liver microsomes by HMG-CoA reductase inhibitors.
Mexazolam (5–50 ␮M) was incubated for 2 min at 37°C with human liver microsomes (one female subject or one male subject, 0.2 mg of protein/ml) in the absence
or presence of HMG-CoA reductase inhibitors (pravastatin, 50 – 400 ␮M; simvastatin, 5–20 ␮M; simvastatin Na⫹, fluvastatin, atorvastatin, and cerivastatin, 20 –200 ␮M).
E, mexazolam 5 ␮M; ⽧, mexazolam 10 ␮M; 䡺, mexazolam 20 ␮M; Œ, mexazolam 50 ␮M; V0, M-1 formation rate (nmol/min/mg). Solid lines, theoretical curves
calculated using the obtained values of Km, Vmax, n, and Ki.
286
ISHIGAMI ET AL.
TABLE 3
Ki of various HMG-CoA reductase inhibitors for the inhibition of mexazolam metabolism by human liver microsomes
Ki values were calculated from data shown in Fig. 5 according to eq. 3.
HMG-CoA Reductase
Inhibitor
Km
Ki
Male
Female
No inhibition
91.9 ⫾ 8.8
3.57 ⫾ 0.48
31.9 ⫾ 4.8
112 ⫾ 14
56.8 ⫾ 5.6
18.7 ⫾ 3.6
No inhibition
90.8 ⫾ 26.4
3.25 ⫾ 0.70
23.0 ⫾ 4.4
90.9 ⫾ 15.0
63.3 ⫾ 11.8
25.1 ⫾ 3.3
Male
Female
No inhibition
10.7 ⫾ 0.7
10.0 ⫾ 2.1
8.57 ⫾ 1.27
10.3 ⫾ 1.0
9.78 ⫾ 1.05
6.53 ⫾ 1.04
No inhibition
10.8 ⫾ 1.3
12.1 ⫾ 0.6
14.2 ⫾ 0.7
15.0 ⫾ 0.8
10.7 ⫾ 0.5
13.8 ⫾ 0.4
␮M
Pravastatin (acid)
Pravastatin lactone
Simvastatin (lactone)
Simvastatin acid
Fluvastatin (acid)
Atorvastatin (acid)
Cerivastatin (acid)
inhibition of mexazolam metabolism in male and female human liver
microsomes by HMG-CoA reductase inhibitors were determined by a
Dixon plot (Fig. 5, a and b; Table 3). The formation of M-1 from
mexazolam in human liver microsomes was not inhibited by pravastatin (acid form), but it was inhibited competitively by other HMGCoA reductase inhibitors. Also, there were no significant differences
in the Ki values of various inhibitors between males and females. The
extent of inhibition was in the following order: simvastatin (lactone
form), cerivastatin (acid form), simvastatin acid, atorvastatin (acid
form) and fluvastatin (acid form).
The effects of HMG-CoA reductase inhibitors (lactone and acid
forms) on the formation of M-1 in human liver microsomes (female)
are shown in Fig. 6. In this figure, the extent of inhibition of the
formation of M-1 was plotted against the concentrations of inhibitors,
and the results were analyzed by regression analysis. The calculated
Ki values are summarized in Table 4. When the effect on mexazolam
metabolism in human liver microsomes was compared for various
HMG-CoA reductase inhibitors, it was found that the inhibitory
activity of the lactone forms was higher than that of the acid forms for
all HMG-CoA reductase inhibitors tested.
Correlation between logD7.0 and Ki for CYP3A4 Inhibition of
HMG-CoA Reductase Inhibitors. Table 4 summarizes the logD7.0
values of HMG-CoA reductase inhibitors, including the values measured in this study [pravastatin (acid form), atorvastatin (acid form),
atorvastatin lactone, fluvastatin (acid form), fluvastatin lactone, and
cerivastatin lactone] and previously reported logD7.0 values for other
HMG-CoA reductase inhibitors (Watanabe, 1990). The logD7.0 values
of all HMG-CoA reductase inhibitors examined were positive, except
for the negative value of pravastatin (acid form). All the lactone forms
examined showed higher logD7.0 values compared with their corresponding acid forms. The difference in logD7.0 values between the
lactone form and the corresponding acid form ranged from 2.9 to 1.9
units, and the mean difference was 2.54 units.
The logD7.0 values of HMG-CoA reductase inhibitors and Ki values
for CYP3A4 inhibition by HMG-CoA reductase inhibitors are shown
in Table 4, and the correlation between logD7.0 and Ki values are
plotted in Fig. 7. The extent of inhibition of mexazolam metabolism
increased with the inhibitor’s lipophilicity among the acid forms,
except for fluvastatin, and water-soluble pravastatin produced no
inhibition at all. The same tendency of stronger inhibition with higher
lipophilicity was observed among the lactone forms, and the Ki value
of 110 ␮M for pravastatin lactone was much higher than that of 10
␮M or less for atorvastatin lactone, cerivastatin lactone, lovastatin
(lactone), simvastatin (lactone), and fluvastatin lactone.
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FIG. 6. Effects of HMG-CoA reductase inhibitors on the formation of M-1 from mexazolam in female human liver microsomes.
Mexazolam (20 ␮M) was incubated for 2 min at 37°C with pooled human liver microsomes (10 female subjects, 0.2 mg of protein/ml) in the absence or presence of
HMG-CoA reductase inhibitors (0.1–200 ␮M). Control activities were obtained in the absence of HMG-CoA reductase inhibitors. Results (mean ⫾ S.E.) were based on
triplicate determinations. 䡺, acid form; ⽧, lactone form. Solid lines, theoretical curves calculated using the obtained values of Km, n, and Ki.
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CYP3A4 INHIBITION BY HMG-CoA REDUCTASE INHIBITORS
TABLE 4
logD7.0 and Ki values of various HMG-CoA reductase inhibitors for the inhibition of mexazolam metabolism by female human liver microsomes
Ki values (mean ⫾ calculated S.D.) were calculated from data shown in Fig. 6 according to eq. 4.
Ki value
logD7.0
Inhibitor
Lactone
Acid
Lactone
2.42
4.40
3.91
3.65
4.2
5.2
⫺0.47
1.88
1.51
1.75
1.53
2.32
115 ⫾ 9
2.13 ⫾ 0.14
2.98 ⫾ 0.18
7.10 ⫾ 0.39
2.54 ⫾ 0.12
2.18 ⫾ 0.10
Acid
␮M
Pravastatin
Simvastatin
Lovastatin
Fluvastatin
Atorvastatin
Cerivastatin
Ki values were calculated from data shown in Fig. 6 according to eq. 4. 䡺, acid
form; ⽧, lactone form. Solid line, linear regression line.
Discussion
It has been reported from a study with cDNA-expressed human
P450 isoforms (CYP1A2, -2A6, -2B6, -2C8, -2C9, -2D6, -2E1, -3A3,
-3A4, and -3A5) in HepG2 cells that mexazolam is mainly metabolized by the CYP3A family (Ono et al., 1993). In the present study, the
inhibitory activity by anti-CYP1A, -2C, -2D6, and -3A4 antisera on
mexazolam metabolism in human liver microsomes was investigated,
and it was shown that CYP3A4 was the main CYP isoform responsible for mexazolam metabolism (Fig. 3). Furthermore, an EadieHofstee plot for the formation of mexazolam metabolite, M-1, showed
a convex curve (Fig. 4), which is characteristic of metabolism by
CYP3A4 (Ueng et al., 1997). The metabolic activities (testosterone
6␤-hydroxylation) of pooled human liver microsomes used in the
present study were 3.7 nmol/min/mg of protein and 2.1 nmol/min/mg
of protein for female and male human liver microsomes, respectively,
i.e., about 1.8 times higher in females than in males (data from
International Institute for the Advancement of Medicine). These data
are consistent with our finding that the intrinsic metabolic clearance of
mexazolam was about 2.3 times higher in females than in males
(Table 2). These results indicate that mexazolam is a suitable probe
for monitoring CYP3A4 activity.
HMG-CoA reductase inhibitors (Fig. 1) are classified into the
following two groups: those administered as a prodrug, i.e., the
lactone form (simvastatin, lovastatin, etc.) and those administered in
the active form, i.e., the acid form (pravastatin, fluvastatin, atorvastatin, cerivastatin, etc.). The inhibition of CYP3A4 activity by the
lactone forms is much higher than that by the acid forms. This
correlated with the higher lipophilicity of the lactone form (logD7.0
value of the lactone forms was 2.54 units higher on average). Among
the acid forms, only pravastatin (acid) showed a negative logD7.0
value, while all the other HMG-CoA reductase inhibitors, such as
fluvastatin (acid), atorvastatin (acid), and cerivastatin (acid), had
positive logD7.0 values, indicating the high lipophilicity of these
compounds. This may explain the finding that only pravastatin (acid)
did not inhibit CYP3A4 activity (Table 4). The Ki values for the
inhibition of CYP3A4 activity were plotted against the logD7.0 values
of HMG-CoA reductase inhibitors for every lactone and acid form to
determine the correlation between logD7.0 and Ki values (Fig. 7).
Consequently, with the exception of fluvastatin (acid) and pravastatin
lactone, the extent of inhibition of CYP3A4 activity increased with the
inhibitor’s lipophilicity (Fig. 7). Although pravastatin lactone and
cerivastatin (acid) have a similar lipophilicity, the inhibition by pravastatin was lower than that by cerivastatin (acid), possibly because the
pyridine in cerivastatin (acid) may play a key role in increasing the
ability to inhibit CYP3A4 activity, or because the decalin ring structure and 6⬘-hydroxy group in pravastatin lactone may be responsible
for reducing the inhibition activity.
Simvastatin (lactone), lovastatin (lactone), atorvastatin (acid), and
cerivastatin (acid), which were reported to be the substrates for
CYP3A4 (Vickers et al., 1990; Wang et al., 1991; Boberg et al., 1997;
Prueksaritanont et al., 1997; Boyd et al., 2000), inhibit mexazolam
metabolism. Simvastatin (lactone), lovastatin (lactone), and atorvastatin (acid) have been reported to exhibit a significant drug interaction
with itraconazole, a well known inhibitor of CYP3A4, when both
drugs are coadministered (Neuvonen and Jalava, 1996; Kantola et al.,
1998; Neuvonen et al., 1998). Therefore, the potential for drug interactions between HMG-CoA reductase inhibitors and other drugs may
be related to the metabolism of HMG-CoA reductase inhibitors by
CYP3A4. Fluvastatin (acid) has been reported to be an inhibitor of
CYP2C9 (Transon et al., 1995, 1996), and in the present study it was
shown that fluvastatin (acid) also inhibited microsomal CYP3A4
activity.
Among the HMG-CoA reductase inhibitors examined in the present
study, inhibitors with a higher lipophilicity generally tended to be
stronger inhibitors of CYP3A4. P450s are involved in the conversion
of a lipophilic drug to a more hydrophilic metabolite and excrete it
from the body. Taking this into consideration, the present finding may
reasonably be explained by the fact that lipophilic drugs have a high
affinity for P450s and, as a result, produce inhibition of P450. Therefore, pravastatin is free from a drug interaction due to its high water
solubility and its absence of metabolism by P450.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2017
FIG. 7. logD7.0 of HMG-CoA reductase inhibitor and Ki for mexazolam
metabolism in human liver microsomes.
No inhibition
69.6 ⫾ 5.2
179 ⫾ 18
289 ⫾ 44
189 ⫾ 30
46.3 ⫾ 2.1
288
ISHIGAMI ET AL.
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