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Vol. 29, Issue 8, 1102-1109, August 2001
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine; and the Division of Clinical Pharmacology, New England Medical Center Hospital, Boston, Massachusetts
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Transformation of escitalopram (S-CT), the pharmacologically active S-enantiometer of citalopram, to S-desmethyl-CT (S-DCT), and of S-DCT to S-didesmethyl-CT (S-DDCT), was studied in human liver microsomes and in expressed cytochromes (CYPs). Biotransformation of the R-enantiomer (R-CT) was studied in parallel. S-CT was transformed to S-DCT by CYP2C19 (Km = 69 µM), CYP2D6 (Km = 29 µM), and CYP3A4 (Km = 588 µM). After normalization for hepatic abundance, relative contributions to net intrinsic clearance were 37% for CYP2C19, 28% for CYP2D6, and 35% for CYP3A4. At 10 µM S-CT in liver microsomes, S-DCT formation was reduced to 60% of control by 1 µM ketoconazole, and to 80 to 85% of control by 5 µM quinidine or 25 µM omeprazole. S-DDCT was formed from S-DCT only by CYP2D6; incomplete inhibition by quinidine in liver microsomes indicated participation of a non-CYP pathway. Based on established index reactions, S-CT and S-DCT were negligible inhibitors (IC50 > 100 µM) of CYP1A2, -2C9, -2C19, -2E1, and -3A, and weakly inhibited CYP2D6 (IC50 = 70-80 µM). R-CT and its metabolites, studied using the same procedures, had properties very similar to those of the corresponding S-enantiomers. Thus S-CT, biotransformed by three CYP isoforms in parallel, is unlikely to be affected by drug interactions or genetic polymorphisms. S-CT and S-DCT are also unlikely to cause clinically important drug interactions via CYP inhibition.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Previous
studies have evaluated the properties of biotransformation of racemic
citalopram (CT) to monodesmethylcitalopram (DCT) in vitro by human
liver microsomes and by heterologously expressed individual human
cytochromes (Kobayashi et al., 1997
; Rochat et al., 1997; von Moltke et
al., 1999b). These studies indicate that CYP3A4, -2C19, and -2D6 all
contribute to this reaction, with approximate relative contributions at
low concentrations of CT estimated at 34% for CYP3A, 36% for CYP2C19,
and 30% for CYP2D6. Stereoselective clearance has also been suggested,
in that intrinsic clearance of S-citalopram (approved
generic name of Escitalopram) may exceed that of
R-citalopram (R-CT; Olesen and Linnet, 1999).
Clinical studies indicate that steady-state concentrations of
R-CT may exceed those of S-CT during chronic administration of racemic CT to humans, and that the elimination half-life of R-citalopram exceeds that of
S-citalopram (Bondolfi et al., 1996; Foglia et al., 1997;
Voirol et al., 1999). These differences are of potential clinical
importance, since the S-isomers of CT and DCT have greater
intrinsic antidepressant activity than their respective
R-isomers (Hyttel et al., 1992). S-Citalopram itself is currently under investigation as an antidepressant agent.
The present study evaluated the biotransformation of the individual R- and S-isomers of CT to DCT, and of R- and S-DCT to didesmethylcitalopram (DDCT), by human liver microsomes, and by microsomes containing pure human cytochromes expressed by cDNA-transfected human lymphoblastoid cells. The inhibitory actions of R- and S-CT, -DCT, and -DDCT versus index reactions representing activity of specific human cytochromes were also evaluated.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In Vitro Procedures. Liver samples from individual human donors with no known liver disease were provided by the International Institute for the Advancement of Medicine, Exton, PA, the Liver Tissue Procurement and Distribution System, University of Minnesota, Minneapolis, MN, and the National Disease Research Interchange, Philadelphia, PA. All samples were of the CYP2D6 and CYP2C19 normal metabolizer phenotype based on prior in vitro phenotyping studies.
Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 M potassium phosphate buffer containing 20% glycerol and stored at
80°C until use. Microsomes containing individual human cytochromes expressed by cDNA-transfected human lymphoblastoid cells (Gentest, Woburn, MA) were similarly stored at
80°C until use. Pure samples of R- and S-CT,
-DCT, and -DDCT were provided by H. Lundbeck A/S (Copenhagen-Valby,
Denmark) through Forest Laboratories (New York, NY). Other chemical
reagents and drug entities were purchased from commercial sources or
kindly provided by their pharmaceutical manufacturers.
Incubation mixtures contained 50 mM phosphate buffer, 5 mM
Mg2+, 0.5 mM NADP+, and an
isocitrate/isocitric dehydrogenase regenerating system. Appropriate
substrates, with and without inhibitor in methanol solution, were added
to a series of incubation tubes. The solvent was evaporated to dryness
at 40°C under conditions of mild vacuum. Reactions were initiated by
addition of microsomal protein (0.25 mg/ml for liver microsomes, 1.0 mg/ml for expressed CYPs). After an appropriate incubation duration at
37°C, reactions were stopped by cooling on ice and addition of 100 µl of acetonitrile. Internal standard was added, the incubation
mixture was centrifuged, and the supernatant was transferred to an
autosampling vial for high performance liquid chromatography analysis.
The mobile phase consisted of a combination of acetonitrile and 50 mM
phosphate buffer as appropriate for each assay. The analytical column
was stainless steel, 30 cm × 3.9 mm, containing reverse-phase
C-18 µBondapak, or 15 cm × 3.9 mm, containing reverse-phase
C-18 Novapak (Waters Associates, Milford, MA). Column effluent was
monitored by ultraviolet absorbance at the appropriate wavelength. All
biotransformation reactions were verified to be in the linear range
with respect to incubation duration and protein concentration.
Inhibition Studies.
The potential inhibitory effect of the stereoisomers of CT, DCT, and
DDCT on the activity of six human cytochromes was evaluated using index
reactions and methods as follows (Table
1): CYP1A2, phenacetin (100 µM) to
acetaminophen (von Moltke et al., 1996a; Venkatakrishnan et al.,
1998b); CYP2C9, tolbutamide (100 µM) to hydroxytolbutamide
(Venkatakrishnan et al., 1998c); CYP2C19, S-mephenytoin (25 µM) to 4'-OH-mephenytoin (Venkatakrishnan et al., 1998c); CYP2D6,
dextromethorphan (25 µM) to dextrorphan (Schmider et al., 1997; von
Moltke et al., 1998c); CYP2E1, chlorzoxazone (50 µM) to
6-OH-chlorzoxazone (Court et al., 1997); CYP3A, triazolam (250 µM) to
-OH-triazolam (von Moltke et al., 1996b, 1998a, 2001; Perloff et
al., 2000). Substrate specificity in the potency of CYP3A inhibitors
has been suggested (Kenworthy et al., 1999); triazolam is an index
CYP3A substrate reported to be representative of the most common
substrate category.
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).
Enzyme Kinetic Studies.
Varying concentrations of R-CT, S-CT,
R-DCT, and S-DCT (0-1000 µM) were added to
incubation tubes, and the solvent evaporated to dryness. Incubations
with human liver microsomes or with heterologously expressed human
cytochromes (Crespi, 1995; Crespi and Penman, 1997; Crespi and Miller,
1999) were performed as described above and as reported previously (von
Moltke et al., 1999b). Reactions were stopped by addition of
acetonitrile and by cooling on ice. Internal standard (phenacetin) was
added, the mixtures were centrifuged, and supernatants were transferred
to high performance liquid chromatography autosampling vials. The
mobile phase was 70% 50 mM
KH2PO4 and 30%
acetonitrile at 2 ml/min. Column effluent was monitored by ultraviolet
absorbance at 230 nm. Concentrations of DCT (following incubation with
R- or S-CT) or of DDCT (following incubation with R- or S-DCT) were determined using calibration
curves containing known amounts of DCT or DDCT. Reaction velocities
were calculated in units of picomoles per minute per milligram of
microsomal protein for liver microsomal studies, or picomoles per
minute per picomole of CYP in expressed cytochrome studies.
Liver Microsomes.
Biotransformation of R- or S-CT to DCT was
consistent with Michaelis-Menten kinetics in four of eight instances.
The following equation was fitted to data points using nonlinear
regression:
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(1) |
;
von Moltke et al., 1996b; Ueng et al., 1997; Harlow and Halpert, 1998;
Witherow and Houston, 1999).
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(2) |
).
Biotransformation of R- and S-DCT to DDCT was
consistent with a two-enzyme process, with the high-affinity (low
Km) component consistent with a
Michaelis-Menten process. The low-affinity process required
approximation by a linear function. The following equation was fitted
to data points.
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(3) |
Heterologously Expressed Cytochromes. Initial screening studies were performed to evaluate the possible formation of DCT from R- and S-CT, and of DDCT from R- and S-DCT, using expressed CYP1A2, -1A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4. Formation of DCT from the enantiomers of CT was mediated by CYP2C19, -2D6, and -3A4; other CYPs did not yield detectable metabolite formation. Transformation of the enantiomers of DCT to DDCT was mediated only by CYP2D6, with no detectable metabolic activity from other cytochromes.
Accordingly, transformation of R- and S-CT to DCT was characterized as described above using varying concentrations of R- and S-CT incubated with heterologously expressed CYP2C19, -2D6, and -3A4 in separate experiments. Transformation of R- and S-DCT to DDCT was characterized using heterologously expressed CYP2D6. The relative activity factor approach was used to estimate relative contributions of CYP2C19, -2D6, and -3A4 to net clearance of R-CT and S-CT. Average relative in vivo abundances, equivalent to the relative activity factors, were estimated using methods described in detail previously (Crespi, 1995;
Venkatakrishnan et al., 1998a,c, 1999, 2000, 2001; von Moltke et al.,
1999a,b; Störmer et al., 2000). These were 13.1% for CYP2C19,
3.8% for -2D6, and 83.1% for -3A4. Vmax
and intrinsic clearance values for the individual cytochromes were
multiplied by the average relative abundances and the values were
normalized to 100%. The resulting values were used to estimate the
contribution of each cytochrome to net reaction velocity in relation to
concentration of R-CT or S-CT.
Chemical Inhibition of R-CT, S-CT,
R-DCT, and S-DCT Biotransformation in
Microsomes.
Human liver microsomes were incubated with fixed concentrations (10 µM or 100 µM) of R-CT or S-CT. These were
performed in the control condition (without inhibitors), or with
coaddition of index inhibitors corresponding to the three CYP isoforms
participating in CT biotransformation (Table 1). These were omeprazole
(25 µM) versus CYP2C19, quinidine (5 µM) versus CYP2D6, and
ketoconazole (1 µM) versus CYP3A. We recognize that 25 µM
omeprazole may not be fully specific as a CYP2C19 inhibitor (Ko et al.,
1997; Giancarlo et al., 2001). The effect of quinidine on
biotransformation of R-CT and S-CT to DDCT was
studied using similar methods. Reaction velocities with coaddition of
inhibitor were expressed as a percentage ratio of the control velocity
with no inhibitor present.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibition Studies.
In all systems the positive control inhibitors produced the expected degree of inhibition of their respective index reactions (Table 1).
CYP1A2.
R- and S-CT and metabolites all were negligible
inhibitors of phenacetin O-deethylation, the index reaction
for CYP1A2. None of these compounds produced 50% inhibition. The mean
IC50 for -naphthoflavone was 0.2 µM, and the
mean IC50 for fluvoxamine was 0.3 µM.
CYP2C9. R-CT, S-CT, R-DCT, and S-DCT were weak inhibitors of CYP2C9, represented by tolbutamide hydroxylation, with less than 50% inhibition produced even at 250 µM. R-DDCT and S-DDCT produced a moderate degree of inhibition, with IC50 values of 30.7 (±6.3) µM and 25.7 (±8.0) µM, respectively. Sulfaphenazole was a strong inhibitor (IC50 = 1.3 µM), and the SSRI fluvoxamine also was a moderately strong inhibitor (IC50 = 9.4 µM).
CYP2C19. R- and S-CT were very weak inhibitors, with less than 50% inhibition of S-mephenytoin hydroxylation even at 100 µM. R- and S-DCT also were weak inhibitors. R- and S-DDCT were moderate inhibitors, with mean IC50 values of 18.7 and 12.1 µM, respectively. Omeprazole was a strong inhibitor of CYP2C19, as was the SSRI fluvoxamine (see Table 2).
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CYP2D6. Only R-DCT had potentially important inhibiting potency versus CYP2D6, represented by dextromethorphan O-demethylation. The mean IC50 was 25.5 (± 2.1) µM. This is very close to the inhibitory potency of sertraline and is consistent with clinical data suggesting that racemic citalopram and sertraline have comparably weak CYP2D6 inhibitory potency. The SSRI paroxetine was at least an order of magnitude more potent (IC50 = 2.6 µM) than R-DCT as a CYP2D6 inhibitor (see Table 2). Fluoxetine and norfluoxetine (mean IC50 = 2.0 and 2.7 µM, respectively) also were strong CYP2D6 inhibitors.
CYP3A.
CT and metabolites all were very weak or negligible inhibitors of
CYP3A, as indicated by triazolam hydroxylation. None of the compounds
(at 100 µM) produced more than 50% inhibition. Fluvoxamine and
nefazodone were moderately strong inhibitors (von Moltke et al., 1996b,
1999c).
CYP2E1. CT and metabolites were weak or negligible inhibitors of chlorzoxazone 6-hydroxylation, producing less than 20% inhibition at 250 µM.
Enzyme Kinetic Studies: Liver Microsomes. The mean Km for biotransformation of R-CT to R-DCT was higher than for S-CT (256 versus 165 µM) (Fig. 1). Using the Vmax/Km ratio (intrinsic clearance) as an approximation of metabolic activity at low substrate concentrations, the mean ratio for S-CT exceeded that for R-CT (6.1 versus 4.9 µl/min/mg protein), but the difference was not statistically significant based on Student's paired t test.
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Enzyme Kinetic Studies: Individual Human Cytochromes.
Table 3 shows enzyme kinetic values for
formation of DCT from R- and S-CT by
heterologously expressed human CYP2C19, -2D6, and -3A4 (Fig.
3). Consistent with prior studies of
racemic CT (von Moltke et al., 1999b), CYP2D6 had the highest affinity
(lowest Km), CYP3A4 had the lowest
affinity, and CYP2C19 fell in between. This was true for both
R-CT and S-CT.
Vmax/Km ratios
corresponding to each of the isoforms were higher for S-CT
than for R-CT.
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Chemical Inhibition of CT and DCT Biotransformation in Microsomes. At 10 µM R- or S-CT, ketoconazole reduced reaction velocity to 55 to 60% of control, quinidine to 80% of control, and omeprazole to 80 to 85% of control (Fig. 6). When the R- and S-CT concentration was increased to 100 µM, the degree of inhibition by ketoconazole increased, while inhibition by quinidine decreased (Fig. 6). These findings are consistent with the data from heterologously expressed CYP isoforms.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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R-CT, S-CT, R-DCT, and
S-DCT are weak or negligible inhibitors of human CYP1A2,
-2C9, -2C19, -2E1, and -3A in human liver microsomes. R- and
S-CT also are weak or negligible inhibitors of CYP2D6. The
R-isomer of DCT is a weak to moderate inhibitor of CYP2D6,
comparable in potency to sertraline. R- and
S-DDCT are moderate inhibitors of CYP2C9 and -2C19. However,
this is unlikely to be of clinical importance due to the low plasma
levels (less than 0.05 µM) of DDCT achieved clinically (Baumann and
Larsen, 1995).
Transformation clearance of S-CT to DCT in liver microsomes
is higher than that of R-CT, accounting for the trend to
higher plasma levels of R-CT during clinical use of racemic
citalopram and the shorter elimination half-life of S-CT
(Bondolfi et al., 1996; Foglia et al., 1997; Voirol et al., 1999).
Formation clearance of DCT from CT exceeds elimination clearance of DCT
to DDCT. Since plasma levels of DCT do not exceed those of CT during
clinical use of racemic citalopram, the findings suggest that another
metabolic pathway (in addition to formation of DDCT) or another
mechanism of elimination may contribute to DCT clearance (Rochat et
al., 1998; Dalgaard and Larsen, 1999).
Studies with heterologously expressed human CYP isoforms indicate that CYP2D6, -2C19, and -3A all contribute to formation of DCT from R- or S-CT, with CYP3A accounting for 35 to 46% of net intrinsic clearance. The contribution of CYP3A is predicted to increase at higher concentrations of CT, while the contribution of CYP2D6 is predicted to decrease. This was verified by studies of chemical inhibition of this reaction in liver microsomes by index inhibitors. As in the case of liver microsomes, intrinsic clearance of S-CT by the three CYP isoforms was higher than that of R-CT.
CYP2D6 was the only identified CYP isoform mediating formation of DDCT from R- or S-DCT. However, these were high-affinity (low Km) reactions in expressed CYP2D6, with Km values lower than the high-affinity component in liver microsomes. Furthermore, the reaction was incompletely inhibited by quinidine (5 µM) in liver microsomes but was extensively inhibited by quinidine in expressed CYP2D6. This again suggests participation of some other process mediating net biotransformation of DCT in liver microsomes and in vivo.
Thus S-CT is biotransformed to its principal demethylated
metabolite by three distinct human CYP isoforms in parallel. As such,
impaired activity of any one of these isoforms due to a drug
interaction or a genetic "poor metabolizer" polymorphism is
unlikely to have a large effect on net metabolic clearance. As
examples, clearance of racemic CT among individuals identified as
phenotypic poor metabolizers of CYP2D6 or CYP2C19 was only modestly
lower than in normal metabolizer controls (Sindrup et al., 1993).
Coadministration of ketoconazole with racemic CT produced a reduction
in CT clearance of approximately 10%, which was not statistically
significant (Gutierrez and Abramowitz, 2001). Cimetidine, an inhibitor
of multiple CYP isoforms, reduced clearance of racemic CT by 30%
(Priskorn et al., 1997a). S-CT and its metabolites
themselves are weak or negligible inhibitors of human CYP isoforms,
indicating a low likelihood of clinically important drug
interactions due to impaired CYP activity (Gram et al., 1993; Priskorn
et al., 1997b; Avenoso et al., 1998; Taylor et al., 1998; Nolting and Abramowitz; 2000; von Bahr et al., 2000).
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Acknowledgments |
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We are grateful for the collaboration and assistance of Karthik Venkatakrishnan, Ph.D.
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Footnotes |
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Received January 2, 2001; accepted April 16, 2001.
This work was supported by Grants MH-01237, MH-58435, MH-34223, DA-13209, DK/AI-58496, and DA-05258 from the Department of Health and Human Services, and by a grant from Forest Laboratories, New York, NY.
Dr. Lisa von Moltke, Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. E-mail: lisa.vonmoltke{at}tufts.edu
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Abbreviations |
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Abbreviations used are: CT, citalopram; DCT, desmethylcitalopram; DDCT, didesmethylcitalopram; CYP, cytochrome P450; SSRI, selective serotonin reuptake inhibitor.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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state of the art and prospects for the future.
Pharmacol Ther
84:
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