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DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 9
Printed in U.S.A.
CYTOCHROME P-450 3A4 AND 2C8 ARE INVOLVED IN ZOPICLONE METABOLISM
LAURENT BECQUEMONT, SAID MOUAJJAH, OLIVIER ESCAFFRE, PHILIPPE BEAUNE, CHRISTIAN FUNCK-BRENTANO,
PATRICE JAILLON
AND
Clinical Pharmacology Unit (L.B., S.M., O.E., C.F.-B., P.J.), Saint Antoine University Hospital, School of Medicine Paris 6, France; and Institut
National de la Santé et de la Recherche Médicale U 490 (P.B.), Saint-Pères University, School of Medicine Paris 5, France
(Received December 31, 1998; accepted May 7, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
phenazole (CYP2C inhibitor) inhibited the formation of ND-Z,
whereas a-naphtoflavone (CYP1A), quinidine (CYP2D6), and chlorzoxazone (CYP2E1) did not affect zopiclone metabolism. The generation of ND-Z and NO-Z were highly correlated to testosterone
6b-hydroxylation (CYP3A activity, r 5 0.95 and 0.92, respectively;
p 5 .0001), and ND-Z was highly correlated to CYP2C8 activity
(paclitaxel 6a-hydroxylase; r 5 0.76, p 5 .004). Recombinant
CYP2C8 had the highest enzymatic activity toward zopiclone metabolism into both its metabolites, followed by CYP2C9 and 3A4.
CYP3A4 is the major enzyme involved in zopiclone metabolism in
vitro, and CYP2C8 contributes significantly to ND-Z formation.
Zopiclone is a g-aminobutyric acid receptor agonist that is widely
prescribed for its hypnotic properties (Noble et al., 1998) in insomniac
patients. As benzodiazepines agents, the duration of its pharmacological effect and the occurrence of side effects, such as morning hypnotic residual effects (Allain et al., 1991), are mainly dependent on its
biological half-life and clearance.
In humans, zopiclone elimination is mainly dependent on its hepatic
clearance because only 5% of the drug is excreted unchanged in the
urine (Noble et al., 1998). Indeed, zopiclone is extensively metabolized by the human liver into two major metabolites (Fig. 1): N-oxidezopiclone (NO-Z)1, which retains a low pharmacologic activity; and
N-desmethyl-zopiclone (ND-Z), which is pharmacologically inactive
(Gaillot et al., 1982, 1983; Le Liboux et al., 1987). The enzymes
involved in zopiclone metabolism have not yet been identified (Noble
et al., 1998), but cytochrome P-450 (CYP) isoforms may be suspected
because some drug interactions in humans with CYP inhibitors or
inducers have been reported (Aranko et al., 1994; Jalava et al., 1996;
Villikka et al., 1997). Because the pharmacological effects of this drug
and its morning residual hypnotic effects may be modulated by some
others drugs known to interfere with CYP activity and expression, it
is important to identify the enzymes involved in zopiclone metabolism
to predict and to prevent some drug interactions in humans. Therefore,
the aim of the present study was to identify the human CYP isoforms
involved in zopiclone metabolism in vitro.
Materials and Methods
Drugs, Chemicals, and Reagents. Zopiclone, NO-Z, and ND-Z were
kindly provided by Rhône-Poulenc Rorer (Antony, France) and ketoconazole
by Jansen (Beerse, Belgium). Hydroquinidine was purchased from Fluka
(Buchs, Switzerland), and sulfaphenazole, quinidine, chlorzoxazone, and
a-naphtoflavone were obtained from Sigma Chemical Co. (St. Louis, MO).
Glucose 6-phosphate, glucose 6-phosphate deshydrogenase, and NADP were
purchased from Boehringer Mannheim (Meylan, France); reagents for protein
assays were obtained from Pierce Chemical Co. (Beigerland, the Netherlands).
All the other reagents and solvents were of the highest grade commercially
available.
Human Liver Microsomes. Human liver microsomes from 12 different
donors were provided by Gentest (Woburn, MA). Additional microsomes were
prepared from liver samples of 10 human donors, collected, and stored as
described previously (Becquemont et al., 1998).
Yeast-Expressed Recombinant Human CYP (rH-CYP) Enzymes. Human CYP 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4 were cloned and
expressed in yeast strains that overexpress endogenous NADPH-P-450 reductase, as described previously (Gautier et al., 1996). Microsomes from the
different yeast cultures were prepared by mechanical lysis, followed by differential ultracentrifugation (Renaud et al., 1990; Gautier et al., 1996).
Proteins and CYP Concentration. Human liver and yeast-expressed CYP
microsomal concentrations were measured by a spectrophotometric method as
described by Schoene et al. (1972). Total protein concentration was assayed by
1
Abbreviations used are: NO-Z, N-oxide-zopiclone; ND-Z, N-desmethyl-zopithe bicinchoninic acid method (Pierce Chemical Co.) according to the suppliclone; CYP, cytochrome P-450; rH-CYP, recombinant human cytochrome P-450.
er’s recommendation and using serum albumin as the standard.
Quantification of CYP Activities. CYP3A, CYP2C9, and CYP2D6 enzySend reprint requests to: Dr. Laurent Becquemont, Faculté de Médecine,
matic activities (testosterone 6b-hydroxylation, diclofenac 49-hydroxylation,
Saint Antoine Paris VI, Service de Pharmacologie, 27 Rue de Chaligny, 75012
and dextromethorphan O-demethylation) for the 22 liver samples were perParis, France. E-mail: [email protected]
formed as described previously (Langouët et al., 1995; Funck-Brentano et al.,
1068
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Zopiclone is a widely prescribed, nonbenzodiazepine hypnotic that
is extensively metabolized by the liver in humans. The aim of the
present study was to identify the human cytochrome P-450 (CYP)
isoforms involved in zopiclone metabolism in vitro. Zopiclone metabolism was studied with different human liver microsomes and a
panel of heterologously expressed human CYPs (CYP1A2, 2C8,
2C9, 2C18, 2C19, 2D6, 2E1, and 3A4). In human liver microsomes,
zopiclone was metabolized into N-desmethyl-zopiclone (ND-Z) and
N-oxide-zopiclone (NO-Z) with the following Km and Vm of 78 6 5
and 84 6 19 mM, 45 6 1 and 54 6 5 pmol/min/mg for ND-Z and
NO-Z generation, respectively. Ketoconazole (CYP3A inhibitor) inhibited ;40% of the generation of both metabolites, sulfa-
1069
IN VITRO ZOPICLONE METABOLISM
1997; Becquemont et al., 1998). Other CYP enzymatic activities were determined previously by the manufacturer on the 12 samples provided from
Gentest.
Zopiclone Metabolism. The kinetics of zopiclone oxidation and demethylation were studied in the presence of 1 mg of human liver microsomes or 100
pmol of the different rH-CYP isoforms in a final volume of 1 ml. Zopiclone
was used at eight different concentrations ranging from 5 to 400 mM. Each
incubation was carried out at 37°C in Tris-EDTA buffer in the presence of an
NADPH-generating system consisting of 0.15 mM NADP, 2.5 mM glucose
6-phosphate, and 1.7 U/ml glucose 6-phosphate deshydrogenase. After 5-min
preincubation, the reaction was started by adding the glucose 6-phosphate
deshydrogenase and stopped 60 min later on ice and by adding 500 ml of
NaH2PO4 (70 mM, pH 8) buffer. After the addition of 50 ml of the internal
standard (hydroquinidine, 1 mM in methanol) and 2 ml of CH2Cl2, the
preparation was mixed for 15 min and centrifuged during 5 min at 3000g to
remove the protein pellet. The organic phase was dried and dissolved in 200 ml
of the HPLC mobile phase. HPLC analysis was performed on a 4.6 3 250-mm
Symmetry C18 column (Waters, Milford, CT). Fluorescence detection was
performed with an excitation wavelength of 300 nm and an emission wavelength of 470 nm. The isocratic mobile phase, consisting of 50 mM NaH2PO4
(pH 3.7) and acetonitrile 80:20 (v/v), was maintained at 1 ml/min during 30
min. The quantification limit of the method was 10 nM for ND-Z and 30 nM
for NO-Z with an intraday coefficient of variation varying from 11 to 7%.
NO-Z and ND-Z formation rates were shown to be linear with time up to 60
min and with human microsomal protein and rH-CYP concentrations up to 2
mg/ml and 100 pmol/ml, respectively.
Inhibition studies of zopiclone metabolism in human liver microsomes were
performed in triplicate in the presence of a single zopiclone concentration of 50
mM and a single concentration of sulfaphenazole (10 mM), quinidine (10 mM),
chlorzoxazone (100 mM), a-naphtoflavone (10 mM), or ketoconazole (0.5
mM). These experiments were performed on three different donor samples.
Correlation studies with the 22 human microsomal samples were performed
in duplicate under the same conditions at two zopiclone concentrations of 25
and 200 mM.
Determination of the CYP isoforms involved in zopiclone metabolism in the
presence of the different rH-CYP isoforms (100 pmol) was performed in
quadruplicate under the same conditions at a single zopiclone concentration of
50 mM.
Data Analysis. Km, the apparent affinity constant, and Vmax, the maximum
initial enzyme velocity, were initially evaluated by graphical examination of
Eadie-Hofstee plots. These values were taken as initial parameters for the
estimation of the Michaelis-Menten parameters and S.E.s by nonlinear leastsquares regression curve fitting as described previously (Funck-Brentano et al.,
1997; Becquemont et al., 1998).
To extrapolate the results of zopiclone metabolism obtained with rH-CYP to
human liver microsomes, we determined the product of the turnover numbers
obtained from each rH-CYP by the specific contents in human liver microsomes of each CYP isoform (Becquemont et al., 1998). This P-450 content was
estimated based on available data (Guengerich and Turvy, 1991; Shimada et
al., 1994; Belloc et al., 1996; Gautier et al., 1996) as being in the range of 69
pmol/mg for CYP1A2, 150 pmol/mg for CYP3A4, 90 pmol/mg for CYP2C9,
35 pmol/mg for CYP2E1, 20 pmol/mg for CYP2D6, and 10 pmol/mg for
CYP2C8, CYP2C18, and CYP2C19. Results are presented as mean 6 S.D.
Results
Zopiclone Metabolism Kinetic Constants in Human Liver Microsomes. We observed that zopiclone was metabolized into NO-Z
and ND-Z in all of the liver microsomes from the 22 different human
donors. There was a 45- and 30-fold extent variability in ND-Z and
NO-Z generation rate, respectively, from one donor to another. We
determined the enzymatic kinetics of zopiclone metabolism in two
liver samples that were chosen among the 22 liver samples for their
predetermined CYP3A activity. One liver had an intermediate CYP3A
activity, and the other showed the lowest CYP3A activity. Enzymatic
constants are presented in Table 1 and illustrated in Fig. 2. These two
liver samples were found to have medium and very low turnover
numbers toward the generation of both zopiclone metabolites (Fig. 3).
Intrinsic clearance (Vm/Km) of ND-Z and NO-Z were, respectively,
10- and 3-fold lower in the liver with the lowest CYP3A activity
compared with the liver with medium CYP3A activity.
In both liver samples, the NO-Z and ND-Z formation rates were
found to be monophasic on Eadie-Hofstee plots (Fig. 2), suggesting
that a single enzyme mainly contributed to their respective generation.
Furthermore, among the 22 liver samples, the generation of both
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FIG. 1. Pathways of zopiclone metabolism in humans.
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BECQUEMONT ET AL.
TABLE 1
Enzymatic constants of zopiclone metabolism
Experiments were performed on two liver samples chosen for their predetermined CYP3A
activity: one with a medium CYP3A activity, and the other for its lowest CYP3A activity.
ND-Z
Vm (pmol/min/mg)
Km (mM)
Intrinsic clearance (ml/min/mg)
NO-Z
Vm (pmol/min/mg)
Km (mM)
Intrinsic clearance (ml/min/mg)
Medium CYP3A
Activity Liver
Sample
Lowest CYP3A
Activity Liver
Sample
45 6 1
78 6 5
0.58
5.6 6 0.1
98 6 5
0.06
54 6 5
84 6 19
0.64
15.4 6 0.6
85 6 9
0.18
The experiment was performed with a single final concentration of zopiclone (25
mM) among liver microsomes provided by 22 different human donors. The two liver
samples used for the determination of zopiclone enzymatic kinetics constants are
shown (F) and indicated by arrows and text.
FIG. 2. Kinetics of NO-Z and ND-Z generation rates in one human liver
microsome with medium CYP3A activity.
This liver sample was chosen among the 22 liver samples of this study for its
medium turnover number toward CYP3A enzymatic. The inset represents the
Eadie-Hofstee plots of the same experiment. E, ND-Z generation rates; F, NO-Z
generation rates.
metabolites was correlated, indicating that their metabolism may be
performed by the same enzyme (Fig. 3).
Screening of Zopiclone Metabolism with rH-CYP. To identify
the CYP isoform(s) involved in zopiclone metabolism, we incubated
zopiclone (50 mM) with a panel of rH-CYP (Fig. 4). CYP2C8 was the
isoform that displayed the highest enzyme activity for the formation
rates of both zopiclone metabolites (Fig. 4A). However, when we
calculated from these data the expected contribution of each CYP
isoform in human liver microsomes the metabolism of zopiclone (Fig.
4B), we observed that CYP3A4 was the major enzyme involved in the
NO-Z formation rate, followed by CYP2C9 and CYP2C8, whereas
CYP2C8 remained the major CYP isoform involved in ND-Z, followed by CYP2C9 and CYP3A4.
Determination of zopiclone enzymatic kinetic constants could be
obtained from rH-CYP2C8 (Fig. 5). Apparent Km and Vm reached,
respectively, 71 6 6 mM and 2.5 6 0.1 pmol/min/pmol CYP2C8 for
ND-Z generation and 59 6 9 mM and 1.0 6 0.1 pmol/min/pmol
CYP2C8 for NO-Z generation. When the intrinsic clearance of ND-Z
(0.035 ml/min/pmol CYP2C8) was extrapolated to human liver, assuming that 1 mg of human liver microsome contains an average of 10
pmol of CYP2C8, it was found to represent ;60% of the intrinsic
clearance obtained in the liver sample with medium CYP3A activity
(0.35 versus 0.58 ml/min/mg). NO-Z intrinsic clearance (0.017 ml/
min/pmol CYP2C8) obtained from recombinant CYP2C8, when extrapolated to human liver, represented 26% of the intrinsic clearance
obtained in the liver sample with medium CYP3A activity (0.17
versus 0.64 ml/min/mg). Similar determinations could not be obtained
from rH-CYP3A4 because of the low turnover number of this isoform
toward the generation of both zopiclone metabolites.
Effects of CYP Inhibitors on Zopiclone Metabolism in Human
Liver Microsomes. To clarify the contribution of the CYP isoforms
outlined previously, we incubated zopiclone with three different human liver microsomes in the presence of different prototypic CYP
inhibitors (Fig. 6). ND-Z generation was mainly inhibited by ketoconazole and sulfaphenazole, whereas NO-Z generation was only
significantly inhibited by ketoconazole, suggesting the involvement of
CYP3A and CYP2C in ND-Z formation and CYP3A in NO-Z formation (Table 1). Surprisingly, a-naphtoflavone, a CYP3A activator,
did not increase the generation of zopiclone metabolites. CYP1A,
CYP2D6, and CYP2E1 inhibitors had only minor effects on the extent
of zopiclone metabolism.
Correlation of Zopiclone Metabolite Generation to Different
CYP Enzymatic Activities in Human Liver Microsomes. To confirm the previous results, we correlated among the different human
liver samples the generation of both zopiclone metabolites to classic
CYP activities. We first observed for both metabolites a unique
significant correlation with testosterone 6-b-hydroxylation (CYP3A
activity) when zopiclone was incubated with the 22 liver samples at a
final concentration of 200 mM (r 5 0.965 and r 5 0.859 for ND-Z and
NO-Z, respectively; p 5 .0001; data not shown). Because zopiclone
concentrations in humans never reach such high levels, we performed
the same experiment at a more relevant concentration (25 mM; Table
2). The generation of both metabolites was always highly correlated to
CYP3A4 activity, but the ND-Z formation rate was also correlated to
CYP2C8 activity.
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FIG. 3. Correlation of ND-Z and NO-Z generation rates in human liver
microsomes.
IN VITRO ZOPICLONE METABOLISM
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FIG. 5. Kinetics of NO-Z and ND-Z generation rates in rH-CYP2C8.
E, ND-Z generation rates; F, NO-Z generation rates.
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FIG. 4. Screening of zopiclone metabolism with rH-CYP.
Fifty mM zopiclone was incubated with 100 pmol of each different recombinant
CYP microsomes. The results are the mean of one experiment performed in
quadruplicate. A, contribution of each CYP isoform expressed per picomoles of
rH-CYP. B, expected contribution of each CYP isoform in human liver microsomes
calculated from the relative content of each CYP isoform available in the literature
(see Materials and Methods). M, ND-Z formation rate; f, NO-Z formation rate.
Discussion
In the present study, we report for the first time the in vitro
metabolism of zopiclone in the presence of human liver microsomes
and rH-CYP. We observed that CYP3A4 was the major enzyme
involved in the generation of both zopiclone metabolites and that
CYP2C8 was also involved in the ND-Z formation rate.
To obtain these results, we used the three in vitro main approaches,
which can be used to determine the enzymes involved in the metabolism of a drug (Rodrigues, 1994): 1) selective inhibition of the main
CYP enzymatic activities in human liver microsomes with specific
CYP inhibitors; 2) correlation of the generation rates of the metabolites to classic predetermined CYP activities in different human liver
samples; and 3) screening the metabolism of the drug with a panel of
different heterologously expressed human CYP.
Zopiclone Metabolism in Human Liver Microsomes. We observed that zopiclone was metabolized into two major metabolites,
ND-Z and NO-Z; this is consistent with the observation in humans
(Goa and Heel, 1986). There was a very large interindividual vari-
FIG. 6. Inhibition of zopiclone metabolism in human liver microsomes.
Results represent the mean (6 S.D.) of the values obtained in three human liver
microsomes and are expressed as the percentage of remaining enzymatic activity
(compared with the same experiment performed in the absence of inhibitor considered as 100% activity). M, ND-Z; f, NO-Z. Sulfaphenazole, CYP2C inhibitor;
a-naphtoflavone, CYP1A inhibitor; quinidine, CYP2D6 inhibitor; chlorzoxazone,
CYP2E1 inhibitor; ketoconazole, CYP3A inhibitor.
ability in zopiclone metabolism from one liver sample to another, a
characteristic that is in agreement with most of the drugs metabolized
by CYP in humans and with the large variability of expression of most
of the different human liver CYPs (Guengerich and Turvy, 1991;
Shimada et al., 1994).
The metabolism of zopiclone into its two metabolites was found to
be monophasic on Eadie-Hofstee plots, suggesting that its metabolism
was probably mainly dependent on a single enzyme. CYP3A seemed
to influence the generation of both zopiclone metabolites because the
liver sample with the lowest CYP3A activity had a 10- and 3-fold
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BECQUEMONT ET AL.
TABLE 2
Correlation of NO-Z and ND-Z formation rates to different predetermined
classical CYP activities
Results are expressed as correlation coefficients. Levels of statistical significance are
indicated when #.05. CYP content indicates the spectrophotometric content of CYP per mg
of microsomal protein. Final concentration of zopiclone in the incubate was 25 mM. There
were no spurious correlations between CYP3A activity and other CYP activities or the total
CYP content.
Correlation Coefficient
NO-Z formation
rate
0.12
0.23
0.95
p 5 .0001
0.76
p 5 .004
0.04
0.92
p 5 .0001
0.19
0.35
0.33
0.07
0.10
0.27
0.291
0.21
0.21
0.02
lower intrinsic clearance of ND-Z and NO-Z, respectively, compared
with another liver sample with a medium CYP3A activity.
CYP inhibitors indicated that CYP3A was responsible for at least
40% of the metabolism of zopiclone into its two metabolites and that
the CYP2C subfamily accounted for about 40% of ND-Z formation.
Correlation studies confirmed the major involvement of CYP3A4 in
both zopiclone metabolites generation and the significant contribution
of CYP2C8 in ND-Z formation.
Zopiclone Metabolism in the Presence of rH-CYP. Screening of
zopiclone metabolism with a panel of different heterologously expressed human CYPs indicated that CYP2C8 had the highest enzymatic activity for the generation of both zopiclone metabolites. These
results were adjusted to the relative content of the different CYP
isoforms in human liver microsomes and indicated that: 1) CYP3A4
could be identified as the major isoform involved in NO-Z formation
but not in ND-Z formation; 2) CYP2C8 was found to be the major
CYP in ND-Z formation and had a lower contribution to NO-Z
generation; and 3) CYP2C9 contributed significantly to the formation
of both metabolites. However, our results cannot exclude the concomitant participation of other CYP isoforms, such as CYP2A6 and
CYP2B6 or flavin monooxygenases, in zopiclone metabolism.
These results obtained with rH-CYPs are not in total agreement
with those obtained with human liver microsomes; there was no
involvement of CYP2C9 from the correlations studies, and CYP3A4
is the major isoform that metabolizes zopiclone into its two metabolites from inhibition and correlation studies. We have no clear explanation for such discrepancies, but we believe that the lack of validation of the different recombinant CYP isoforms may be one of the
major hypotheses. Indeed, their enzymatic activity and affinity toward
the substrate may be extremely different, depending on the ratio of
recombinant CYP to recombinant cytochrome b5 and recombinant
CYP reductase in each preparation (Rodrigues, 1994; Shet et al.,
1995; Yamazaki et al., 1996a,b). Therefore, until complete validation
of such recombinant devices by adapting their enzymatic activities to
those found in human hepatic tissues, human liver microsomes should
remain the gold standard for determining the enzymes involved in the
metabolism of drugs. However, the intrinsic clearance of ND-Z determined with recombinant CYP2C8, representing 60% of the one
Acknowledgments. We thank the Bioavenir research program
(supported by the French Ministry of Research, Rhône-Poulenc Rorer
and Roussel-Uclaf) for providing the recombinant human CYP isoforms.
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CYP1A2 (n 5 12)
Phenacetin O-deethylase
CYP3A4 (n 5 22)
Testosterone 6 b-hydroxylase
CYP2C8 (n 5 12)
Paclitaxel 6a-hydroxylase
CYP2C9 (n 5 22)
Diclofenac 49-hydroxylase
CYP2C19 (n 5 12)
(S)-Mephenytoin 49-hydroxylase
CYP2D6 (n 5 22)
Bufuralol 19-hydroxylase
CYP2E1 (n 5 12)
Chlorzoxazone 6-hydroxylase
CYP content (n 5 22)
nmol/mg
ND-Z formation
rate
obtained from the human liver sample with medium CYP3A activity,
is in agreement with the results obtained with specific inhibitors; this
concluded that CYP2C would represent ;40% of zopiclone metabolism.
Altogether, recombinant CYP studies confirmed the significant
contribution of CYP3A4 and CYP2C8 in zopiclone metabolism.
Zopiclone CYP-Dependent Drug Interactions in Humans. Our
in vitro results are in agreement with previous studies performed in
humans that outlined the role of CYP3A in the metabolism of zopiclone. Itraconazole and erythromycin, two classical CYP3A inhibitors, were shown to significantly decrease the clearance of zopiclone
(Aranko et al., 1994; Jalava et al., 1996). Rifampin, a classical
CYP3A inducer, significantly increased zopiclone clearance (Villikka
et al., 1997).
Thus far, CYP2C8 has never been involved in drug interactions
concerning zopiclone. However, because we still do not know specific
inhibitors of this isoform and because only very few drugs are presently identified as CYP2C8 substrates, such drug interactions may
have been difficult to identify. Therefore, clinical relevance of the in
vitro CYP2C8 involvement in zopiclone metabolism remains to be
determined.
In conclusion, the present study characterizes for the first time the
human CYP involved in vitro in zopiclone metabolism. Our results
may help to prevent possible drug interactions associating zopiclone
with other potent CYP3A inhibitors, such as ritonavir or clotrimazole
(Quinn and Day, 1995; Bertz and Granneman, 1997).
IN VITRO ZOPICLONE METABOLISM
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