Download sertraline is metabolized by multiple cytochrome p450 enzymes

0090-9556/05/3302-262–270$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics
DMD 33:262–270, 2005
Vol. 33, No. 2
2428/1193208
Printed in U.S.A.
SERTRALINE IS METABOLIZED BY MULTIPLE CYTOCHROME P450 ENZYMES,
MONOAMINE OXIDASES, AND GLUCURONYL TRANSFERASES IN HUMAN:
AN IN VITRO STUDY
R. Scott Obach, Loretta M. Cox, and Larry M. Tremaine
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, Connecticut
Received September 20, 2004; accepted November 12, 2004
ABSTRACT:
alyzed sertraline deamination with comparable Km values (230–270
␮M). Monoamine oxidase B catalyzed the reaction approximately
3-fold faster than did monoamine oxidase A. Sertraline N-carbamoyl glucuronidation was measured in human liver microsomes
in bicarbonate buffer and under a CO2 atmosphere (Km ⴝ 50 ␮M)
and was catalyzed at the fastest rate by recombinant human
UGT2B7. The observation that multiple enzymes appear to be
involved in sertraline metabolism suggests that there should be no
single agent that could substantially alter the pharmacokinetics of
sertraline, nor should there be any single drug-metabolizing enzyme genetic polymorphism (e.g., CYP2D6, CYP2C19, CYP2C9,
UGT1A1) that could profoundly impact the pharmacokinetics of
sertraline.
Sertraline is an effective and highly utilized drug for the treatment
of depression and mania. It is one example of a class of drugs referred
to as selective serotonin reuptake inhibitors. The members of this class
of drugs are predominantly cleared by oxidative metabolism by the
cytochrome P450 (P450) enzymes. For example, fluoxetine is primarily metabolized via N-demethylation by CYP2D6, 2C9, and 3A (von
Moltke et al., 1997; Margolis et al., 2000), paroxetine by demethylenation of its methylenedioxy group by CYP2D6 (Bloomer et al.,
1992), venlafaxine by O-demethylation by CYP2D6 (Otton et al.,
1996; Fogelman et al., 1999), and citalopram by CYP3A4-, 2D6-, and
2C19-catalyzed N-demethylation (Kobayashi et al., 1997; von Moltke
et al., 1999, 2001; Olesen and Linnet, 1999). As such, these drugs
have been shown to be subject to drug interactions by various inhibitors of cytochrome P450 enzymes such as quinidine and ketoconazole (Lessard et al., 1999; Eap et al., 2003; Lindh et al., 2003). Also,
some of these agents have been shown to exhibit substantial differences in pharmacokinetics in subjects who lack CYP2D6 or
CYP2C19, such as fluoxetine, paroxetine, and venlafaxine (Hamelin
et al., 1996; Lessard et al., 1999; Liu et al., 2001; Charlier et al., 2003;
Lindh et al., 2003; Yu et al., 2003).
Previous reports have attempted to address the identities of cyto-
chrome P450 enzymes responsible for sertraline N-demethylation
(Greenblatt et al., 1999; Kobayashi et al., 1999; Xu et al., 1999).
N-Demethylation is a major route of sertraline metabolism (Fig. 1). In
vivo, the predominant metabolites of sertraline in excreta include
entities that could arise via the further oxidative metabolism of the
N-desmethyl metabolite or via initial deamination of the methylamino
substituent (data on file, Pfizer, Inc.). Also, sertraline undergoes
N-carbamoyl glucuronidation, an unusual reaction in drug metabolism, albeit that this pathway appears to be minor in human but major
in the dog (Tremaine et al., 1989). The reports on sertraline Ndemethylation in vitro have been conflicting, likely due to a complex
number of cytochrome P450 enzymes that can catalyze the reaction.
In the report of Kobayashi et al. (1999), recombinant heterologously
expressed human P450 enzymes were used in determining that five
enzymes all appeared to be significantly involved in sertraline Ndemethylation: CYP2B6, 2C9, 2C19, 2D6, and 3A4. The report of
Greenblatt et al. (1999) also identified CYP2C9, 2C19, 3A4, and 2D6
each as being partially involved in sertraline N-demethylation, with a
very minor role for CYP2B6. Alternately, the report of Xu et al.
(1999) claimed that CYP2C19 and CYP2C9 were the major P450
enzymes responsible for sertraline metabolism. These investigators
utilized human liver microsomes phenotyped for CYP2C19 as well as
chemical inhibitors to support their conclusions. However, in the
studies of Xu et al. (1999), a role for CYP2B6 was not addressed.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.104.002428.
ABBREVIATIONS: P450, cytochrome P450; MAO, monoamine oxidase; PPP, 2-phenyl-2-(1-piperdinyl)propane; RAF, relative activity factor;
UDPGA, uridine diphosphoglucuronic acid; UGT, uridine diphosphoglucuronic acid transferase; HPLC, high-performance liquid chromatography;
rP450, recombinant cytochrome P450; HLM, human liver microsome(s); CP-105,162, 4-(4-chlorophenyl)-1,2,3,4-tetrahydro-1-naphthalenamine.
262
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The oxidative and conjugative metabolism of sertraline was examined in vitro to identify the enzymes involved in the generation of
N-desmethyl, deaminated, and N-carbamoyl-glucuronidated metabolites in humans. In human liver microsomes, sertraline was
N-demethylated and deaminated by cytochrome P450 (P450) enzymes with overall Km values of 98 and 114 ␮M, respectively, but
the intrinsic clearance for N-demethylation was approximately 20fold greater than for deamination. Using P450 isoform-selective
inhibitors and recombinant heterologously expressed enzymes, it
was demonstrated that several P450 enzymes catalyzed sertraline
N-demethylation, with CYP2B6 contributing the greatest extent,
and lesser contributions from CYP2C19, CYP2C9, CYP3A4, and
CYP2D6. For deamination, data supported a role for CYP3A4 and
CYP2C19. Purified human monoamine oxidases A and B also cat-
METABOLISM OF SERTRALINE IN VITRO
263
Furthermore, sertraline has been shown to exhibit no differences in
pharmacokinetics in CYP2D6 extensive versus poor metabolizers
(Hamelin et al., 1996) and minor differences in CYP2C19 extensive
versus poor metabolizers (Wang et al., 2001), suggesting that neither
of these enzymes predominates in the metabolic clearance of sertraline.
The study of drug metabolism in vitro has emerged as a powerful
approach to address the potential for drug-drug interactions in vivo as
well as addressing the potential for interpatient variability in pharmacokinetics that are due to metabolism (Venkatakrishnan et al., 2003).
Such data can be used both retrospectively, to provide a mechanistic
understanding of pharmacokinetic observations, and prospectively, to
predict drug interactions or pharmacokinetics that are highly variable
because of metabolism by enzymes subject to genetic polymorphism.
The objective of the study described in this report was to provide
greater clarity to our understanding of the enzymes involved in
sertraline metabolism, and to reconcile the apparent conflicting information in the previous reports (Greenblatt et al., 1999; Kobayashi et
al., 1999; Xu et al., 1999). Additionally, since sertraline contains a
benzylamine moiety, a structure associated with monoamine oxidase
substrates, the potential for MAO-catalyzed oxidation was also explored. Finally, the unusual metabolic reaction of sertraline N-carbamoyl glucuronidation was explored in the first systematic and
quantitative study of this type of drug metabolism reaction in vitro.
Materials and Methods
Materials. Sertraline, N-desmethylsertraline, sertraline ketone, PPP, (⫹)N-3-benzylnirvanol, and CP-105,162 were obtained from Pfizer, Inc. (Groton,
CT). Other reagents used were from the following sources: furafylline, quinidine, omeprazole, diethyldithiocarbamate, sulfaphenazole, UDPGA, and quercetin from Sigma-Aldrich (St. Louis, MO); ketoconazole and NADPH, ICN
(Aurora, OH). (⫹)-N-3-Benzylnirvanol was prepared according to the method
of Suzuki et al. (2002). Sertraline N-carbamoyl glucuronide was biosynthe-
sized using dog liver microsomes and isolated by sequential liquid extraction,
solid phase extraction, and preparative HPLC. Human liver microsomes were
prepared under contract to Pfizer by BD Gentest (Woburn, MA), and recombinant heterologously expressed human UGT enzymes were from this same
source. HL-MIX-101 represents a pool of liver microsomes from 60 individual
donors. Recombinant human cytochrome P450 enzymes heterologously expressed using a baculovirus expression system were obtained from PanVera
Corp. (Madison, WI; for CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4) or BD
Gentest (CYP1A1, 2A6, 2C8, 2E1, and 3A5). Purified human MAO-A and -B
were generously provided by Dr. Dale Edmonson, Emory University (Atlanta,
GA).
Incubation Conditions. Desmethylsertraline Formation. Incubations containing sertraline (at various concentrations), human liver microsomes (0.1
mg/ml), NADPH (1.3 mM), and MgCl2 (3.3 mM) in a total volume of 0.2 ml
of 25 mM potassium phosphate buffer (pH 7.4) were conducted at 37°C in a
shaking water bath. Initial reaction velocity conditions were established such
that product formation was linear over time to 20 min; thus, all subsequent
incubations were conducted for this time period. The incubations were terminated by addition of 0.2 ml of 1 M NaOH. All incubations were conducted in
triplicate.
Enzyme kinetic experiments in pooled human liver microsomes (MIX 101;
combination of 60 individual livers) were conducted at sertraline concentrations ranging from 0.25 to 200 ␮M. In cases in which human recombinant
heterologously expressed cytochrome P450 enzymes were examined, a total
P450 concentration of 20 pmol of P450/ml was utilized, with protein concentrations of 0.5 mg/ml maintained by addition of microsomes from nontransfected cells. Sertraline concentrations were 0.5 or 50 ␮M. In incubations in
which the effects of chemical inhibitors in human liver microsomes were
examined, the sertraline concentration examined was 0.5 ␮M and the inhibitors
were added in H2O/CH3CN (50:50) such that the final solvent concentration
was 0.5%. Control incubations had solvent without inhibitor. For furafylline,
methyl phenethyl piperidine, and diethyldithiocarbamate, the inhibitor was
preincubated with microsomes and NADPH for 15 min at 37°C before addition
of sertraline and additional NADPH.
Sertraline Ketone Formation by P450. Incubations containing sertraline (at
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FIG. 1. The metabolism of sertraline.
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OBACH ET AL.
which allowed visualization of both N-desmethylsertraline and sertraline.
Under these conditions, the retention times for N-desmethylsertraline, sertraline, and internal standard were 9.8, 10.8, and 7.7 min, respectively. Quantitation was accomplished using a linear standard curve of N-desmethylsertraline
ranging from 1.0 to 1000 ng/ml with 1/X2 weighting. In some experiments,
concentrations of N-desmethylsertraline in incubation mixtures were anticipated to be well below 100 ng/ml, and in these cases, the standard curve was
run from 1.0 to 100 ng/ml. In experiments in which high sertraline substrate
concentrations were used (i.e., ⬎20 ␮M), the incubation samples were diluted
10 times before analysis to avoid the excess of sertraline overwhelming the
signal for N-desmethylsertraline.
Analysis of Sertraline Ketone. To terminated incubation mixtures was
added 3 ml of methyl t-butyl ether followed by extraction by agitation on a
multitube vortex mixer. The mixtures were spun in a centrifuge (2500g), after
which the organic layer was transferred into a fresh test tube. The solvent was
removed by evaporation under N2 at 35°C and reconstituted in 0.04 ml of
water/acetonitrile (50:50).
The reconstituted extracts were analyzed by HPLC-mass spectrometry. The
HPLC system consisted of an Agilent 1100 quaternary pump with membrane
degasser, a CTC PAL autosampler (LEAP Technologies, Inc.), and a PE Sciex
API-100 single quadrupole mass spectrometer (PerkinElmerSciex Instruments,
Boston, MA) with TurboIon spray interface. Samples were injected (30 ␮l)
onto a Metasil AQ C18 column (2.0 ⫻ 50 mm; 5-␮m particle size) equilibrated
in a mobile phase consisting of 20 mM acetic acid (pH adjusted to 4.0 with
NH4OH) containing 55% CH3CN at a flow rate of 0.5 ml/min. This mobile
phase composition was maintained for a run time of 7 min.
The effluent was introduced into the ionspray source of the mass spectrometer operated in the positive ion mode. The following settings on the mass
spectrometer were established to optimize the signal for sertraline ketone: ion
spray voltage, 4500 V; orifice, 25 V; source temperature, 450°C; with other
potentials and settings adjusted to optimize the signal. Detection was accomplished in the selected ion monitoring mode following m/z 291. Additionally,
the effluent was monitored by UV at 245 nm, which allowed visualization of
sertraline ketone. Under these conditions, the retention time for sertraline
ketone was 3.4 min. Quantitation was accomplished using an external linear
standard curve of sertraline ketone routinely ranging from 20 to 2000 ng/ml
with 1/X weighting. In some cases, the limit of quantitation was extended down
to 6.32 ng/ml.
Analysis of Sertraline N-Carbamoyl Glucuronide. Terminated incubation samples (0.2 ml) were extracted with methyl t-butyl ether (3 ml), and the
organic fraction was collected by freezing the aqueous layer in dry ice/acetone
and evaporated under N2. The residue was reconstituted in 0.04 ml of water/
CH3CN (50:50) and injected (0.01 ml) onto the aforementioned HPLC- tandem
mass spectrometry system. The column was a Varian Basic 2.0 ⫻ 50 mm
narrow bore column (Varian, Inc., Palo Alto, CA) with material of 3-␮m
particle size. The mobile phase consisted of 0.1% formic acid containing 2 mM
NH4OH at 65% and CH3CN at 35%, at a flow rate of 0.4 ml/min. After 2 min,
a linear gradient to 65% CH3CN at 8 min was applied, followed by reequilibration. The effluent was introduced into the source of a Micromass Ultima
tandem quadrupole mass spectrometer operated in the positive ion mode.
Detection was accomplished by monitoring the mass transition m/z 543 (ammoniated molecular ion of sertraline N-carbamoyl glucuronide) to m/z 350.
The following settings on the mass spectrometer were established to optimize
the signal for sertraline N-carbamoyl glucuronide: capillary, 2.0 kV; cone, 30;
source temperature, 100°C; desolvation temperature. 350°C; cone gas. 182 l/h;
desolvation gas, 774 l/h; collision energy, 15. The retention time was 5.1 min.
Quantitation was accomplished by extrapolation from a linear standard curve
of N-carbamoyl glucuronide ranging from 10 to 1000 ng/ml (human liver
microsomes) or 1.0 to 100 ng/ml (recombinant UGT enzymes).
Data Analysis. Enzyme kinetic data were fit using the Sigma Plot Enzyme
Kinetics Module (v 1.0; SPSS Inc., Chicago, IL). The sertraline N-demethylation and N-carbamoyl glucuronidation data were best fit using the substrate
inhibition equation:
v⫽
V max 䡠 关S兴
Km ⫹ 关S兴
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various concentrations), human liver microsomes (1.0 mg/ml), NADPH (1.3
mM), and MgCl2 (3.3 mM) in a total volume of 1.0 ml of 25 mM potassium
phosphate buffer (pH 7.4) were conducted at 37°C in a shaking water bath.
Initial reaction velocity conditions were established such that product formation was linear over time to 10 min; thus, all subsequent incubations were
conducted for this time period. The incubations were terminated by addition of
0.5 ml of 1 M HCl. All incubations were conducted in triplicate.
Enzyme kinetic experiments in pooled human liver microsomes (HL-MIX
101) were conducted at sertraline concentrations ranging from 5 to 500 ␮M. In
cases in which human recombinant heterologously expressed cytochrome P450
enzymes were examined, a total P450 concentration of 40 pmol of P450/ml
was utilized, with protein concentrations of 1.0 mg/ml maintained by addition
of microsomes from sham transfected cells. Sertraline concentrations were 50
␮M. Chemical inhibitors were utilized in the same manner as described above.
Identical incubation conditions were applied when examining N-desmethylsertraline as a substrate, with the substrate concentration set at 50 ␮M for all
experiments.
Sertraline Ketone Formation by MAO. Incubations containing sertraline (at
various concentrations) and purified MAO (0.1 nmol/ml) in a total volume of
1.0 ml of 25 mM potassium phosphate buffer (pH 7.4) were conducted at 37°C
in a shaking water bath. Initial reaction velocity conditions were established
such that product formation was linear over time to 4 h; thus, all subsequent
incubations were conducted for this time period. The incubations were terminated by addition of 0.5 ml of 1 M HCl. All incubations were conducted in
triplicate. Enzyme kinetic experiments were conducted at sertraline concentrations ranging from 10 to 1000 ␮M. Identical incubation conditions were
applied when examining N-desmethylsertraline as a substrate, with the substrate concentration set at 50 ␮M for all experiments.
Sertraline N-Carbamoyl Glucuronidation. Microsomes were first incubated
with alamethacin, MgCl2, and sodium bicarbonate (pH adjusted to 7.5) on ice
for 15 min in a total volume of 0.145 ml. This was followed by addition of
sertraline and saccharolactone and warming to 37°C over 5 min under CO2.
Incubations were commenced by addition of UDPGA in a final incubation
volume of 0.5 ml. Final assay concentrations were: microsomes, 0.02 mg/ml;
alamethacin, 0.05 mg/ml; NaHCO3, 100 mM; MgCl2, 5 mM; saccharolactone,
5 mM; sertraline, 2 to 200 ␮M; and UDPGA, 5 mM. Incubations were
conducted in a Lucite container submerged in a heated water bath that permitted the continuous flow of CO2. The CO2 was passed through a gas warmer
to prevent cooling of the incubations. After 45 min, incubations were terminated by the addition of 0.5 ml of HCl (1 M). The incubation time and protein
concentration used were determined to be linear in initial experiments.
Analysis of N-Desmethylsertraline. To terminated incubation mixtures
was added internal standard (20 ␮l of a 0.5 ␮g/ml solution of CP-105,162, the
monochloro analog of desmethylsertraline), followed by 3 ml of methyl t-butyl
ether and extraction by agitation on a multi-tube vortex mixer. The mixtures
were spun in a centrifuge (2500g), after which the aqueous layer was frozen in
a dry ice-acetone bath and the organic layer was decanted into a fresh test tube.
The solvent was removed by evaporation under N2 at 35°C and reconstituted
in 0.1 ml of water/acetonitrile (75:25) containing 0.1% formic acid.
The reconstituted extracts were analyzed by HPLC-tandem mass spectrometry. The HPLC system consisted of two Shimadzu LC-10ADvp pumps,
DGU-14 solvent degasser, and SCL-10ADvp controller (Shimadzu, Columbia,
MD), a CTC PAL autosampler (LEAP Technologies Inc., Carrboro, NC), and
a Micromass Quattro Ultima tandem quadrupole mass spectrometer with
ionspray interface (Waters, Milford, MA). Samples were injected (75 ␮l) onto
a Metachem Polaris C18 column (4.6 ⫻ 250 mm; 5-␮m particle size) equilibrated in a mobile phase consisting of 55% H2O/45% CH3CN containing 0.1%
formic acid at a flow rate of 0.8 ml/min. This mobile phase composition was
maintained for a run time of 14 min.
The effluent was introduced into the ionspray source of the mass spectrometer operated in the positive ion mode. The following settings on the mass
spectrometer were established to optimize the signal for N-desmethylsertraline:
capillary, 3.0 kV; cone, 20; source temperature, 135°C; desolvation temperature, 350°C; cone gas, 190 l/h; desolvation gas, 790 l/h; collision energy, 20
eV. Other potentials were adjusted to optimize the signal. Detection was
accomplished in the multiple reaction monitoring mode following the transitions m/z 2923159 (N-desmethylsertraline) and m/z 2413125 (internal standard). Additionally, the mass transition m/z 2753159 was also monitored,
METABOLISM OF SERTRALINE IN VITRO
265
For sertraline deamination, the data were best fit to the standard MichaelisMenten equation:
v⫽
V max 䡠 关S兴
关S兴2
Km ⫹ 关S兴 ⫹
KS
冉 冊
Inhibition data were fit to the equation:
% of control ⫽ A ⫺
B 䡠 关I兴
关I兴 ⫹ IC50
in which 100 ⫺ (A ⫺ B) represents the maximum inhibition and the IC50 is the
inflection point of the curve on a plot of percentage of control versus log[I]
(representing the potency of the inhibitor).
Percentage contribution by P450s using the relative activity approach (RAF;
Venkatakrishnan et al., 2001) was determined using RAF values calculated
from Vmax of rP450s and pooled human liver microsomes. RAF values for the
pooled human liver microsomes used were 22 (CYP1A2; phenacetin Odeethylation), 32 (CYP2B6; bupropion hydroxylation), 23 (CYP2C9; tolbutamide methyl hydroxylation), 7 (CYP2C19; mephenytoin 4⬘-hydroxylation), 6
(CYP2D6; bufuralol 1⬘-hydroxylation), 86 (CYP2E1; chlorzoxazone 6-hydroxylation), and 48 (CYP3A4; average of testosterone 6␤-hydroxylation,
midazolam 1⬘-hydroxylation, and felodipine dehydrogenation).
Results
Sertraline N-Demethylation. Enzyme Kinetics in Liver Microsomes. The enzyme kinetics of sertraline N-demethylation in pooled
human liver microsomes were measured to ascertain the Michaelis
constant and ensure that subsequent reaction phenotyping experiments
were conducted at a substrate concentration below Km. A plot of the
substrate saturation curve is shown in Fig. 2A. The data analysis best
fit a model of substrate inhibition occurring at high substrate concentrations. The enzyme kinetic parameters were: Km ⫽ 98 ␮M, Vmax ⫽
1920 pmol/min/mg microsomal protein, and KS ⫽ 63 ␮M. Thus, all
subsequent phenotyping experiments could be conducted at substrate
concentrations below 98 ␮M. A decrease in sertraline N-demethylase
activity at high sertraline concentrations was also observed previously
(Greenblatt et al., 1999), and the Km value reported (84 ␮M) was
similar to the one measured in the present report.
Incubations with rP450 Enzymes. Sertraline was incubated with
recombinant human cytochrome P450s 1A1, 1A2, 2A6, 2B6, 2C8,
2C9, 2C19, 2D6, 2E1, 3A4, and 3A5, at substrate concentrations of
0.5 and 50 ␮M. Of the enzymes tested, N-desmethylsertraline was
detected in most (Fig. 3, A and B). At [S] ⫽ 0.5 ␮M, the fastest rate
was observed for CYP2C19, followed by CYP2D6, 2B6, 3A4, and
2C9. At [S] ⫽ 50 ␮M, the rank order was CYP2C19, 2D6, 2C9, 2B6,
and 3A4. Trace quantities of N-desmethylsertraline were observed in
incubations with CYP1A1, 1A2, 2A6, 2C8, 2E1, and 3A5. Application of relative activity factors (Venkatakrishnan et al., 2001) for the
P450 activities in recombinant P450s and human liver microsomes
suggest that at low concentrations, CYP2B6 ⬎ CYP2C19 ⬇
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FIG. 2. Substrate saturation plots for sertraline metabolism in pooled human liver microsomes and by purified human monoamine oxidases. Plot A, N-demethylation in HLM; plot
B, deamination in human liver microsomes; plot C, N-deamination by MAO-A; plot D, N-deamination by MAO-B. Each point represents the mean from three determinations.
266
OBACH ET AL.
CYP2D6 ⬇ CYP3A4 ⬎ CYP2C9, whereas at high concentrations, the
rank order changes to CYP2C9 ⬇ CYP3A4 ⬇ CYP2B6 ⬇
CYP2C19 ⬎ CYP2D6 (Table 1).
P450-Specific Inhibitors. The activity of human liver microsomes
to catalyze sertraline N-demethylation was examined in the presence
of chemical inhibitors and inactivators that are selective for specific
cytochrome P450 enzymes. Of those tested, 2-phenyl-2-(1-piperdinyl)propane (CYP2B6-selective inactivator) demonstrated the greatest
inhibition at a sertraline concentration of 0.5 ␮M (Fig. 4). Only mild
inhibition (ca. 10% or less) was observed for ketoconazole (CYP3A),
sulfaphenazole (CYP2C9), and N-benzylnirvanol (CYP2C19).
To better delineate the effects of these inhibitors and to ensure that
concentrations used were adequate, they were also tested using their
respective recombinant P450 enzymes (Fig. 4). For quinidine, sulfaphenazole, and ketoconaozle, inhibitor concentrations used in human
liver microsomes (1.0, 10, and 1.0 ␮M, respectively) demonstrated
potent inhibition in rP450s for sertraline N-demethylation, indicating
that the inhibitor concentrations used for liver microsomes were
appropriate. For N-benzylnirvanol, 10 ␮M yielded 65% inhibition of
CYP2C19-catalyzed sertraline N-demethylation. For PPP, preincuba-
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FIG. 3. Metabolism of sertraline and N-desmethylsertraline in human rP450 enzymes. A, sertraline N-demethylation at [Sertraline] ⫽ 0.5 ␮M; B, sertraline
N-demethylation at [Sertraline] ⫽ 50 ␮M; C, sertraline (solid bars) and N-desmethylsertraline (open bars) N-deamination at substrate concentrations of 50 ␮M. Each
bar represents the mean ⫾ S.D. for three determinations.
tion of 10 ␮M inhibitor yielded potent inhibition of CYP2B6 (89%)
consistent with this inhibitor acting as a mechanism-based inactivator
of CYP2B6 (Chun et al., 2000).
A full inhibitor concentration range was tested for N-benzylnirvanol and PPP, to aid in better delineating the relative contributions of
CYP2B6 and CYP2C19 to sertraline N-demethylation. Inhibitor concentrations were tested ranging from 0.1 to 100 ␮M (with PPP
undergoing preincubation with microsomes). The results are plotted in
Fig. 5 and show that PPP yields a maximum inhibition of approximately 50%, indicating that in pooled human liver microsomes,
CYP2B6 contributes approximately half the sertraline N-demethylation observed. N-Benzylnirvanol yielded a maximum inhibition of
approximately 30%. Omeprazole was also tested. Omeprazole has
been claimed as a CYP2C19 inhibitor, and a previous investigation
had used this compound as an inhibitor to claim a role for CYP2C19
in sertraline N-demethylation (Xu et al., 1999). The data obtained with
pooled human liver microsomes suggest that omeprazole does not
cause a substantial amount of inhibition of sertraline N-demethylase
except for when high inhibitor concentrations are tested (100 ␮M)
(Fig. 5).
To further delineate the contributions of CYP2B6, 2C9, 2C19, 2D6,
and 3A to sertraline N-demethylation in human liver microsomes,
specific inhibitors of these five enzymes were tested in five lots of
microsomes from individual donors. These lots were selected because
each represented high activity for one of the enzymes, with moderate
or low activity for the other four. The results are presented in Fig. 6.
PPP demonstrated a range of inhibition from 23 to 66%. The lot of
microsomes with the greatest CYP2B6 activity (HH75; as assessed
with S-mephenytoin N-demethylase activity) was the one most affected by PPP (66% inhibition). The lot with the greatest CYP2C19
activity (HH100; assessed with S-mephenytoin 4-hydroxylase activity) demonstrated the greatest sensitivity to N-benzylnirvanol, with
63% inhibition. The sensitivity of the five lots to benzylnirvanol
inhibition ranged from 11 to 63%. Corresponding results were found
for the other three lots of human liver microsomes: ketoconazole had
the greatest effect on HH8 (highest of the five lots for CYP3A
activity), sulfaphenazole had the greatest effect on HH91 (highest of
the five lots for CYP2C9 activity), and quinidine had the greatest
effect on HH80 (highest of the five lots for CYP2D6 activity).
Relative contributions of the P450 enzymes estimated from the inhibition data in these individual donors are listed in Table 1.
Enzyme Kinetics in Liver Microsomes. The enzyme kinetics of
sertraline N-deamination in pooled human liver microsomes were
determined. The data demonstrated inhibition at the highest sertraline
concentration, but inclusion of the 500 ␮M data point did not allow
for fitting of the kinetics. Exclusion of this point yielded enzyme
kinetic parameters of Km ⫽ 114 ␮M and Vmax ⫽ 106 pmol/min/mg
microsomal protein. Thus, all subsequent phenotyping experiments
could be conducted at substrate concentrations below 114 ␮M. A plot
of the substrate saturation curve is shown in Fig. 2B.
Incubations with rP450 Enzymes. Sertraline N-deamination was
measured in rP450 at a substrate concentration of 50 ␮M. (Due to
assay sensitivity limitations, a substrate concentration of 0.5 ␮M
could not be examined as it was for the N-demethylation reaction.) Of
the 11 rP450s tested, measurable activity was detected for CYP1A1,
1A2, 2C19, 3A4, and 3A5 (Fig. 3C). Application of RAF as described
above, yielded estimates of 63%, 35%, and 2% contribution from
CYP3A4, 2C19, and 1A2, respectively (Table 2).
P450-Specific Inhibitors. Following demonstration that sertraline
deamination was catalyzed by rCYP1A2, 2C19, and 3A4, incubations
were conducted in HLM-101 using furafylline, N-benzylnirvanol, and
ketoconazole as selective inhibitors for these three enzymes. Whereas
267
METABOLISM OF SERTRALINE IN VITRO
TABLE 1
Estimations of percentage contribution of human cytochrome P450 enzymes to sertraline N-demethylation
Microsomes
From inhibition data
HH8 (high CYP3A)
HH75 (high CYP2B6)
HH80 (high CYP2D6)
HH91 (high CYP2C9)
HH100 (high CYP2C19)
Average
Pooled (HL-MIX-101)
From rP450s using the relative activity factor approach
关Sertraline兴 ⫽ 0.5 ␮M
关Sertraline兴 ⫽ 50 ␮M
CYP2B6
CYP2C9
CYP2C19
CYP2D6
CYP3A
40
65
42
36
15
40
59
10
10
7
26
19
14
11
9
11
10
13
42
17
15
19
7
31
9
14
16
0
22
8
10
16
10
15
16
36
16
4
21
22
16
16
5
18
18
FIG. 4. Inhibition of sertraline N-demethylase in pooled human liver microsomes
and recombinant human P450 enzymes. Solid bars, human liver microsomes; open
bars, recombinant human P450s. Concentrations of inhibitors used were: furafylline,
10 ␮M; PPP, 10 ␮M; quercetin, 10 ␮M; sulfaphenazole, 10 ␮M; N-benzylnirvanol,
10 ␮M; quinidine, 1 ␮M; diethyldithiocarbamate, 30 ␮M; ketoconazole, 1 ␮M.
Each bar represents the mean ⫾ S.D. for three determinations.
furafylline had no effect on the activity, N-benzylnirvanol (10 ␮M)
and ketoconazole (1 ␮M) demonstrated 9% and 53% inhibition,
respectively (Fig. 7). This is consistent with a role for CYP2C19 and
CYP3A in this metabolic reaction.
Sertraline N-Deamination by MAO. Sertraline N-deamination was
catalyzed by purified human MAO-A and MAO-B. Enzyme kinetics
were determined (Fig. 2, C and D) with Km values of 230 and 270
␮M, and Vmax values of 39.9 and 120 pmol/min/nmol MAO for
MAO-A and MAO-B, respectively. Thus, the intrinsic clearance for
MAO-B is approximately 2.7 times greater than that for MAO-A,
when normalized on a per nanomole of enzyme basis. The relative
contributions of these enzymes to the N-deamination of sertraline, and
relative to the P450 contribution to this reaction, will depend on the
relative expression levels of these enzymes in various human tissues.
N-Desmethylsertraline N-Deamination. N-Desmethylsertraline
was also demonstrated to generate sertraline ketone when incubated
with purified MAO-A and MAO-B, as well as with human liver
microsomes. At a substrate concentration of 50 ␮M, rates of N-
desmethylsertraline N-deamination were lower than those for sertraline N-deamination for all three enzyme systems (Table 3).
For P450 enzymes, CYP1A1, CYP2C19, CYP3A4, and, to a very
small extent, CYP2E1 demonstrated generation of sertraline ketone
from N-desmethylsertraline (Fig. 3C). In human liver microsomes,
N-desmethylsertraline was inhibited by N-benzylnirvanol by 35%,
ketoconazole by 33%, and diethyldithiocarbamate by 38%, suggesting
roles for CYP2C19, CYP3A, and CYP2E1 in this reaction (Fig. 7).
Sertraline N-Carbamoyl Glucuronidation. Incubation of pooled
human liver microsomes with sertraline, along with other reagents
needed for observation of in vitro glucuronidation reactions, yielded
sertraline N-carbamoyl glucuronide. The incubations were conducted
in bicarbonate buffer and under a continuous flow of CO2 to generate
the carbamoyl glucuronide. Under these conditions, the enzyme kinetics of this reaction showed substrate inhibition by sertraline at
concentrations above 100 ␮M (Fig. 8), and values determined for Km,
Vmax, and KS were 50 ␮M, 960 pmol/min/mg, and 58 ␮M, respectively. In incubations with recombinant heterologously expressed
human UGT enzymes, sertraline N-carbamoyl glucuronidation was
measurable in UGT1A3, 1A6, 2B4, and 2B7, with the greatest activity
observed with the latter enzyme (Fig. 8). Product formation was under
the limit of quantitation for UGT1A1, 1A4, 1A7, 1A8, 1A9, 1A10,
and 2B15.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 14, 2017
FIG. 5. Inhibition of sertraline N-demethylation by PPP (CYP2B6-selective inactivator), N-benzylnirvanol (CYP2C19-selective inhibitor), and omeprazole (a
CYP2C19-nonselective inhibitor) in pooled human liver microsomes. Squares, PPP;
circles, (⫹)-N-3-benzylnirvanol; triangles, omeprazole. Each point represents the
mean ⫾ S.D. from three determinations. The data were fitted to the inhibition
function described under Materials and Methods,“Data Analysis.”
268
OBACH ET AL.
FIG. 6. Inhibition of sertraline N-demethylase in human liver microsomes from five individual donors. Each bar represents the mean ⫾ S.D. for three determinations.
TABLE 2
Microsomes
CYP1A2
CYP2C19
CYP3A
From inhibition data
Pooled (HL-MIX-101)
From rP450s using the relative activity factor
approach
⬍1
2.2
15
35
85
63
FIG. 7. Inhibition of sertraline and N-desmethylsertraline N-deamination in pooled
human liver microsomes. Solid bars, sertraline; open bars, N-desmethylsertraline.
Concentrations of inhibitors used were: furafylline, 10 ␮M; N-benzylnirvanol, 10
␮M; diethyldithiocarbamate, 30 ␮M; ketoconazole, 1 ␮M. Each bar represents the
mean ⫾ S.D. for three determinations.
Discussion
To this point, there have been three published reports of in vitro
data that address identification of the cytochrome P450 enzymes
involved in sertraline metabolism, with focus only on the N-demethylation reaction (Greenblatt et al., 1999; Kobayashi et al., 1999; Xu et
al., 1999). In the report of Kobayashi et al. (1999), the enzyme
kinetics of sertraline N-demethylation were examined in recombinant
human P450s, and the conclusion made was that several P450s
(CYP2B6, 2C9, 2C19, 2D6, and 3A4) were involved in this reaction.
In the work of Greenblatt et al. (1999), four of these enzymes, 2C9,
2C19, 2D6, and 3A4, were stated to be nearly equally contributing to
sertraline N-demethylation. However, in the report of Xu et al. (1999),
a different conclusion was made using human liver microsomes and
chemical inhibitors: CYP2C19 and 2C9 were claimed to be the
enzymes involved. However, in this latter example, examination of
CYP2B6 was not made. When it appears that multiple P450 enzymes
are involved in a metabolic transformation, as is the case with sertraline N-demethylation, ascertaining the relative contributions of each is
more challenging. The present work described in this report was
undertaken to attempt to clarify this transformation and included the
use of inhibitors selective for CYP2B6 and CYP2C19 that have been
described since the earlier reports were published. The data in this
report support the idea that sertraline N-demethylation is catalyzed by
CYP2B6 along with lesser roles for CYP2C19, 2C9, 2D6, and 3A,
and that different expression levels of these enzymes among individuals will contribute to different percentages of contribution of these
enzymes in different subjects.
The enzyme kinetics of sertraline N-demethylation were consistent
with a “single enzyme model,” i.e., that just a single Km value could
be observed, in this case representing a hybrid of several enzymes
with Km values that are kinetically indistinguishable. This is consistent with the values measured in rP450s by Kobayashi et al. (1999)
and the value measured by Greenblatt et al. (1999), in human liver
microsomes. These data are in contrast to those of Xu et al. (1999), in
which a two-enzyme model was observed in liver microsome samples
containing CYP2C19 and a single-enzyme model in microsomes
deficient in CYP2C19 activity. The low Km value in CYP2C19
extensive metabolizer microsomes was reported to be approximately
1 to 2 ␮M (Xu et al., 1999). The data in the present report showed no
sign of a low Km enzyme, even with sertraline substrate concentrations as low as 0.25 ␮M being tested in the substrate saturation
experiment. Furthermore, in the reports of Kobayashi et al. (1999) and
Greenblatt et al. (1999), Km values of 9 and 33 ␮M were measured for
rCYP2C19, which are well above the value claimed to be attributed to
CYP2C19 in the report of Xu et al. (1999).
2-Phenyl-2-(1-piperdinyl)propane and (⫹)-N-3-benzylnirvanol are
recently reported inhibitors of CYP2B6 and CYP2C19, respectively
(Chun et al., 2000; Suzuki et al., 2002). These inhibitors were not
reported at the time of the previous publications on sertraline Ndemethylation, and they were successfully applied in the present
work. PPP was shown to inhibit sertraline N-demethylation 15 to
65%, depending on the activity of CYP2B6 in the liver microsome
preparation. N-Benzylnirvanol demonstrated inhibition between 9 and
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Estimations of percentage contribution of human cytochrome P450 enzymes to
sertraline N-deamination
METABOLISM OF SERTRALINE IN VITRO
269
TABLE 3
Comparison of rates of N-deamination for sertraline and N-desmethylsertraline
in MAO and human liver microsomes
Substrate concentration used was 50 ␮M.
Reaction Velocity
Enzyme System
Pooled human liver microsomes
(pmol/min/nmol P450)
MAO-A (pmol/min/nmol MAO)
MAO-B (pmol/min/nmol MAO)
Sertraline
N-Desmethylsertraline
95.8 ⫾ 8.2
29.5 ⫾ 3.6
6.04 ⫾ 0.86
14.4 ⫾ 0.8
3.37 ⫾ 0.76
8.88 ⫾ 0.76
FIG. 8. Sertraline N-carbamoyl glucuronidation in pooled human liver microsomes
and human UGT enzymes. A, substrate saturation plot in pooled human liver
microsomes; B, reaction velocities for sertraline N-carbamoyl glucuronidation in
recombinant human UGT enzymes at a sertraline concentration of 10 ␮M.
made, and values range, depending on the tissue examined, from 1 to
30 pmol/mg tissue protein (O’Carroll et al., 1989; Riley and Denney,
1991; Saura et al., 1992, 1996). Nevertheless, quantitative comparison
of the role of MAO versus P450 in sertraline metabolism would
require further study, such as the measurement of sertraline metabolism in preparations of other tissues besides liver (e.g., brain, kidney,
lung, etc.) and application of specific P450 versus MAO inhibitors, or
in vivo studies in humans with concomitant administration of selective P450 versus MAO inhibitors.
N-Carbamoyl glucuronidation is an unusual metabolic reaction
exhibited by some primary and secondary amines. Sertraline N-carbamoyl glucuronide, along with tocainide N-carbamoyl glucuronide,
was one of the first examples of this type of metabolite (Ronfeld et al.,
1982; Tremaine et al., 1989). Subsequent investigations of the Ncarbamoyl glucuronidation of carvedilol described the first means by
which such metabolism could be observed in an in vitro system and
demonstrated that the incubation needed to be conducted under a CO2
atmosphere and in carbonate buffer (Schaefer, 1992). The data reported in this paper describe the first observations of the generation of
sertraline N-carbamoyl glucuronide in vitro, and this is the first time
that this type of reaction was characterized for its enzyme kinetics and
the identity of UGT enzymes involved. In order for the reaction to
occur, it is hypothesized that the amine spontaneously forms a transient carbamic acid intermediate with the dissolved CO2 followed by
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 14, 2017
42%, again depending on the activity of CYP2C19 in the liver
microsome preparation tested. The use of these inhibitors provides the
most convincing in vitro evidence of the roles of CYP2B6 and
CYP2C19 in sertraline N-demethylation. A minor contribution from
CYP2C9, 2D6, and 3A4 is also apparent from these data.
The finding that sertraline N-demethylation is catalyzed by several
P450 enzymes in humans is consistent with in vivo data. In clinical
drug interaction studies, no drug has been identified that can cause a
large increase in sertraline exposure by inhibiting sertraline metabolism (DeVane et al., 2002). Sertraline pharmacokinetics are not different in CYP2D6 extensive and poor metabolizers, suggesting that
CYP2D6 does not contribute substantially to sertraline clearance in
vivo (Hamelin et al., 1996). In CYP2C19 poor metabolizers, sertraline
exposure was about 40% greater than in extensive metabolizers
(Wang et al., 2001). This suggests a minor role for CYP2C19 in
sertraline metabolism. If this enzyme contributed a major role, then
the difference between CYP2C19 extensive and poor metabolizer
subjects would be great, and a bimodal distribution of sertraline
exposure values would have been observed in the population, as has
been observed for drugs for which CYP2C19 plays a considerable
role, such as omeprazole (Desta et al., 2002). CYP2B6 has not been
studied as extensively as other human P450 enzymes with regard to
clinical pharmacokinetics and drug interactions. In vitro studies have
established a few drugs, such as bupropion, ifosphamide, and cyclophosphamide, as CYP2B6 substrates, but there have been no in vivo
studies in which a profound increase in the exposure to these drugs
has been demonstrated. CYP2B6 is also subject to genetic polymorphisms that can manifest themselves in a poor metabolizer phenotype,
as well as observed gender differences in expression (Lang et al.,
2001; Lamba et al., 2003). However, since sertraline appears to be
metabolized by several enzymes, an effect of this variability in
CYP2B6 activity among individuals on sertraline pharmacokinetics
may not be observable.
In humans, the major excretory metabolite is hydroxyl sertraline
ketone (data on file, Pfizer, Inc.). This metabolite could arise from
three initial possible parallel pathways: N-demethylation followed by
N-deamination and hydroxylation; N-deamination of the methylamine
substituent followed by hydroxylation, and/or initial hydroxylation
followed by oxidative metabolism of the methylamine moiety. The
latter seems unlikely because hydroxylated sertraline was not observable in incubations of sertraline, liver microsomes, and NADPH (data
not shown). In this report, we did demonstrate that sertraline can be
directly N-deaminated to sertraline ketone. This reaction was catalyzed by both P450 enzymes (CYP3A4 and 2C19) as well as monoamine oxidases (MAO-A and MAO-B). Whereas P450 enzymes are
predominantly expressed in the liver, MAO enzymes are abundantly
expressed in numerous tissues. This difference complicates any attempt to determine a role for MAO versus P450 in sertraline metabolism and also complicates the determination of whether the main
initial metabolic pathway for sertraline is N-demethylation or Ndeamination. Some estimates of MAO expression levels have been
270
OBACH ET AL.
glucuronidation. However, proof that the carbamic acid is the actual
UGT substrate and that UGT does not also catalyze the formation of
the carbamic acid would require that this intermediate is stable and
isolable. Sertraline N-carbamoyl glucuronidation was primarily
formed by UGT2B7, which is also responsible for numerous acyl
glucuronidation reactions of carboxylic acid drugs (Tephly and Green,
2000). This might suggest that sertraline N-carbamic acid is the actual
UGT substrate; however, such a claim remains speculative without
mechanistic proof. Exploring this metabolic reaction remains the
object of an ongoing investigation.
In conclusion, because it appears that sertraline has multiple enzymes involved in its initial metabolic pathways, it would be difficult
for any single agent to cause a meaningful drug interaction via
inhibition of the metabolic clearance of sertraline. To date, there have
been no agents identified that can give rise to a marked increase in
sertraline exposure upon concomitant administration (DeVane et al.,
2002). The findings in this study are consistent with these observations.
Address correspondence to: R. Scott Obach, MS 4088, Groton Laboratories,
Pfizer, Inc., Groton, CT 06340. E-mail: [email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 14, 2017
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