An in vitro approach to potential methadone metabolic-inhibition interactions

Article abstract of DOI:10.1007/s00228-007-0327-z

Objective: The aim of this study was to assess the drug interaction potential of psychotropic medication on methadone N-demethylation using cDNA-expressed cytochrome P450 CYP enzymes. Methods: Methadone was incubated with various drugs (n = 10) and cDNA-e

Full text of DOI:10.1007/s00228-007-0327-z

Eur J Clin Pharmacol (2007) 63:821–827
DOI 10.1007/s00228-007-0327-z
PHARMACOKINETICS AND DISPOSITION
An in vitro approach to potential methadone
metabolic-inhibition interactions
Stephanie Bomsien & Gisela Skopp
Received: 2 May 2007 /Accepted: 22 May 2007 /Published online: 28 June 2007
#
Springer-Verlag 2007
Abstract
of drug-methadone interactions should help to improve patient
care during methadone maintenance treatment.
Objective The aim of this study was to assess the drug
interaction potential of psychotropic medication on metha-
done N-demethylation using cDNA-expressed cytochrome
P450 CYP enzymes.
.
.
Keywords Methadone metabolism In vitro interaction
.
.
Psychotropic co-medication CYP3A4 CYP2D6
Methods Methadone was incubated with various drugs (n=
10) and cDNA-expressed CYP3A4, CYP2D6, CYP2B6,
CYP2C19 and CYP1A2 enzymes to screen for their
inhibition potency. The nature of enzyme selective activity
for inhibition was further investigated for potent inhibitors.
To test for a mechanism-based component in inhibition, all
substances were tested with preincubation and without. 2-
Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)
concentration was determined by liquid chromatography/
tandem mass spectrometry following liquid/liquid extraction.
Results Formation of EDDP was catalysed by CYP3A4,
CYP2D6 and CYP2C19. The N-demethylation of metha-
done was preferentially inhibited by amitriptyline, bupre-
norphine, methylenedioxymethamphetamine (MDMA) and
zolpidem. Both amitriptyline and buprenorphine were
strong, reversible inhibitors of CYP3A4. Similarly, amitrip-
tyline and MDMA were identified as inhibitors of CYP2D6.
Zolpidem revealed a mechanism-based inhibition of
CYP3A4.
Introduction
Methadone is the most accepted and established substitute
for treating heroin dependence worldwide [1]. Neverthe-
less, some controversy has emerged due to reports of
overdose deaths [2–4]. Deaths often occurred in relation to
the risks associated with the first few weeks, take-home
doses or the concurrent consumption of prescription or
illegal drugs [5, 6]. Co-administration of drugs can lead to
important interactions which also may be caused by
metabolic inhibition of cytochrome P450 (CYP) activity.
For many drugs that are regularly prescribed or misused
during methadone maintenance treatment, such data are not
available.
Methadone is extensively metabolized by CYP enzymes
in the liver. First reports indicate that CYP3A4 is the major
isoform involved in the formation of 2-ethylidene-1,5-
dimethyl-3,3-diphenylpyrrolidine (EDDP), which is the main
metabolite of methadone. EDDP is further N-demethylated
to 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP). p-Hy-
droxylation of one aromatic ring in all three compounds
has been reported, and small amounts of methadol and
normethadol are produced in addition. Aside from methadol
and normethadol, none of the metabolites has any apparent
pharmacologic activity, and none except EDDP is found to
any significant extent in blood [7–10]. Data for CYP2D6
seem to be controversial. Some studies ruled out a moderate
involvement on the N-demethylation of methadone [7, 8,
Conclusion Amitriptyline, MDMA and zolpidem are likely to
slow down conversion of methadone and to increase its area
under the curve (AUC). A consideration of the in vitro evidence
:
S. Bomsien G. Skopp (*)
Institute of Legal Medicine and Traffic Medicine,
Ruprecht-Karls University,
Voßstr. 2,
69115 Heidelberg, Germany
e-mail: gisela_skopp@med.uni-heidelberg.de

822
Eur J Clin Pharmacol (2007) 63:821–827
10–12], while others could not prove any participation in
methadone metabolism [9] or even demonstrated inhibition
of CYP2D6 by methadone [13]. Gerber et al. [10] reported
CYP2B6 and CYP2C19 also to take part in methadone N-
demethylation. Limited data indicate contribution of
CYP1A2, which also may take part in another metabolic
pathway than the N-demethylation [8].
In the present study, 10 psychotropic drugs undergoing
metabolism by the enzymes mentioned above were investi-
gated for their influence on methadone conversion. The data
should help to assess the likelihood of drug-drug interactions
in individuals on methadone maintenance treatment.
Study design
First, the involvement of particular enzymes in N-demeth-
ylation of methadone was investigated. Other than EDDP, no
further metabolites were detectable from incubations. Reac-
tion velocity linearity with regard to both time and enzyme
concentration was ensured. Estimates of Km (Michaelis-
Menten constant: substrate concentration that yields a
reaction velocity that is half of maximum) were derived
from preceding experiments (data not shown). Initial
inhibition screening experiments on three different concen-
trations were performed with and without a 30-min
preincubation of each inhibitor to test for a mechanism-
based inhibition. In order to accurately determine inhibitor
concentrations corresponding to a 50% inhibition of sub-
strate metabolism (IC50) at Km concentration of methadone,
further data points were established for those drugs that
significantly inhibited the formation of EDDP (at least 40%
of control velocity at the highest inhibitor concentration).
Following addition of the internal standard (EDDP-d3) and
liquid-liquid extraction (pH 8.5, ethyl acetate), the concentra-
tion of EDDP was determined from the samples by high-
pressure liquid chromatography/tandem mass spectrometry
(API 365, Applied Biosystems, Toronto, Canada) in a validated
assay [14]. All experiments were carried out in duplicate.
The inhibition constant Ki was determined based on the
assumption of reversible inhibition when similar results were
obtained with or without preincubation. The inhibitor concen-
tration KI that yields half the maximum rate of inactivation
and the enzyme inactivation rate constant kinact were deter-
mined if there was evidence of a mechanism-based inhibition.
Materials and methods
Supersomes and chemicals
NADPH-regenerating systems A (26.1 mM NADP+, 66 mM
glucose-6-phosphate, 66 mM magnesium chloride) and B
(40 U/mL glucose-6-phosphate dehydrogenase in 5 mM
sodium citrate), which generate NADPH in situ, and human
Supersomes were obtained from BD Gentest (Woburn, MA,
USA). Supersomes enzymes are recombinant cDNA-
expressed cytochrome P450 enzymes prepared from the
baculovirus-infected insect cell system with supplemental
cDNA-expressed reductase. Insect cell microsomes infected
with wild-type baculovirus (control Supersomes) as well as
Supersomes with a representative P450 content of
1,000 pmol/mL were delivered: human CYP3A4 (total pro-
tein concentration: 4.5 mg/mL), human CYP2D6 (total
protein concentration: 4.5 mg/mL), human CYP2B6 (total
protein content: 6.3 mg/mL), CYP2C19 (total protein
content: 3.6 mg/mL) and human CYP1A2 (total protein
content: 4.5 mg/mL). Methadone hydrochloride, amitripty-
line, atomoxetine, buprenorphine, citalopram, clobazam,
clozapine, methylenedioxymethamphetamine (MDMA), nic-
otine, zolpidem, zopiclone and potassium phosphate were
purchased from Sigma/Aldrich (Steinheim, Germany). Meth-
adone, EDDP and EDDP-d3 as standard solutions were
provided by Promochem (Wesel, Germany), and ethyl
acetate, acetonitrile and methanol were obtained from Roth
(Karlsruhe, Germany).
Screening of inhibitory potencies
A 1 mg/mL solution of substrate was prepared in 0.1 M
potassium phosphate buffer, pH 7.4. Substrate concentra-
tion for each enzyme was chosen to be equal to the
apparent Km value determined in kinetic studies (data not
shown), which was 13.3 μM for CYP3A4, 2.98 μM for
CYP2D6, 69.6 μM for CYP2B6, 72.7 μM for CYP2C19
and 44.2 μM for CYP1A2. All inhibitors under investiga-
tion were prepared in methanol and evaporated to dryness
prior to reconstitution in the incubate. In the initial
inhibition screening, final inhibitor concentrations were
equal to 1/2·Km, Km and 2·Km for the particular enzyme,
using Km values of each inhibitor from the Drug Interaction
Database of the University of Washington [15].
For assays performed without preincubation, a 250-μL
incubation mixture consisting of the NADPH-regenerating
system, substrate and inhibitor in 0.1 M potassium phosphate
buffer of pH 7.4, and 10 pmol enzyme was incubated at 37°C
for 20 min in a shaking water bath. The same incubation
mixture and reaction conditions were used in the mechanism-
Information on psychotropic substances frequently ob-
served in patients on methadone treatment was available from
a local substitution centre, where urine samples were regularly
screened for prescribed and illicitly used drugs by high
pressure liquid chromatography/UV detection (Remedi Drug
Profiling System, Hercules, CA, USA). A choice of 10
potential inhibitors was made from these drugs including
amitriptyline, atomoxetine, buprenorphine, citalopram, cloba-
zam, clozapine, MDMA, nicotine, zolpidem, zopiclone.

Eur J Clin Pharmacol (2007) 63:821–827
823
based inhibition experiments except that methadone was added
after a 30-min preincubation at 37°C [16]. Subsequently, the
reaction was stopped with 125 μL ice-cold acetonitrile.
Control enzymes and incubation mixtures without
inhibitor were used as controls. The metabolite formation
rate with co-addition of an inhibitor was expressed as a
percentage ratio relative to the control velocity without
inhibitor. Ratios were analysed using non-linear regression.
following incubation, a 25-μL aliquot of the first incubation
mixture was transferred to a second vial containing methadone
at Km concentration and NADPH-regenerating system (final
volume: 250 μL). Reactions were carried out at 37°C for
20 min and then terminated as described above. All experi-
ments were carried out four times. Data were analysed by
Kitz-Wilson plots [18].
Data analysis
Determination of Ki
Statistical analysis and determination of Ki, KI and kinact
were performed using Microsoft Excel and nonlinear
regression on Sigma Plot 9.0 (Point Richmond, CA, USA).
Experiments with three different substrate concentrations
(1/2·Km, Km, 2·Km) and four different inhibitor (1/4·Km,
1/2·Km, Km, 2·Km) concentrations were conducted (total
reaction volume 300 μL). Incubation mixture and reaction
conditions were the same as described for the IC50
screening. A 250-μL sample of the incubation mixture
was added to 10 pmol of enzyme and incubated at 37°C for
20 min. The reaction was terminated by addition of 125 μL
ice-cold acetonitrile. All experiments were carried out four
times, and mean values were analysed by Dixon plots [17].
Results
Screening of inhibitory potencies
Substances significantly inhibiting production of EDDP are
shown in Table 1. In addition, results following a 30-min
preincubation are given.
Determination of KI and kinact
When studied with and without preincubation, conversion of
methadone via CYP3A4 considerably decreased to approxi-
mately 10% of control velocity at maximum inhibitor
concentration in the presence of amitriptyline and buprenor-
phine. Also, zolpidem revealed a mechanism-based inhibition.
Only two out of four drugs tested on CYP2D6 demon-
strated an important influence on EDDP formation. Without
preincubation, N-demethylation of methadone was inhibited
KI and kinact were determined in time-dependent two-step
experiments at four different inhibitor concentrations
(1/4·Km, 1/2·Km, Km, 2·Km) and a uniform substrate
concentration (Km). In a first incubation mixture (150 μL),
phosphate buffer as a blank or the inhibitor was added to
60 pmol enzyme. Prior to incubation and every 5 min
Table 1 Results for the determination of relative IC50 values (n=2, range <10%) of inhibitors of methadone metabolism at three different
inhibitor concentrations (1/2·Km, Km, 2·Km),
CYP
Inhibitor
% Ratio relative to control velocity without inhibitor
Without preincubation
30-min preincubation
a
a
1/2·Km
Km
2·Km
1/2·Km
Km
2·Km
3A4
3A4
3A4
2D6
2D6
2D6
2D6
2B6
2C19
2C19
Buprenorphine
Zolpidem
Amitriptyline
Citalopram
Clozapine
Amitriptyline
MDMA
Atomoxetine
Amitriptyline
Atomoxetine
24.0
33.8
38.0
76.9
76.5
58.9
39.3
59.4
47.1
21.8
15.1
22.9
17.9
60.6
59.8
38.2
21.7
46.3
32.2
15.3
8.5
15.5
9.8
41.9
43.0
23.6
14.2
36.7
21.6
11.6
24.9
18.6
42.9
46.5
46.6
48.3
76.4
32.4
12.0
9.1
16.0
15.3
24.7
43.6
30.2
61.4
22.9
21.1
9.8
12.6
9.2
8.1
32.3
31.0
38.0
13.8
10.9
8.7
6.6
4.1
IC50 Inhibitor concentration that yields 50% of the control activity, Km Michaelis Menten constant: the substrate concentration that yields a
reaction velocity that is half of maximum
a Amitriptyline (3A4) Km=92.0 μM, amitriptyline (2D6) Km=14.7 μM, amitriptyline (2C19) Km=13.45 μM, atomoxetine (2B6) Km=29.0 μM,
atomoxetine (2C19) Km=96.0 μM, buprenorphine (3A4) Km=21.2 μM, citalopram (2D6) Km=20.1 μM, clozapine (2D6) Km=15.0,
methylenedioxymethamphetamine (MDMA) (2D6) Km=4.1 μM, zolpidem (3A4) Km=140.0 μM (Drug Interaction Database, University of
Washington [15])

824
Eur J Clin Pharmacol (2007) 63:821–827
Fig. 1 Dixon plot of the revers-
ible inhibitors amitriptyline and
methylenedioxymethamphet-
amine (MDMA) [17]. EDDP
formation in the presence of the
respective inhibitor is expressed
as nanograms per millilitre per
minute versus inhibitor concen-
tration expressed as a multiple
of its IC50 value, methadone
Km=2.98 μM, amitriptyline
Ki amitriptyline (CYP2D6)
0,003
0,0025
0,002
0,0015
0,001
0,0005
0
IC50=9.48 μM, MDMA IC50
=
-1
-0,8
-0,6
-0,4
-0,2
-0,0005
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
2,2
1.46 μM. Each point is the mean
of values determined in four
separate experiments
multiple of IC50 [µM]
1/2 Km according to S1 1 Km according to S2 2 Km according to S3
Ki MDMA (CYP2D6)
0,005
0,004
0,003
0,002
0,001
0
-0,8
-0,6
-0,4
-0,2
-0,001
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
2,2
-0,002
multiple of IC50 [µM]
1/2 Km according to S1 1 Km according to S2 2 Km according to S3
by amitriptyline (23.6% of control velocity at the highest
inhibitor concentration) and MDMA (14.2% of control
velocity at the highest inhibitor concentration). Both sub-
stances demonstrated reversible inhibition. Citalopram and
clozapine revealed moderate inhibition potency.
The most dominant inhibition of CYP2B6 at maximum
inhibitor concentration was caused by atomoxetine after
preincubation (10.9% of control velocity at the highest
inhibitor concentration). Nicotine and buprenorphine did not
show any important inhibitory influence, while amitriptyline
displayed moderate inhibition potency.
CYP3A4 and CYP2D6 and demonstrated reversible inhibi-
tion with apparent IC50 values of 32.43 and 9.48 μM,
respectively. MDMA was also tested via CYP2D6 with a
reduced IC50 value of 1.46 μM. Buprenorphine and zolpidem
inhibited methadone N-demethylation via CYP3A4 with
apparent IC50 values of 3.31 and 34.85 μM. As expected,
the IC50 value of zolpidem (9.24 μM) was lower following a
30-min preincubation period.
Ki values
Following a 30-min preincubation period, amitriptyline
and atomoxetine demonstrated a strong inhibitory effect on
CYP2C19. Only a moderate inhibitory potential was indicated
for zolpidem, clozapine, and citalopram.
The inhibition constant Ki was determined for selected
inhibitors suggesting no mechanism-based inhibition of
EDDP formation. Ki values for amitriptyline were 12.85
For CYP1A2, amitriptyline was the only substance
reasonably inhibiting N-demethylation of methadone (about
67% of control velocity at the highest inhibitor concentration,
data not shown).
Table 2 Data for the Kitz-Wilson plot of zolpidem [18]
Inhibitor concentration (μM)
Linear equation
R
20
35
70
140
y=−0.019x+0.906
y=−0.021x+0.873
y=−0.024x+0.750
y=−0.029x+0.690
0.952
0.989
0.950
0.940
IC50 values
Four drugs were chosen to further specify the kinetic
parameters of inhibition. Amitriptyline was tested via
R Coefficient of correlation

Eur J Clin Pharmacol (2007) 63:821–827
Kitz-Wilson plot of zolpidem
825
in the inhibition of methadone metabolism (CYP3A4) by
zolpidem. The mechanism-based inhibition process may
cause a severe increase in the AUC of methadone. EDDP
formation via CYP3A4 was also inhibited by buprenor-
phine and amitriptyline, and both amitriptyline and MDMA
inhibited conversion of methadone via CYP2D6.
100
80
60
40
20
0
y = 379,02x + 34,277
R = 0.951
The in vivo interpretation of kinetic constants is difficult
because there are no standardised methods available in the
literature to date. Approaches to predict pharmacokinetic drug
interactions based on in vitro data continue to be controversial
[19]. The potential for interaction is high when concomitant
drugs are metabolized by the same enzyme. The present
results are restricted to enzyme-selective activities for
inhibition. However, delineation of drug interaction through
an in vitro approach is considered to be helpful in patients on
unusual high-dose regimens or co-medication or with a high
risk of illicit drug use, and where evidence of metabolic
interaction is inevitably restricted to case reports.
The IC50 value is a relative value that depends on the
individual substrate concentration. It can be used to screen
for the presence of a mechanism-based component in
inhibition and to directly compare the inhibition potency
among tested inhibitors in one experiment.
-0,2
-0,15
-0,1
-0,05
0
0,05
0,1
0,15
0,2
-20
-40
inhibitor concentration-1 [µM-1]
Fig. 2 Kitz-Wilson plot of zolpidem [18], a represents the gradient of
the linear equations. For data see Table 2
1.49 μM (CYP3A4) and 3.52 0.40 μM (CYP2D6). A
significantly reduced value of 0.23 0.07 μM was obtained
for MDMA inhibition of CYP2D6. As an example, the
Dixon plots for amitriptyline and MDMA (CYP2D6) are
shown in Fig. 1. Ki values of 2.46 0.12 and 61.08
3.25 μM were determined for CYP3A4 inhibition by
buprenorphine and zolpidem respectively.
KI and kinact values
KI and kinact were determined via the Kitz-Wilson plot. A
mechanism-based component in the inhibition of CYP3A4
activity was evident for zolpidem (Table 2 and Fig. 2) with KI
and kinact values of 11.06 μM and 0.02917 min−1 respectively.
In Table 3, kinetic constants obtained from all potential
inhibitors are summarised.
Ki theoretically reflects the affinity of an inhibitor towards
the enzyme and is not dependent on substrate concentration.
Venkatakrishnan et al. [19] used Eq. (1) for an estimation of
the change in the AUC due to an inhibitor that is not
believed to operate via a mechanism-based inhibition:
Vmax
AUCI
AUC
CL
½Iꢀ
½SꢀþKm
¼
¼
¼ 1 þ
ð1Þ
Vmax
ꢀ
ꢁ
CLI
Ki
Discussion
½Iꢀ
½SꢀþKm
ꢁ
1þKi
Preceding experiments revealed a major involvement of
CYP3A4 and CYP2D6 in methadone metabolism. We
present data consistent with a mechanism-based component
where AUC is the area under the concentration versus time
curve without and with (AUCI) administration of an
Table 3 Summary of mean kinetic constants (IC50, Ki, KI, kinact) of the most potent inhibitors on EDDP formation
Inhibitor
IC50 (μM) (n=2, range <10%)
Ki (μM) (n=4, mean SD)
KI (μM), kinact (min−1) (n=4, R=0.95)
Amitriptyline
CYP3A4
CYP2D6
32.43
9.48
12.85 1.49
3.52 0.40
Buprenorphine
CYP3A4
MDMA
CYP2D6
Zolpidema
3.31
1.46
2.46 0.12
0.23 0.07
CYP3A4
Without preincubation
With preincubation
34.85
9.24
61.08 3.25
KI=11.06, kinact=0.02917
IC50 Inhibitor concentration that yields 50% of the control activity, Ki inhibition constant, KI inactivator concentration at which the inactivation
rate is half of maximum, kinact enzyme inactivation rate constant, SD standard deviation, R coefficient of correlation
a A mechanism-based component was evident for zolpidem

826
Eur J Clin Pharmacol (2007) 63:821–827
inhibitor, CL is the clearance without and with (CLI)
administration of an inhibitor, Vmax is the maximum
reaction velocity, S the substrate concentration, Km the
Michaelis Menten constant, I the inhibitor concentration,
and Ki the inhibition constant.
as well as benzodiazepines. In a study by Plummer et al.
[24], a decreased methadone clearance was observed with
co-administered amitriptyline. The present results lend
some support to explaining these findings. Amitriptyline
may cause a decreased clearance of methadone due to
inhibition of multiple CYPs. Involvement of multiple CYPs
is a critical consideration in drug-drug interaction studies.
Milroy and Forrest [25] reported on a methadone-
associated death together with MDMA also present in
blood. Because of the severe in vitro interaction caused by
MDMA, monitoring of patients using stimulant agents
seems important.
In 9 out of 35 post-mortem examinations which were
positive for methadone, buprenorphine could be detected
simultaneously [26]. Most fatalities linked to methadone
resulted from overdose or the association of methadone
with other drugs. Although not irrefutably proven, the
potential for pharmacokinetic interaction between metha-
done and buprenorphine should be considered.
Reports on adverse or fatal events with co-ingestion of
zolpidem are not available at present. The in vitro
interaction of zolpidem and methadone should be taken
into account in both the choice of prescription and in any
comparison of methadone toxicity.
Although the authors are quite aware of the limitations
of in vitro-in vivo scaling and the restriction of the results
on enzyme-selective activities for inhibition, the present
initial findings should be able to indicate which drugs are
associated with a potentially higher risk during methadone
maintenance treatment. Also, further in vitro studies, e.g.
approaches involving relative activity factors, seem neces-
sary to refine and extend the results obtained in the present
investigation [27].
Interactions are considered to be likely with values
greater than 2, possible between 1.1 and 2 and unlikely
with values lower than 1. According to Eq. (1), buprenor-
phine and zolpidem did not significantly increase the AUC
of methadone. In the upper therapeutic range (0.05–0.30 μg
amitriptyline/mL plasma), amitriptyline is expected to cause
a small change in the AUC of methadone (AUCI/AUC:
1.01–1.08) via CYP3A4. In contrast, both amitriptyline and
MDMA may affect metabolic clearance of methadone via
CYP2D6. So, interactions seem possible with amitriptyline
(AUCI/AUC: 1.05–1.30) at therapeutic concentrations and
are very likely with MDMA (AUCI/AUC: 3.25–8.88) after
average single doses (0.10–0.35 μg MDMA/mL plasma).
In addition to reversible inhibition, mechanism-based
inhibition was observed for some drugs under investigation.
In most cases, these kinds of inhibition lead to CYP
inactivation or complexation [20]. Knowledge of time-
dependent inhibition due to mechanism-based processes is
of great concern, as many drugs that cause profound drug-
drug interactions do so by this mechanism [19]. It is
assumed that prolonged or repeated in vivo exposure to
mechanism-based enzyme inactivators may result in a more
distinct inhibition [21]. For prediction of the magnitude of a
mechanism-based inhibition, the following equation has
been proposed:
ꢀ
ꢁ
½Iꢀꢁkinact
½IꢀþKI
kdeg rad
kdeg rad
þ
AUCI
AUC
CL
¼
¼
ð2Þ
CLI
Acknowledgements We appreciate the kind assistance of Ms.
Hoenekopp and her team from a local maintenance treatment centre
in Mannheim, Germany.
where AUC is the area under the concentration versus time
curve without and with (AUCI) administration of an
inhibitor, CL is the clearance without and with (CLI)
administration of an inhibitor, kdegrad is the degradation rate
constant for protein in vivo, I is the inhibitor concentration,
kinact the enzyme inactivation rate constant, and KI the
inactivator concentration that yields half the maximum rate
of inactivation.
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