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Vol. 297, Issue 3, 1044-1050, June 2001
Department of Drug Disposition, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The formation of R- and
S-norfluoxetine was analyzed in vitro in human liver
microsomes. Low apparent Km values for
R-norfluoxetine formation of 
8 µM and
S-norfluoxetine of <0.2 µM were determined. R-Norfluoxetine formation rates in a characterized
microsomal bank correlated with the catalytic activities for cytochrome
P450 (CYP) 2D6, CYP2C9, and CYP2C8. Expressed CYP2C9, CYP2C19, and CYP2D6 formed R-norfluoxetine following incubation with
1 µM R-fluoxetine and exhibited apparent
Km values of 9.7, 8.5, and 1.8 µM,
respectively. Multivariate correlation analysis identified CYP2C9 and
CYP2D6 as significant regressors with R-norfluoxetine
formation. Antibodies to the CYP2C subfamily and CYP2D6 each exhibited
moderate inhibition of R-norfluoxetine formation.
Therefore, CYP2D6 and CYP2C9 contribute to this biotransformation. At
pharmacological concentrations of S-fluoxetine,
S-norfluoxetine formation rates in the bank of
microsomes were found to correlate only with CYP2D6 catalytic activity
and only expressed CYP2D6 was found to be capable of forming
S-norfluoxetine. Thus, it would appear that both CYP2D6
and CYP2C9 contribute to the formation of
R-norfluoxetine, whereas only CYP2D6 is responsible for
the conversion to S-norfluoxetine. Since the enantiomers
of fluoxetine and norfluoxetine are inhibitors of CYP2D6, upon chronic dosing, the CYP2D6-mediated metabolism of the fluoxetine enantiomers would likely be inhibited, resulting in R-norfluoxetine
formation being mediated by CYP2C9 and S-norfluoxetine
formation being mediated by multiple high Km enzymes.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Fluoxetine, a racemic mixture of
R- and S-fluoxetine, is a selective serotonin
reuptake inhibitor currently marketed for the treatment of depression
and other disorders. R-fluoxetine was a drug candidate in
development for use in psychiatric illness. The major route of
metabolism of the enantiomers of fluoxetine is
N-demethylation (Wong et al., 1995
). Identification of the enzymes involved in the formation of norfluoxetine would help explain
interindividual differences observed in the metabolic clearances of
these compounds. To date, however, the definitive identification of the
cytochrome P450s (CYPs) responsible for the metabolism of R-
and S-fluoxetine to R- and
S-norfluoxetine has proven to be elusive.
It has long been recognized that the enantiomers of fluoxetine and
norfluoxetine are inhibitors of CYP2D6-mediated reactions. S-Fluoxetine and S-norfluoxetine are
approximately 5-fold more potent in their ability to inhibit
CYP2D6-mediated reactions than R-fluoxetine and
R-norfluoxetine (Ki values
of 0.22, 0.31, 1.38, and 1.48 µM, respectively) (Stevens and
Wrighton, 1993). In spite of this CYP2D6 inhibitory potential, previous
studies performed in vitro suggested that CYP2D6 plays only a partial
role the biotransformation of R- and S-fluoxetine
to their respective N-desmethyl metabolites (Stevens and
Wrighton, 1993). A similar conclusion was reached by von Moltke et al.
(1997) who determined that although CYP2D6, CYP2C19, and CYP3A
partially contributed to the formation of racemic norfluoxetine from
racemic fluoxetine, CYP2C9 was the primary CYP responsible for
norfluoxetine formation. Recently a third study concluded that CYP2D6,
CYP2C9, and CYP3A were the greatest contributors to fluoxetine
N-demethylation (Margolis et al., 2000).
There have been a few studies in humans that have examined the
clearance of the enantiomers of fluoxetine following both single and
multiple doses of racemic fluoxetine. In a study examining the
pharmacokinetics of a single dose of racemic fluoxetine a major role
for CYP2D6 in S-fluoxetine metabolism was proposed, for
S-fluoxetine clearance was 12-fold slower in poor
metabolizers (PMs) of CYP2D6-mediated reactions than that observed in
extensive metabolizers (EMs) (Fjordside et al., 1999). However,
the clearance of R-fluoxetine was only 2-fold slower in PMs
compared with EMs. These differences in the clearances of S-
and R-fluoxetine in PMs after a single dose indicated that
the formation of S-norfluoxetine is highly dependent on
CYP2D6, whereas other CYPs in addition to CYP2D6 participate in the
formation of R-norfluoxetine. This differential dependence
on CYP2D6 for S- and R-norfluoxetine formation was also demonstrated by the pharmacokinetic profiles of S-
and R-fluoxetine after multiple dosing of racemic fluoxetine
(Bergstrom et al., 1991). These researchers found that when
compared with a single dose of fluoxetine, after multiple dosing of
fluoxetine the clearance of S-fluoxetine was substantially
decreased, but that of R-fluoxetine was only slightly
decreased. Thus, it appears that self-inhibition of the CYP2D6-mediated
metabolism by the enantiomers of S- and
R-fluoxetine occurred and had a greater effect on the
clearance of S-fluoxetine compared with the clearance of
R-fluoxetine. Therefore, although fluoxetine disposition is different in PMs and EMs following single dosing, these differences are
significantly diminished upon multiple dosing. With this information as
background, and noting the conflicting conclusions in the previous studies performed in vitro, the aim of this study was to definitively identify the enzymes involved in the N-demethylation of the
enantiomers of fluoxetine to help explain population variability in the
pharmacokinetics of both racemic and R-fluoxetine.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials. R-fluoxetine, R-norfluoxetine, S-fluoxetine, S-norfluoxetine, and LY110086 (internal standard) were synthesized by Eli Lilly and Co. (Indianapolis, IN). Diclofenac, phenacetin, chlorzoxazone, zoxazolamine, 7-hydroxy coumarin, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). Midazolam was obtained from Hoffmann La Roche (Nutley, NJ) and 4'-hydroxy diclofenac was obtained from GENTEST (Woburn, MA). S-Mephenytoin, 4'-hydroxy mephenytoin, and 1'-hydroxy midazolam were purchased from Ultrafine (Manchester, UK). 6-Hydroxy chlorzoxazone was obtained from Sigma/RBI (Natick, MA) and acetaminophen was obtained from Kodak (Rochester, NY). Coumarin and trolox were obtained from Aldrich Chemical Co. (Milwaukee, WI). Bufuralol and 1'-hydroxy bufuralol were obtained from GENTEST.
Monoclonal antibodies to CYP2D6 and CYP2C in ascites fluid were obtained from Panvera (Madison, WI) and control ascites fluid was obtained from ICN Biochemicals (Aurora, OH). The specificity of these antibodies was shown by the observation that the CYP2C monoclonal antibody inhibited by 90% the form-selective CYP2C19 biotransformation of S-mephenytoin 4'-hydroxylase and CYP2C9-specific diclofenac 4'-hydroxylation. The CYP2D6 antibody inhibited >90% of the CYP2D6 form-selective biotransformation of bufuralol 1'-hydroxylation (Panvera).Microsomes.
Human liver samples designated HLA through HLT
were obtained from the Medical College of Wisconsin (Milwaukee, WI),
Medical College of Virginia (Richmond, VA), or Indiana University
School of Medicine (Indianapolis, IN), under protocols approved by the appropriate committee for the conduct of human research. Hepatic microsomes were prepared by differential centrifugation (van der Hoeven
and Coon, 1974) and characterized for their relative levels of CYPs and
flavin-containing monooxygenase (FMO) via immunoquantification or
through the use of form-selective catalytic activities (see below).
Microsomes prepared from a human 
-lymphoblastoid cell line
engineered to express CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, or CYP3A4) were obtained from GENTEST.
Examination of R- or S-Norfluoxetine Formation. The conversions of R-fluoxetine to R-norfluoxetine and S-fluoxetine to S-norfluoxetine were accomplished under linear rate conditions. Incubations were performed for 10 min at 37°C, after a 3-min preincubation, with incubation mixtures containing the indicated concentrations of substrate with human hepatic microsomes (0.5 mg/ml) and 1 mM NADPH in 100 mM sodium phosphate buffer, pH 7.4. The reactions were stopped with an equal volume of acetonitrile followed by the addition of internal standard. The denatured protein was removed by centrifugation and the supernatant analyzed for metabolite formation.
Estimations of apparent enzyme kinetic parameters for the formation of R-norfluoxetine were determined by human liver microsomal samples HLM, HLG, HLK, and HLO. Samples HLM and HLG were chosen as representative of livers that contain an average complement of CYPs, and HLO was chosen for its high level of CYP2D6, CYP3A, and CYP2A6 (Table 1). Sample HLK was chosen because it is deficient in CYP2D6 as determined by immunoblot analysis (Wrighton et al., 1993b). In these studies concentrations of
R-fluoxetine ranged from 1 to 300, 0.25 to 100, 2.5 to 300, and 0.2 to 100 µM, respectively. Similarly, to examine the enzyme
kinetics for the formation of S-norfluoxetine concentrations
of S-fluoxetine ranged from 0.5 to 150, 0.11 to 120, and 5 to 150 µM with HLG, HLM, and HLK, respectively.
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-lymphoblastoid cells transfected
with human CYP cDNA were examined for their ability to form R- or S-norfluoxetine following 60-min, 37°C
incubations with 2 mM NADPH and 1 and 30 µM R-fluoxetine
or 1 and 75 µM S-fluoxetine. For the estimation of
apparent enzyme kinetic parameters for the formation of
R-norfluoxetine by expressed CYP2C9, CYP2C19, or CYP2D6,
incubations were performed under initial rate conditions with
R-fluoxetine concentrations ranging from 0.5 to 75, 1 to 300, and 0.05 to 50 µM, respectively. The enzyme kinetic parameters by expressed CYP2D6 were determined for S-norfluoxetine
formation under linear rate conditions following incubations with
S-fluoxetine concentrations ranging from 0.05 to 25 µM.
Monoclonal antibodies to the CYP2C subfamily or CYP2D6 were used with
human liver microsomal samples HLM, HLG, and HLS to assess their effect
on R-norfluoxetine formation. In these studies, microsomes,
2 µM R-fluoxetine, and ascites fluid containing antibodies or control ascites fluid were preincubated for 5 min at 37°C prior to
the initiation of the 10-min reaction with 1 mM NADPH. Preliminary experiments to determine maximum inhibition of
R-norfluoxetine formation by each of these antibodies were
performed in incubations with HLM containing 1, 2, 4, or 8 µl of each
of the ascites preparation. Maximum inhibition for both anti-2C and
anti-2D6 occurred with the addition of 4 µl of ascites fluid (data
not shown). Therefore, 4 µl of these inhibitory antibodies was used
to examine their ability to inhibit the formation of
R-norfluoxetine.
Phosphate buffer (0.1 M) was added to supernatants from the centrifuged
samples and they were loaded onto Isolute HCX solid phase extraction
cartridges (130 mg, 3 ml) (Jones Chromatography, Lakewood, CO)
preconditioned with sequential washes of methanol and 0.1 M potassium
phosphate buffer, pH 6.0. Cartridges were washed sequentially with
methanol, acetonitrile and 50:50 (v/v) hexane/ethyl acetate, and eluted
with methylene chloride/methanol/concentrated ammonium hydroxide
(78.4:19.6:0.2, v/v/v). The eluted samples were dried at 50°C under
nitrogen, 1 ml of 2% heptafluorobutyric acid anhydride in hexane
added, and samples heated at 80°C for 30 min. Derivatized samples
were dried at 50°C under nitrogen, solubilized in hexane, and
analyzed for R- or S-norfluoxetine formation by
gas chromatography/mass spectral analysis using a 15-m Restek Rtx-5 MS
column (Restek Corp., Bellefonte, PA) and negative chemical ionization
with methane as the reagent gas. The lower and upper limits of
detection of the analytical assay for both R- and
S-norfluoxetine were 1 and 501 pmol per the 200-µl incubation volume.
CYP Form-Selective Catalytic Activities. The O-deethylation of phenacetin (acetaminophen formation) was used as a marker of CYP1A2-mediated metabolism. Acetaminophen was detected by HPLC with UV detection (254 nm) using an Alltima Phenyl column (Alltech, Deerfield, IL) (5 µm, 4.6 × 150 mm) and a mobile phase of 25 mM sodium phosphate buffer, pH 3.0/methanol (95:5, v/v) delivered at 1.0 ml/min with a retention time of 6 min. To characterize the human liver microsomal bank for acetaminophen formation, microsomes were incubated under initial rate conditions with 50 µM phenacetin (Table 1).
The 4'-hydroxylation of diclofenac was used as a marker of CYP2C9-mediated metabolism. 4'-Hydroxy diclofenac and internal standard (trolox) had retention times 1.8 and 1.3 min, respectively, following HPLC with electrochemical detection using a Zorbax SB-CN column (Mac-Mod Analytical, Inc., Chadds Ford, PA), 3.5 µm, 4.6 × 75 mm and a mobile phase of 100 mM potassium phosphate, pH 3.0/ acetonitrile (60:40, v:v) delivered at 1.5 ml/min. To characterize the human liver microsomal bank, incubations were performed under initial rate conditions with 5 µM diclofenac (Table 1). Chlorzoxazone biotransformation to 6-hydroxy chlorzoxazone was used as a marker of CYP2E1-mediated metabolism. 6-Hydroxy chlorzoxazone and internal standard zoxazolamine were detected by HPLC with UV detection (287 um) with retention times of 11 and 13 min, respectively, using a Zorbax SB-CN column (Mac-Mod Analytical, Inc.), 3.5 µm, 4.6 × 75 mm and a mobile phase of 100 mM potassium phosphate, pH 3.0/acetonitrile (60:40, v/v) delivered at 1.5 ml/min. To characterize the human liver microsomal bank, incubations were performed under initial rate conditions with 400 µM chlorzoxazone (Table 1). Coumarin 7-hydroxy formation, form-selective for CYP2A6 activity, was characterized in the human liver microsomal bank following incubation under initial rate conditions with 100 µM coumarin by a modification of the method of Greenlee and Poland (1978) (Table 1).
Immunoquantification of CYP2B6 in the human liver bank was previously
reported by Ekins et al. (1998). Taxol 6-hydroxylation, form-selective
for CYP2C8 activity, was determined by the method of Harris et al.
(1994) following incubation of the human liver microsomal bank under
initial rate conditions with 10 µM Taxol (Table 1). The
4'-hydroxylation of S-mephenytoin, as determined by the
method of Wrighton et al. (1993a), was used as a marker of
CYP2C19-mediated metabolism. Incubations were performed under initial
rate conditions with 50 µM S-mephenytoin for
characterization of the human liver bank (Table 1). The microsomal bank
was characterized for 1'-hydroxy bufuralol formation, form-selective
for CYP2D6, following incubation under initial rate conditions with 25 µM bufuralol by the method of Ring et al. (1996) (Table 1). The 1'-hydroxylation of midazolam was used as a marker of CYP3A4/5-mediated metabolism and was analyzed by a modification of the method of Wrighton
and Ring (1994) using a 4.6 × 150 mm YMC Basic column (YMC Inc.,
Wilmington, NC) following 1-min incubations. Incubations were performed
with 25 µM midazolam for characterization of the human liver bank
(Table 1).
Calculations.
Enzyme kinetic parameters were determined
following fit of the data to the appropriate kinetic equations using
nonlinear regression analysis (WinNonlin, version 1.5; Statistical
Consultants, Inc., Cary, NC). Formation rates of R- or
S-norfluoxetine were fit to one of the following models
(Segel, 1975; Copeland, 1996):
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0.05) following univariate regression analysis, multivariate regression analysis was performed. In these analyses, a
stepwise analysis was performed in a forward mode. This procedure added
regressors to the correlation that most improve the fit, given that the
added term was significant at least at the p = 0.15 level. Once potential significant regressors were identified, leverage
plots were examined to assess the significance of the added regressors
to the correlation model.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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R-Fluoxetine N-Demethylation to
R-Norfluoxetine.
Enzyme kinetic parameters for the
formation of R-norfluoxetine were determined under initial
rate conditions in four human liver samples: HLO, HLG, HLM, and HLK.
The Eadie-Hofstee plot (data not shown) for the formation of
R-norfluoxetine by microsomal sample HLO was biphasic,
suggesting the involvement of at least two enzymes in this
biotransformation. This microsomal sample demonstrated high activities
of CYP2D6, CYP3A, and CYP2A6 relative to the other human liver
microsomal samples examined (Table 1). The apparent low
Km (1.6 µM) and high
Km (34 µM) values for this reaction
by HLO were determined using eq. 4 (Table
2). The formation of
R-norfluoxetine in the three additional human liver
microsomal samples exhibited Michaelis-Menten kinetics at low substrate
concentrations and product inhibition at high substrate concentrations
(Fig. 1). Therefore, the apparent
kinetic parameters for this biotransformation by these samples were
estimated using eq. 2. Two of these samples, HLG and HLM, contained a
full complement of CYP enzymes and exhibited apparent
Km values of <10 µM (Table 2).
Sample HLK was deficient in CYP2D6 and exhibited an apparent
Km value of 20 µM (Table 2). The
apparent Ki values for these three
microsomal samples ranged from 65 to 294 µM.
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S-Fluoxetine N-Demethylation to
S-Norfluoxetine.
Enzyme kinetic parameters for the
formation of S-norfluoxetine were determined under initial
rate conditions in three human liver samples: HLM, HLG, and HLK.
Eadie-Hofstee plots of the formation of S-norfluoxetine by
HLM and HLG were biphasic in nature, consistent with two enzymes being
responsible for this biotransformation. Therefore, the kinetic
parameters for the formation of S-norfluoxetine by HLG and
HLM were estimated using eq. 4. The low
Km values for this biotransformation
in HLG and HLM were 0.17 and 0.18 µM, respectively (Table
5). The high
Km values for the formation of
S-norfluoxetine were 88 and 67 µM in HLG and HLM,
respectively (Table 5). The Eadie-Hofstee plot of the data generated
for HLK, a liver deficient in CYP2D6, was monophasic in nature, which
suggests that one enzyme was responsible for this biotransformation in
this sample. The kinetic parameters obtained for the formation of
S-norfluoxetine in HLK were determined using eq. 1, resulting in an apparent Km value of
109 µM for this biotransformation (Table 5).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The identification of the enzyme(s) responsible for the oxidative
metabolism of a drug allows one to predict and/or explain interindividual differences in the effects of the drug that are due to
differences in its metabolic clearance. Previous in vitro (Stevens and
Wrighton, 1993; von Moltke et al., 1997, Margolis et al., 2000) and in
vivo studies (Bergstrom et al., 1991; Fjordside et al., 1999) suggested
CYP2D6 contributes to the biotransformation of the enantiomers of
fluoxetine to their N-demethylated metabolites. However,
these studies also suggested that additional enzymes were involved in
these biotransformations. Therefore, the current studies were designed
to definitively identify the enzymes involved in the
N-demethylation of R- and
S-fluoxetine.
An examination of the enzyme kinetics of R-norfluoxetine
formation suggested that at least two enzymes were involved in this biotransformation, which exhibited apparent
Km values ranging from about 1 to 35 µM. It is interesting to note that inhibition of
R-norfluoxetine formation was observed at high substrate
concentrations in three of four microsomal samples examined. This
observation is similar to that reported by Stevens and Wrighton (1993)
and von Moltke et al. (1997). This phenomenon is most likely due to product inhibition (Copeland, 1996). In these studies the apparent affinity of the enzyme-product complex
(Ki values) ranged from 65 to 294 µM. This greatly exceeds the expected in vivo steady-state concentration of <1 µM R-fluoxetine; therefore, product
inhibition would not be expected to be a factor in the in vivo
clearance of R-fluoxetine.
To identify the low Km enzyme(s)
involved in the formation of R-norfluoxetine further studies
were performed using R-fluoxetine concentrations near the
low Km value observed for this
biotransformation. These studies included correlating the rates of
R-norfluoxetine formation to known activities mediated by
the CYPs and FMO in a bank of liver microsomal samples, examination of
the ability of cDNA expressed CYPs to form this metabolite, and the use
of monoclonal antibodies in an attempt to inhibit this
biotransformation. The formation of R-norfluoxetine
following incubation with a pharmacological concentration of
R-fluoxetine correlated to CYP2C9, CYP2D6, and CYP2C8
form-selective catalytic activities. Expressed CYP2C9, CYP2C19, and
CYP2D6 were the only enzymes able to form R-norfluoxetine. Performing multivariate regression analysis using CYPs identified in
these studies as possible coregressors with R-norfluoxetine formation, only CYP2C9 and CYP2D6 were found to be significant regressors with the formation of R-norfluoxetine. Monoclonal
antibodies were used to quantify the role of particular CYPs in the
formation of R-norfluoxetine (Gelboin et al., 1999). Through
the use of these antibodies the role of CYP2C9 and CYP2D6 in this
biotransformation by human liver microsomes was further confirmed.
Finally, kinetic assessments of the formation of
R-norfluoxetine by expressed CYP2D6, CYP2C9, and CYP2C19
determined apparent Km values of 1.8, 9.7, and 8.5 µM, respectively. Interestingly, the kinetic analyses with expressed CYP2D6 and CYP2C9 exhibited product inhibition at high
R-fluoxetine concentrations, which was similar to that observed in three microsomal liver samples. Expressed CYP2C9 also exhibited substrate activation at low concentrations.
Taken together, the data presented indicate that in microsomal samples
containing a full complement of CYPs, the contribution of CYP2C9 and
CYP2D6 to the formation of R-norfluoxetine at low R-fluoxetine concentrations is similar. Although present in
relatively small amounts (~2%, Shimada et al., 1994) in the human
liver, CYP2D6 has a low Km value,
which indicates a high affinity for R-fluoxetine. This
coupled with the antibody inhibition data suggests that CYP2D6 plays an
important role (~40%) in the formation of R-norfluoxetine. Although CYP2C9 appears to have a 5-fold
higher Km value for
R-fluoxetine, results with inhibitory antibodies suggest it
also plays a primary role in R-fluoxetine metabolism (~55%), which is likely due to CYP2C9 levels in the liver that are
about 10-fold greater than those of CYP2D6 (Shimada et al., 1994).
Expressed CYP2C19 and CYP2C9 have a similar affinity for R-fluoxetine; however, CYP2C19 represents only ~1% of the
CYPs in the liver (Inoue et al., 1997), therefore it would not be
expected to play a major role in this biotransformation. Furthermore,
the correlation studies indicated that CYP2C19 levels were not related to the formation of R-norfluoxetine, suggesting that CYP2C19
does not play a significant role in this biotransformation.
Similar studies were performed examining the conversion of S-fluoxetine to S-norfluoxetine. Enzyme kinetic studies in two liver samples containing a full complement of enzymes were consistent with two enzymes being involved in this biotransformation, with the low Km enzyme exhibiting an apparent Km value of about 0.2 µM. Interestingly, a microsomal sample deficient in CYP2D6 (HLK) apparently contained only the high Km enzyme (Km = 109 µM) able to form S-norfluoxetine. In the correlation studies, the only activity that correlated with S-norfluoxetine formation following incubation with 2.5 µM S-fluoxetine was that for CYP2D6. Only expressed CYP2D6 was able to form this metabolite (apparent Km value of 0.58 µM) at a low S-fluoxetine concentration. These results confirm the apparently exclusive role of CYP2D6 in this biotransformation at low, pharmacological S-fluoxetine concentrations.
In the current study, multiple CYPs were found to be capable of forming
both R- and S-norfluoxetine following incubation
with high concentrations of R- and S-fluoxetine.
As reported herein, at high concentrations of substrate, CYP2D6 is only
one of many CYPs that may participate in this biotransformation. This
may explain the conclusions of von Moltke et al. (1997) and Margolis et
al. (2000) who suggested that in addition to CYP2D6 and CYP2C9 playing
a role in norfluoxetine formation, that CYP2C19 and CYP3A may also be
involved. These conclusions were confirmed in the current studies where
CYP2C19 and CYP3A (along with other CYPs) were able to form
R- and S-norfluoxetine at high substrate concentrations.
The involvement of CYP2D6 and CYP2C9 in the metabolism of the
enantiomers of fluoxetine at pharmacological concentrations helps to
explain the pharmacokinetic parameters observed with the administration
of racemic fluoxetine. The identification of CYP2D6 as the principle
enzyme responsible for the formation of S-norfluoxetine is
important since CYP2D6 is polymorphically expressed where 5 to 10% of
the Caucasian population and <1% of the Asian population lack
functional enzyme. Therefore, the involvement of CYP2D6 in
S-norfluoxetine formation explains the observation made in
vivo where following a single dose of racemic fluoxetine the clearance
of S-fluoxetine in PMs of substrates of CYP2D6 was 12-fold
slower than that observed in an EM population (Fjordside et al., 1999).
However, because both CYP2C9 and CYP2D6 contribute to
R-norfluoxetine formation, the clearance of
R-fluoxetine should be less affected in a CYP2D6 PM
population than S-fluoxetine, since
R-norfluoxetine would also be substantially formed by
CYP2C9. This was also confirmed in the Fjordside et al. (1999) in which the change in R-fluoxetine clearance in PMs was reported to
be only 2-fold slower than that observed in EMs. Interestingly, it has
been observed that upon multiple dosing of racemic fluoxetine, the
metabolism of coadministered dextromethorphan, a CYP2D6 substrate, in
EMs was inhibited to a point where a majority of the subjects became
phenotypically PMs of CYP2D6-mediated dextromethorphan O-deethylation (Alfaro et al., 1999). This is apparently due
to the potent inhibition by S-fluoxetine and
S-norfluoxetine of CYP2D6-mediated reactions
[Ki values of 0.22 and 0.31, respectively (Stevens and Wrighton, 1993)]. Furthermore, since CYP2D6
has been identified as the primary enzyme involved in
S-norfluoxetine formation, inhibition of its own metabolism
would be predicted to occur upon multiple dosing. This is exactly what
was observed after chronic dosing of racemic fluoxetine (Bergstrom et
al., 1991). Specifically, upon chronic administration of 60 mg/day
racemic fluoxetine, EM subjects were found to clear
S-fluoxetine similarly to that of PMs. Therefore, with the
chronic use of racemic fluoxetine, patients, no matter what their
CYP2D6 genotype, would be phenotypically PMs of CYP2D6 substrates.
Therefore, CYP2C9 and the other enzymes that exhibit a high
Km value for the conversion of the
enantiomers of fluoxetine to norfluoxetine most likely mediate the
metabolic clearance of racemic fluoxetine upon chronic dosing.
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Acknowledgments |
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We thank J. Harris for screening our human liver microsomal bank for Taxol 6-hydroxylase activity.
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Footnotes |
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Accepted for publication February 19, 2001.
Received for publication September 12, 2000.
Send reprint requests to: Barbara J. Ring, Lilly Corporate Center, Mail Drop 0730, Eli Lilly and Co., Indianapolis, IN 46285. E-mail: ring_barbara_j{at}lilly.com
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Abbreviations |
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CYP, cytochromes P450; PM, poor metabolizer of CYP2D6 substrates; EM, extensive metabolizer of CYP2D6 substrates; HL, human liver; FMO, flavin-containing monooxygenase; HPLC, high-performance liquid chromatography; bql, below quantifiable limit.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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H. Shen, M. M. He, H. Liu, S. A. Wrighton, L. Wang, B. Guo, and C. Li Comparative Metabolic Capabilities and Inhibitory Profiles of CYP2D6.1, CYP2D6.10, and CYP2D6.17 Drug Metab. Dispos., August 1, 2007; 35(8): 1292 - 1300. [Abstract] [Full Text] [PDF]
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J. L. F. Rehmel, J. A. Eckstein, N. A. Farid, J. B. Heim, S. C. Kasper, A. Kurihara, S. A. Wrighton, and B. J. Ring INTERACTIONS OF TWO MAJOR METABOLITES OF PRASUGREL, A THIENOPYRIDINE ANTIPLATELET AGENT, WITH THE CYTOCHROMES P450 Drug Metab. Dispos., April 1, 2006; 34(4): 600 - 607. [Abstract] [Full Text] [PDF]
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 J.-M. Sauer, A. J. Long, B. Ring, J. S. Gillespie, N. P. Sanburn, K. A. DeSante, D. Petullo, M. R. VandenBranden, C. B. Jensen, S. A. Wrighton, et al. Atomoxetine Hydrochloride: Clinical Drug-Drug Interaction Prediction and Outcome J. Pharmacol. Exp. Ther., February 1, 2004; 308(2): 410 - 418. [Abstract] [Full Text] [PDF]
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B. J. Ring, J. S. Gillespie, S. N. Binkley, K. M. Campanale, and S. A. Wrighton The Interactions of a Selective Protein Kinase C Beta Inhibitor with the Human Cytochromes P450 Drug Metab. Dispos., September 1, 2002; 30(9): 957 - 961. [Abstract] [Full Text] [PDF]
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J. M. Hutzler and T. S. Tracy Atypical Kinetic Profiles in Drug Metabolism Reactions Drug Metab. Dispos., April 1, 2002; 30(4): 355 - 362. [Full Text] [PDF]
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B. J. Ring, J. S. Gillespie, J. A. Eckstein, and S. A. Wrighton Identification of the Human Cytochromes P450 Responsible for Atomoxetine Metabolism Drug Metab. Dispos., March 1, 2002; 30(3): 319 - 323. [Abstract] [Full Text] [PDF]
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), HLM (
), and HLK (
).