Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin

Article abstract of DOI:10.1016/S0928-0987(00)00150-0

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R²=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC₅₀ values of a series of five N-substituted analogs of 3,4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6. Copyright (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S0928-0987(00)00150-0

European Journal of Pharmaceutical Sciences 12 (2000) 151–158
www.elsevier.nl/locate/ejps
Evaluation of a novel high-throughput assay for cytochrome P450 2D6
using 7-methoxy-4-(aminomethyl)-coumarin
*
Jennifer Venhorst, Rob C.A. Onderwater, John H.N. Meerman, Nico P.E. Vermeulen ,
Jan N.M. Commandeur
Leiden/Amsterdam Center for Drug Research (LACDR), Department of Pharmacochemistry, Division of Molecular Toxicology, Vrije Universiteit,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Received 6 March 2000; received in revised form 26 April 2000; accepted 26 April 2000
Dedicated to Professor D.D. Breimer on the occasion of the 25th anniversary of his appointment to the Chair of Pharmacology at Leiden University.
Abstract
We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6
(CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes
use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and
human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is
easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been
performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n56) of
CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results
indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the Ki values
reveal a very good correlation (R250.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC50 values
of a series of five N-substituted analogs of 3,4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained
demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and
a higher selectivity for CYP2D6.
2000 Elsevier Science B.V. All rights reserved.
Keywords: Human CYP2D6; High-throughput screening; Microplate reader; Flow injection analysis; Inhibition; Validation
1. Introduction
2D6 (CYP2D6, EC 1.14.14.1) (Spatzenegger and Jaeger,
1995; Tanaka and Breimer, 1997). This is not only due to
its important role in drug metabolism, but also because it is
deficient with varying frequencies in different populations,
as a result of a variety of genetic defects (Kroemer and
Eichelbaum, 1995; Wormhoudt et al., 1999). This so-called
debrisoquine/sparteine polymorphism (Eichelbaum et al.,
1975; Mahgoub et al., 1977) can lead to a reduced
bioactivation capacity towards prodrugs, as well as unde-
sired clinical responses to drugs, due to high plasma levels
and altered metabolic pathways of a therapeutic agent
(Breimer, 1983; Tucker, 1994). High affinity binding to
CYP2D6 may, therefore, be used as a negative selection
criterion in the process of drug discovery and develop-
ment.
The application of combinatorial chemistry in the dis-
covery and development of new drug candidates has led to
an increasing demand for high-throughput screening meth-
ods that are capable of identifying enzymes involved in the
binding (prediction of drug–drug interactions) and, poten-
tially, the metabolism of new ligands (Rodrigues, 1997).
As hepatic cytochromes P450 (CYPs) are the enzymes
mainly responsible for the metabolism of therapeutic
agents, several efforts have been made to develop such
methods for individual CYPs (Crespi et al., 1997). Next to
human CYP3A4 and CYP1A2, one of the isoenzymes of
particular interest in this regard is human cytochrome P450
Recently, we have reported on the design and synthesis
of a novel and selective substrate of CYP2D6, 7-methoxy-
4-(aminomethyl)-coumarin (MAMC) (Onderwater et al.,
*Corresponding author. Tel.: 131-20-4447-590; fax: 131-20-4447-
610.
E-mail address: vermeule@chem.vu.nl (N.P.E. Vermeulen).
0928-0987/00/$ – see front matter
PII: S0928-0987(00)00150-0
2000 Elsevier Science B.V. All rights reserved.

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J. Venhorst et al. / European Journal of Pharmaceutical Sciences 12 (2000) 151–158
previously (Onderwater et al., 1999). Structural analogs of
3,4-methylenedioxyamphetamine were synthesised accord-
ing to Shulgin (1991). Glucose-6-phosphate dehydrogen-
ase was from Boehringer (Mannheim, Germany). NADPH
was obtained from Applichem (Darmstadt, Germany).
SK&F 525A was from SmithKline&French Laboratories
(Herts, UK). All other chemicals were obtained from
Sigma (St Louis, MO, USA).
Fig. 1. O-Demethylation of 7-methoxy-4-(aminomethyl)-coumarin
(MAMC) to its 7-hydroxyl derivative HAMC, mediated by CYP2D6.
1999). Characterisation of this compound revealed that
MAMC was selectively O-demethylated to 7-hydroxy-4-
(aminomethyl)-coumarin (HAMC; Fig. 1) by CYP2D6,
with only CYP1A2 contributing to the formation of this
metabolite to a small extent in human liver microsomes.
The latter contribution could, however, be eliminated
completely by the addition of the highly selective CYP1A2
inhibitor furafylline. As the fluorescent properties of
MAMC and its metabolite HAMC were found to be
significantly different, it was suggested that MAMC could
be an interesting tool for high-throughput screening.
Until recently, only classical assays based on HPLC and
GC techniques have been described for inhibition of
CYP2D6 using substrates such as dextromethorphan,
bufuralol and metoprolol (Kronbach et al., 1987; Lennard
and Silas, 1983). At this moment two high-throughput
assays have been described (Crespi et al., 1997; Rodrigues
et al., 1994). However, the substrate used in the first assay,
7-ethoxy-3-cyano-coumarin, is not selective for CYP2D6
and has a much lower turnover than MAMC (Onderwater
et al., 1999). The second assay has the disadvantage that it
makes use of radioactively labelled dextromethorphan.
In this study, we evaluated a high-throughput microplate
reader assay using MAMC as a novel fluorescent probe for
determining IC50 or Ki values. The method was validated
by comparing the inhibition constants (Ki values) obtained
with the high-throughput assay, with those from the
frequently used O-demethylation assay of dextromethor-
phan. Both specific and non-specific ligands of CYP2D6,
displaying a wide range of binding affinities, were used as
a test-set. In addition, kinetic studies on MAMC O-de-
methylation were performed in human liver microsomes to
investigate the applicability of the microplate reader assay
to this system. To further demonstrate the usefulness of the
high-throughput assay, IC50 values were determined for a
series of N-substituted analogs of 3,4-methylenedioxyam-
phetamine (MDA). These compounds are of interest be-
cause CYP2D6 has been implicated in the neurotoxic
effect of the N-methyl and N-ethyl derivatives of MDA
(MDMA and MDE, respectively) (Ensslin et al., 1996; Lin
et al., 1997).
2.2. Microsomal protein
Human cytochrome P450 2D6 (CYP2D6) microsomal
protein expressed in a human lymphoblastoid cell line
(catalogue no. P171) was purchased from GENTEST
(Woburn, MA, USA). The CYP2D6 content was 175
pmol/mg microsomal protein. Human liver microsomes of
a single individual were a generous gift from Dr. B.
Rademaker (Rephartox, The Netherlands).
2.3. Calibration curve of HAMC
Calibration curves of HAMC were made for quantifica-
tion purposes. To 100 ml of a mixture containing the
appropriate amount of microsomal protein and different
concentrations of HAMC (0.625–40 mM final concen-
tration) in a 100 mM phosphate buffer (pH 7.4), 100 ml of
a preincubated NADPH-regenerating system (see below)
were added. Subsequently, 10 ml 5 M trichloroacetic acid
(TCA) were added. The pH was readjusted to pH 9 by the
addition of 100 ml of a 1.5 M Tris–HCl buffer (pH 9.0).
The fluorescence of HAMC was measured at excitation
and emission wavelengths of 405 nm (bandwidth 8 nm)
and 460 nm (bandwidth 30 nm), respectively, on a Victor2
1420 multilabel counter (Wallac, Turku, Finland).
2.4. Time and protein dependency of HAMC formation
Prior to the inhibition studies, linearity of the formation
of HAMC and its protein dependency were investigated by
incubating 40 mM MAMC at 378C in a 100 mM phosphate
buffer (pH 7.4) containing 20, 40 or 60 nM CYP2D6, and
a NADPH-regenerating system containing a final con-
centration of 0.1 mM NADP1 (see below). The amount of
HAMC formed was assayed as described below at differ-
ent time-points.
2.5. Assay of MAMC O-demethylation for IC50
measurements
IC50 measurements were performed in costar 3595 96-
well plates using a 100 mM phosphate buffer (pH 7.4) and
a final volume of 200 ml. Briefly, a concentration range of
inhibitor was obtained by serially diluting 50 ml of a
solution containing the appropriate amount of inhibitor,
with 100 ml of buffer solution. Subsequently, 50 ml of a
2. Materials and methods
2.1. Chemicals
MAMC and HAMC were synthesised as described

J. Venhorst et al. / European Journal of Pharmaceutical Sciences 12 (2000) 151–158
153
mixture containing 40 nM CYP2D6 and 40 mM MAMC
were added. After 10 min of preincubation at 378C, 50 ml
of a preincubated cofactor solution was added to start the
reaction, resulting in final concentrations of 0.1 mM
NADPH, 0.3 mM glucose-6-phosphate, 0.4 mM MgCl2,
and 0.4 units/ml glucose-6-phosphate dehydrogenase.
After 45 min, the reaction was stopped by adding 10 ml of
a 5 M TCA solution. Prior to analysis, the pH was adjusted
with 100 ml of a 1.5 M Tris–HCl buffer (pH 9.0).
Quantification of HAMC formation was performed using
the standard curve described above.
wavelength of 405 nm (bandwidth 18 nm) and an emission
wavelength of 480 nm (bandwidth 18 nm), with an
analysis time of 1.0 min. For quantification, a calibration
curve of HAMC was used.
2.8. MAMC O-demethylation in human liver microsomes
In addition to heterologously expressed CYP2D6, the
applicability of the microplate assay was also investigated
in human liver microsomes. The incubations were general-
ly carried out as described above; however, the microsomal
fraction and NADPH-regenerating system were pre-incu-
bated for 5 min at 378C in the presence of 30 mM
furafylline (Onderwater et al., 1999), before starting the
reaction by the addition of substrate. Also, microsomal
protein was removed from the stopped incubation mixtures
by centrifugation, and the supernatant transferred into a
96-well plate for analysis by microplate reader as de-
scribed above. Protein-dependency of HAMC formation
was investigated in human liver microsomes by incubating
40 mM of MAMC with varying amounts of microsomal
protein (0.12–0.36 mg/ml). Samples were drawn after 0,
15, 30, and 45 min to determine the linearity of the
reaction. Additional incubations, also containing 1 mM
quinidine, were performed to ensure that the observed
HAMC formation was solely CYP2D6 mediated. Enzyme
kinetics of MAMC O-demethylation were studied using
0.30 mg/ml microsomal protein.
2.6. Assay of MAMC O-demethylation for Ki
measurements
The O-demethylation of MAMC was investigated in
costar 3595 96-well plates, in both the absence and
presence of two different concentrations of inhibitor, to
establish apparent Km and Vmax values. The incubations
were performed in a 100 mM phosphate buffer (pH 7.4)
with a final volume of 200 ml. A concentration range of
MAMC (1.25–160 mM final concentration) was obtained
by serially diluting 100 ml of substrate solution with 100
ml of buffer. Subsequently, 25 ml of a solution containing
the appropriate amount of inhibitor (determined by IC50
measurements), and 25 ml of a solution containing
CYP2D6 (10 nM final concentration) were added. After a
preincubation (378C) of 10 min, 50 ml of a preincubated
solution (378C) containing a NADPH-regenerating system
were added (see above). After 45 min the reaction was
stopped by the addition of 10 ml of a 5 M TCA solution.
Prior to analysis, 100 ml of a 1.5 M Tris–HCl buffer (pH
9.0) was added to the incubation mixture in order to
increase the fluorescent signal of HAMC. The amount of
HAMC formed was quantified by measuring the fluores-
cence at excitation and emission wavelengths of 405
(bandwidth 8 nm) and 460 nm (bandwidth 30 nm),
respectively, on a Victor2 1420 multilabel counter (Wal-
lac).
2.9. Assay of dextromethorphan O-demethylation
The O-demethylation of dextromethorphan to its metab-
olite dextrorphan was investigated both in the presence and
absence of two different concentrations of inhibitor, in a
100 mM phosphate buffer (pH 7.4). A mixture containing
dextromethorphan (2.5–40 mM final concentration),
CYP2D6 (8.3 nM final concentration), and the appropriate
amount of inhibitor (as determined by IC50 measurements),
in a total volume of 100 ml, was pre-incubated for 5 min at
378C. The reaction was started by the addition of 100 ml of
a 2 mM NADPH solution, in a 100 mM phosphate buffer
(pH 7.4). After 10 min the reaction was stopped by the
addition of 100 ml 3 M perchloric acid. The incubation
mixture was subsequently centrifuged for 15 min at 40003
g, and the supernatant was analysed by HPLC.
2.7. Flow injection analysis of HAMC formation
As an alternative to measuring HAMC formation with a
microplate reader, a flow injection analysis (FIA) setup
was used. The incubations were basically performed as
described above. However, after the addition of TCA, the
precipitated protein was first centrifuged for 15 min at
40003g, and 100 ml of 1.5 M Tris–HCl buffer (pH 9.0)
were added to 180 ml of supernatant. HAMC formation
was measured by injecting 100 ml of sample into a HPLC
apparatus, consisting of a model 305 pump (Gilson
Medical Electronics, WI, USA); a Waters 717 plus auto-
sampler (Waters, MA, USA) cooled to 48C; and a model
821-FP fluorescence detector (Separations, The Nether-
lands). As eluent 50:50 (v/v) methanol:H2O was used at a
flow rate of 1 ml/min. Detection was at an excitation
2.10. HPLC analysis of dextrorphan
Chromatographic separation of dextromethorphan and
its metabolite dextrorphan was achieved with a HPLC
system consisting of a model 305 pump (Gilson Medical
Electronics); a Waters 717 plus autosampler (Waters); a
Suplex pKb-100 column (4.6 mm315 cm) and corre-
sponding guard column (Supelco, PA, USA); and a model
821-FP fluorescence detector (Separations). The mobile
phase consisted of 25% acetonitrile, 1% triethylamine, and

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J. Venhorst et al. / European Journal of Pharmaceutical Sciences 12 (2000) 151–158
74% H2O, pH 3.0, delivered at a flow rate of 1 ml/min.
The injection volume was 100 ml. Dextrorphan was
detected at excitation and emission wavelengths of 280 and
311 nm (bandwidth 18 nm), respectively. For quantifica-
tion, a calibration curve of dextrorphan was used (0.1–10
mM). The analysis time was 20 min.
2.11. Kinetic analyses
Apparent Km and Vmax parameters were determined by
using the non-linear least-square fitting method for one site
binding, as implemented in Prism 2.0 (GraphPad Software,
USA). Ki values were subsequently calculated with the
following equation:
9
Ki 5 (Km /(Km 2 Km)) ? [I]
In the equation, Km is the apparent Km value in the
9
absence of inhibitor, Km is the apparent Km value in the
presence of inhibitor, and [I] is the inhibitor concentration.
IC50 values were determined by using the non-linear least-
square fitting method for one site competition, as im-
plemented in Prism 2.0 (GraphPad Software).
3. Results
3.1. Linearity of HAMC formation by heterologously
expressed CYP2D6
Fig. 2. (a) Time-dependent formation of HAMC from MAMC, in the
presence of 20 (j), 40 (m) and 60 (d) nM CYP2D6. Incubations were
performed at 378C in a 100 mM phosphate buffer (pH 7.4). (b) Protein-
dependency of MAMC O-demethylation to HAMC by CYP2D6, shown
for an incubation of 45 min at 378C in a 100 mM phosphate buffer (pH
7.4).
We have investigated the time- and protein-dependent
formation of HAMC from MAMC, at a substrate con-
centration of 40 mM. As can be seen in Fig. 2a, the
formation of HAMC by CYP2D6 was linear in time, for at
least 45 min, at all P450-concentrations investigated.
Metabolite formation was also found to be linear with
respect to protein concentration (Fig. 2b).
3.2. Enzyme kinetics of heterologously expressed
CYP2D6
The metabolism of MAMC to its O-demethylated
metabolite HAMC by CYP2D6 displayed apparent Mich-
aelis–Menten kinetics, as demonstrated by Fig. 3. Appar-
ent Km and Vmax values measured by the microplate assay
on different days were 7.962 mM and 8.160.7 min21
(n57, Table 1), respectively. The detection limit for
HAMC quantification by the microplate reader was 0.3
mM (signal-to-noise ratio, 3:1). Kinetic studies performed
on separate days using the FIA method resulted in similar
Km and Vmax values of 6.460.6 mM and 7.360.2 min21
(n56, Table 1), respectively. This method displayed a
detection limit of 0.1 mM for HAMC.
Fig. 3. Rate of O-demethylation of MAMC to its 7-hydroxyl metabolite
HAMC by CYP2D6 plotted against the substrate concentration. Incuba-
tions were performed in a 100 mM phosphate buffer (pH 7.4).

J. Venhorst et al. / European Journal of Pharmaceutical Sciences 12 (2000) 151–158
155
Table 1
Table 2
Apparent Km and Vmax values for, respectively, the O-demethylation of
MAMC and dextromethorphan by CYP2D6, expressed in a human
lymphoblastoid cell line (n56)a
Apparent Ki values for the inhibition of MAMC O-demethylation by
different ligands of human lymphoblast-expressed CYP2D6a
Ligand
Ki value (mM)
Substrate
Assay
Km (mM)
Vmax (min21
)
Quinidine
0.002960.0004
0.04460.015
0.6660.15
1.060.42
MAMC
MAMC (human liver)
MAMC
Microplate
Microplate
FIA
7.962.0
34.662.0
6.460.6
2.060.6
8.160.7
251614b
7.360.2
4.860.3
SK&F 525A
Dextromethorphan
Triprolidine
Sparteine
Dextromethorphan
HPLC
16.165.6
Codeine
24.063.4
a The enzymatic parameters for MAMC O-demethylation were addi-
tionally determined in human liver microsomes from a single individual
(n53). Values are the mean6S.D.
a Values are the mean6S.D. (n53).
b In pmol/min per mg microsomal protein.
of HAMC formation, with a Ki value of 0.6660.15 mM
(n53).
3.3. MAMC O-demethylation in human liver microsomes
To further validate the microplate assay for MAMC
O-demethylation, Ki values were measured for a number
of known ligands of CYP2D6, with both the dextromethor-
phan and the MAMC O-demethylation assay (Table 2).
For this purpose a selection of specific and non-specific
ligands of CYP2D6 was made, displaying a wide range of
inhibitory potencies. The specific ligands included codeine,
sparteine, and quinidine, whereas the non-specific ligands
were triprolidine and SK&F 525A. All ligands of CYP2D6
studied displayed competitive inhibition towards both
dextromethorphan and MAMC. As shown in Fig. 5, a good
correlation of R250.984 was obtained between the Ki
values from both assays.
IC50 values for CYP2D6 of a series of N-substituted
analogs of 3,4-methylenedioxyamphetamine (MDA) were
determined. The measurements were performed at a final
concentration of MAMC close to the Km value (10 mM).
As can be seen in Table 3, elongation of the N-alkyl chain
initially resulted in an increased inhibitory potency of the
ligand for CYP2D6. This trend is, however, discontinued
when the N-propyl moiety is replaced by a N-butyl group.
HAMC formation in human liver microsomes, pre-incu-
bated for 5 min with 30 mM furafylline, displayed linearity
for at least 45 min. MAMC O-demethylation was also
linear with varying quantities of microsomal protein. In the
presence of both 30 mM furafylline, a selective CYP1A2
inhibitor, and 1 mM quinidine, a selective CYP2D6
inhibitor, no HAMC formation could be detected. The
metabolism of MAMC in the human liver microsomes
displayed apparent Michaelis–Menten kinetics. Apparent
Km and Vmax values were found to be 34.662.0 mM and
251614 pmol/min per mg microsomal protein, respec-
tively.
3.4. Inhibition studies
Inhibition of the CYP2D6 mediated O-demethylation of
dextromethorphan (Km 52.060.6 mM, n56) by MAMC,
showed that MAMC is a relatively potent competitive
inhibitor of this model substrate of CYP2D6, with a Ki
value of 10.863.1 mM (n53, Fig. 4). Additionally,
dextromethorphan was found to be a competitive inhibitor
Fig. 5. Plot of the apparent Ki values obtained with the dextromethorphan
O-demethylation assay versus those obtained with the MAMC O-de-
methylation assay. A correlation of R250.984 was obtained. The trend in
inhibitory potency is: quinidine.SK&F 525A.triprolidine.sparteine.
codeine.
Fig. 4. Lineweaver–Burke plot for dextromethorphan O-demethylation
by CYP2D6, in the presence of 0 mM (d), 40 mM (m) and 80 mM (j)
MAMC. Incubations were performed in a 100 mM phosphate buffer (pH
7.4).

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J. Venhorst et al. / European Journal of Pharmaceutical Sciences 12 (2000) 151–158
Table 3
be determined more easily, the incubation time can be
reduced. Also, the enzymatic reaction does not have to be
stopped. However, to make the assay suitable for use with
a multi-plate reader, we used an end-point measurement of
HAMC formation.
IC50 values for the inhibition of the CYP2D6 mediated O-demethylation
of MAMC by a series of N-substituted MDA analogsa
MDA analog
IC50 value (mM)
R5Methyl
R5Ethyl
R5Propyl
R5Butyl
0.9260.14
0.4460.06
0.09660.014
0.1960.03
190670
We also examined the use of flow injection analysis
(FIA) as an alternative to quantification of HAMC by a
microplate reader. The FIA method was found to require
only some small adaptations of the microplate assay and
represents a significant improvement in analysis time
compared to a classical HPLC method, since no chromato-
graphic separation is required. An advantage of FIA over a
microplate reader is the reduction in background signal,
due to the fact that protein has been removed from the
incubation mixture. This results in a higher sensitivity, as
reflected by the lower detection limit for HAMC. How-
ever, it also makes the FIA assay more laborious. It should
be noted that, in the case of the FIA assay, an emission
wavelength of 480 nm was used, as this was optimal for
reducing the background level (Onderwater et al., 1999).
The incubations containing human liver microsomes
clearly show the advantages of MAMC over other fluores-
cent probes for CYP2D6. The P450 selectivity of MAMC
is such that, in the presence of the specific CYP1A2
inhibitor furafylline, HAMC formation is solely attribut-
able to CYP2D6. This is demonstrated by the fact that
addition of quinidine, combined with furafylline, complete-
ly inhibited MAMC metabolism. Measurements of MAMC
O-demethylation using heterologously expressed CYP2D6
in the presence and absence of 30 mM furafylline demon-
strated that the latter is a weak inhibitor of CYP2D6 at this
concentration, resulting in a factor 1.5 increase in Km
value. The addition of furafylline alone, however, cannot
explain the increase in Km value observed in human liver
microsomes. Instead, the higher Km value obtained in
human liver microsomes may additionally be due to
(aspecific) binding of MAMC to other proteins and/or a
relatively high amount of CYP2D6. Alternatively, as only
one human liver sample was investigated it cannot be
excluded that the microsomes contained a polymorphic
form of CYP2D6 which differs from the heterologously
expressed CYP2D6, resulting in different binding charac-
teristics. The difference in Km value does not, however,
affect the ability of the assay to identify new ligands of
CYP2D6, which could potentially cause drug–drug inter-
actions. When compared to heterologously expressed
CYP2D6, only small modifications to the microplate
reader assay are required in order to obtain a very rapid
method for screening the CYP2D6 activity in human liver
microsomes.
R5Phenethyl
a CYP2D6 was expressed in human lymphoblasts. Values are shown as
the mean6S.D. (n53).
The N-phenethyl substituted analog displayed the lowest
capacity for inhibiting the enzyme.
4. Discussion
The aim of this study was to evaluate a novel and
selective high-throughput assay for measuring inhibition of
CYP2D6, heterologously expressed and in human liver
microsomes, in order to identify potential ligands of
CYP2D6. MAMC was used as the substrate, because this
was recently demonstrated to be a selective substrate of
CYP2D6 potentially suitable for high-throughput screen-
ing, due to the fluorescent properties of its metabolite
HAMC (Onderwater et al., 1999).
Initial kinetic studies on the O-demethylation of MAMC
by CYP2D6 demonstrated that a good signal-to-back-
ground ratio could be obtained with a protein concentration
as low as 10 nM CYP2D6 per well, when an incubation
time of 45 min was used. In order to minimise the amount
of CYP2D6 required, this concentration and incubation
time were used for the inhibition experiments. As noted
previously, NADPH interferes with the fluorescent signal
of HAMC (Onderwater et al., 1999). This interference
could, however, be reduced satisfactorily to background
levels by using a final concentration of 0.1 mM NADP1 in
the NADPH-regenerating system, and an excitation wave-
length of 405 nm. The stop-solution used was a 5 M TCA
solution, as it was found that the addition of 100 ml
acetonitrile–0.1 M Tris, pH 9.0 (60:40, v/v), the stop-
solution used in other high-throughput assays (Crespi et
al., 1997), interferes with our assay. To increase the
fluorescent yield of HAMC, 100 ml of a Tris–HCl buffer,
pH 9.0, was added. It should be noted that when the
samples were not analysed immediately after addition of
this base, the incubation mixtures had to be stored in the
dark at 48C.
Although we have described an end-point measurement
of HAMC formation, we have also performed some real-
time measurements, during which the increase in fluores-
cence was measured every 5 min. An advantage of the
latter method is the fact that the level of the background
signal is less disturbing, as activity is determined by the
slope of the increase in fluorescence. Because the slope can
The present inhibition studies show that MAMC is a
competitive inhibitor of dextromethorphan, and vice versa,
which is in agreement with previous molecular modelling
results (Onderwater et al., 1999; De Groot et al., 1997;
Koymans et al., 1992). Comparison of the Ki values
obtained for the series of low- and high-affinity ligands

J. Venhorst et al. / European Journal of Pharmaceutical Sciences 12 (2000) 151–158
157
with both the MAMC and dextromethorphan assay, fur-
thermore, revealed a very good correlation (R250.984,
Fig. 5) between the two methods. It can also be seen that
the Ki values obtained with the two assays differ in
absolute value, the Ki values from the MAMC O-de-
methylation assay (Table 2) being consistently smaller.
Importantly, this difference constitutes a constant factor.
The observed trend in a 4-order range of inhibitory
potency of the studied ligands is, thus, identical for the two
methods. Theoretically, Ki values should be independent of
the substrate used, provided the binding of the different
substrates is of a truly competitive nature. This ideal
situation is, however, difficult to accomplish experimental-
ly, since no two CYP2D6 substrates, belonging to different
chemical classes, are likely to share the exact same
interaction points within the active site of the enzyme. It is
therefore not surprising that significant differences in Ki
values generally arise between methods that use different
substrates (Tucker, 1998). The fact that the Ki values for
MAMC are smaller than those for the larger substrate,
dextromethorphan, can be rationalised by the ability of the
latter to form more lipophilic interaction points with active
site residues, making its displacement more difficult.
Another explanation may be the use of slightly different
incubation conditions. It can, therefore, be concluded that
MAMC O-demethylation, measured by the present micro-
plate assay, is a valid tool for determining binding
affinities of ligands of CYP2D6.
In order to illustrate the potential use of inhibition of
MAMC O-demethylation for large-scale screening pur-
poses, we have determined IC50 values for a series of
N-substituted analogs of 3,4-methylenedioxyamphetamine
(MDA) with the microplate reader. As can been seen from
Table 3, IC50 values were determined reproducibly. The
microplate assay thus also provides an adequate screening
tool for identifying structural analogs, which display a
lower potential for drug–drug interactions. In the case of
the MDA analogs, substitution at the basic nitrogen with a
phenethyl-group resulted in the highest IC50 value for
CYP2D6 and, by inference, the lowest risk of drug–drug
interactions.
MAMC O-demethylation assay can be viewed as a signifi-
cant improvement over the existing 7-ethoxy-3-cyano-
coumarin O-deethylation assay (Km 567 mM, Vmax 50.017
min21; Crespi et al., 1997) as MAMC is a much more
selective substrate of CYP2D6, with a more than 400-fold
higher turnover. Recently, GENTEST introduced 3-[2-
(N, N -diethyl-N -methylamino)ethyl]-7-methoxy-4-me-
thylcoumarin (AMMC) as a new CYP2D6 substrate,
suitable for high-throughput screening (Crespi, 1999).
However, the fluorescent characteristics of AMMC and its
metabolite, and the turnover number of AMMC by
CYP2D6 (Vmax 51 min21), are inferior to those of MAMC.
The use of AMMC thus either requires a higher con-
centration of CYP2D6, or a lower concentration of
NADPH in order to decrease background fluorescence.
In conclusion, a novel high-throughput microplate
reader assay is available for determining the inhibition and
activity of CYP2D6 in heterologously expressed systems
and human liver microsomes, which is easy to use, rapid
and sensitive. The assay makes use of the selective
CYP2D6 substrate 7-methoxy-4-(aminomethyl)-coumarin
(MAMC) as a fluorescent probe, and represents a signifi-
cant improvement over previous assays, with a higher
selectivity for CYP2D6 and higher turnover of MAMC. In
addition to the microplate reader-based assay, flow in-
jection analysis can also be used. In the case of human
liver microsomes, pre-incubation with furafylline fully
eliminates the contribution to HAMC formation by
CYP1A2, which is the only P450 slightly active towards
MAMC besides CYP2D6.
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