Methadone metabolism by human placenta

Article abstract of DOI:10.1016/j.bcp.2004.04.011

Methadone pharmacotherapy is considered the standard for treatment of the pregnant heroin/opioid addict. One of the factors affecting the transfer kinetics of opioids across human placenta and their levels in the fetal circulation is their metabolism by t

Full text of DOI:10.1016/j.bcp.2004.04.011

Biochemical Pharmacology 68 (2004) 583–591
Methadone metabolism by human placenta
Tatiana N. Nanovskaya, Sujal V. Deshmukh, Ilona A. Nekhayeva,
*
Olga L. Zharikova, Gary D.V. Hankins, Mahmoud S. Ahmed
Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555 0587, USA
Received 12 February 2004; accepted 16 April 2004
Abstract
Methadone pharmacotherapy is considered the standard for treatment of the pregnant heroin/opioid addict. One of the factors affecting
the transfer kinetics of opioids across human placenta and their levels in the fetal circulation is their metabolism by the tissue. The aim of
this investigation is to identify the enzyme(s) responsible for the metabolism of methadone, determine the kinetics of the reaction and the
metabolites formed utilizing placental tissue obtained from term healthy pregnancies. Microsomal fractions of trophoblast tissue
homogenates had the highest activity in catalyzing the metabolism of methadone. The product formed was identified by HPLC-UVas 2-
ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Inhibitors selective for cytochrome P450 (CYP) isozymes were used to identify
the enzyme catalyzing the biotransformation of methadone. Aminoglutethimide and 4-hydroxyandrostenedione inhibited EDDP
formation by 88 and 70%, respectively, suggesting that CYP19/aromatase is the enzyme catalyzing the reaction. This was confirmed
by the effect of monoclonal antibodies raised against CYP19 that caused an 80% inhibition of the reaction. The apparent Km and V
ꢁ
max
ꢁ1
1
values for the CYP19 catalyzed metabolism of methadone to EDDP were 424 ꢀ 92 mM and 420 ꢀ 89 pmol (mg protein) min
,
respectively. Kinetic analysis of a cDNA-expressed CYP19 for the metabolism of methadone to EDDP was identical to that by placental
microsomes. Taken together, these data indicate that CYP19/aromatase is the major enzyme responsible for the metabolism of methadone
to EDDP in term human placentas obtained from healthy pregnancies.
#
2004 Elsevier Inc. All rights reserved.
Keywords: Human placenta; Aromatase; Methadone; Metabolism; Pregnancy
1
. Introduction
pyrroline (EMDP) as shown (Fig. 1). Both EDDP and
EMDP do not have analgesic activity [1]. Cytochrome
P450 (CYP) 3A4 was identified as the major enzyme
catalyzing the metabolism of methadone in human
liver and intestine though the involvement of other CYP
isoforms (namely CYP2D6, CYP1A2, CYP2C9, and
CYP2C19) was not ruled out [2–5].
Several reports indicated that the metabolism of metha-
done in pregnant women is different from the non-preg-
nant. The differences were attributed to the changes in
maternal physiology that accompany the onset of preg-
nancy to accommodate the growth and development of the
feto-placental unit. During pregnancy, the placenta func-
tions as an additional extra-hepatic site for biotransforma-
tion of drugs though the amount and activity of its
metabolic enzymes is lower than that in the liver [6–9].
Therefore, the metabolic activity of the placenta could
contribute to the changes in the pharmacokinetic-pharma-
codynamic profile (bio-disposition) of drugs administe-
red to the pregnant women. Indeed, methadone plasma
Methadone is the only approved pharmacotherapy for
treatment of the pregnant heroin/opioid addict. Its use,
since the late 1960s, has been the subject of numerous
clinical trials, reports, and review articles that, for the most
part, agree on the benefits of methadone maintenance
programs in improving maternal and neonatal outcome.
A review of this literature would be out of the scope of
this manuscript.
Methadone is a synthetic, long-acting m-agonist that
undergoes first pass metabolism by its sequential N-
demethylation to 2-ethylidene-1,5-dimethyl-3,3-diphenyl-
pyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-
Abbreviations: EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrroli-
dine; EMDP, 2-ethyl-5-methyl-3,3-diphenylpyrroline; CYP, cytochrome
P450; LAAM, L-a-acetylmethadol; TCA, trichloroacetic acid; BUP,
buprenorphine
*
Corresponding author. Tel.: þ1 409 772 8708; fax: þ1 409 747 1669.
E-mail address: maahmed@utmb.edu (M.S. Ahmed).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.04.011

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T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
Fig. 1. The structure of the products formed by the sequential N-demethylation of methadone.
concentrations during the second and third trimesters were
lower than after delivery and elimination of methadone
in pregnant patients was significantly more rapid than in
the non-pregnant [10,11]. These reports suggested that
biotransformation of methadone during pregnancy may
be accelerated as a result of enhanced maternal and fetal
hepatic metabolism and (or) biotransformation of the drug
by the placenta. As a result, the dose of methadone during
pregnancy may have to be adjusted to enhance compliance
and safety of the mother and newborn [10,11].
Pregnant women in methadone maintenance programs
may require therapeutic interventions due to infections or
other pregnancy-associated complications that dictate con-
sideration of drug interactions. Changes in methadone
clearance were observed in patients treated with cimeti-
dine, erythromycin, ketoconazole, rifampin, and barbitu-
rates [12,13]. Other sites for drug interactions can include
key enzymes in placental biosynthesis of steroid hormones
namely, CYP11A1 and CYP19, that are also involved in
the metabolism of xenobiotics [14,15], buprenorphine, and
L-a-acetylmethadol (LAAM) [16,17].
Taken together, these reports emphasize the need for
information on the enzyme metabolizing methadone in
human placenta and the products formed. This information
will also allow a comparison of placental bio-disposition of
methadone and buprenorphine as the latter is emerging as
an alternative to treatment of the pregnant opioid-depen-
dent woman. Therefore, the goal of this investigation is to
identify and characterize the enzyme responsible for the
metabolism of methadone in term human placenta.
previously reported [18]. The cDNA-expressed CYP19
‘‘supersomes’’ is commercially available from Gentest,
and their characterization was described earlier [19].
2.2. Human placentas and derived preparations
Full-term human placentas from uncomplicated preg-
nancies were obtained from the labor and delivery ward
at the University of Texas Medical Branch Hospital,
Galveston, Texas, according to a protocol approved by
the Institutional Review Board. Placentas of women who
abused drugs during pregnancy were excluded from this
investigation.
Approximately 40–50 g of villus tissue was excised at
random from the placenta and rinsed with cold saline. The
tissue was then minced and homogenized in two volumes
of cold 0.1 M potassium phosphate buffer, pH 7.4 using a
Tekmar SDT-1810, Tissumizer. The homogenate was sub-
jected to differential centrifugation to prepare the follow-
ing subcellular fractions: mitochondrial, 10,000 ꢂ g pellet;
microsomal, 104,000 ꢂ g pellet; and the supernatant for
the cytosolic. The mitochondrial and microsomal pellets
were resuspended in 0.1 M potassium phosphate buffer
(pH 7.4). Protein content of the preparations was deter-
mined by a kit from Bio-Rad (Bio-Rad laboratories) using
bovine serum albumin as a standard. Aliquots of the
subcellular fractions were stored at ꢁ70 8C until analyses
were performed within 2–3 weeks, and the enzymatic
activity was not affected under these storage conditions.
2.3. Methadone metabolism
2
. Materials and methods
The activity of placental mitochondrial, microsomal,
and cytosolic fractions in catalyzing the N-demethylation
of methadone to EDDP was determined. Unless otherwise
stated, the reaction components contained 100 mM
potassium phosphate buffer, pH 7.4, 1 mg protein of the
subcellular fraction and methadone at its final concentra-
tion of 1.25 mM (i.e., approximately 3ꢂ of its apparent
2
.1. Chemicals and other supplies
All chemicals were purchased from Sigma Chemical Co.
unless otherwise mentioned. Acetonitrile was purchased
from Fisher Scientific Co. Methadone, its metabolites, and
L-a-acetylmethadol (LAAM) were a gift from the National
Institute on Drug Abuse drug supply unit. Monoclonal
antibodies to CYP isoforms were a generous gift from Dr.
Andrew Parkinson (Xenotech LLC). Rabbit antiserum to
human placental aromatase was purchased from Haupt-
man–Woodward Institute. Properties of the antibodies and
their effect on the activity of placental aromatase have been
K ) in a total volume of 1 mL. The addition of the
m
subcellular fraction to the components initiated the pre-
incubation period of 5 min at 37 8C and the reaction was
initiated by adding the NADPH-regenerating system made
of the following: 0.4 mM NADP, 4 mM glucose-6-phos-
phate, 1 U/mL glucose-6-phosphate dehydrogenase, and
2 mM MgCl . This system contains glucose-6-phosphate
2

T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
585
dehydrogenase that will convert, in presence of glucose-6-
þ
2.4.2. Inhibition of EDDP formation by monoclonal
antibodies to CYP isozymes
phosphate, the NADP formed to NADPH required for the
oxidase enzyme. The reaction components were then
shaken at 37 8C for 20 min and reaction was terminated
by the addition of 100 mL of 15% (w/v) trichloroacetic
acid (TCA) containing 1 mg/mL LAAM as an internal
standard. The reaction components were then centrifuged
at 12,000 ꢂ g for 10 min and the amounts of EDDP formed
was analyzed by HPLC as described below. The conversion
of EDDP to EMDP was determined under identical experi-
mental conditions. The control reaction was carried out
utilizing TCA-denatured placental fraction. The effect of
methadone, in the concentration range of 50–2000 mM, on
reaction velocity was used to construct the saturation curve
used to calculate the apparent Km and Vmax values. The
remaining experimental conditions ensured that the rate of
methadone N-demethylation was linear with protein con-
centration and incubation time.
Monoclonal antibodies against human liver CYP1A2,
2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1 and 3A4, and rabbit
antiserum to human placental aromatase were used to
identify the CYP isozyme responsible for the metabolism
of methadone by placental microsomes. Each monoclonal
antibody was added to the reaction components at its
concentration causing 80% inhibition of the CYP isoform
it was raised against. Microsomal protein, 0.2 mg, was pre-
incubated with the antibody at room temperature for
15 min followed by the addition of methadone at its final
concentration of 1.25 mM. The addition of the NADPH-
regenerating system initiated the reaction that was incu-
bated for 20 min at 37 8C then terminated by the addition
of TCA. In the control reaction, mouse IgG replaced the
monoclonal antibodies.
2.5. Metabolism of methadone by a cDNA-expressed
CYP19 preparation
2.4. Identification of the enzyme catalyzing the
metabolism of methadone
The kinetics for a cDNA-expressed CYP19 preparation
for catalysis of the N-demethylation of methadone to
EDDP was determined. The concentration of CYP19
was 40 pmol/mL of reaction volume. The effect of a range
of methadone concentrations between 50 and 2000 mM on
the velocity of EDDP formation was used to obtain the
saturation curves necessary to calculate the apparent Km
and Vmax for the reaction. The incubation conditions were
identical to those mentioned above for determining the
kinetics for N-demethylation of methadone by placental
microsomal fractions. The rates of EDDP formation
obtained were expressed as pmol of EDDP/pmol of
CYP19.
Identification of the enzyme was achieved by utilizing
1) chemicals known as selective inhibitors of CYP
(
isoforms and (2) monoclonal antibodies raised against
purified human liver CYP isoforms.
2.4.1. Effect of chemical inhibitors on the
biotransformation of methadone to EDDP
The effect of inhibitors selective for the different CYP
isoforms on EDDP formation by microsomal fractions was
determined by adding each to the reaction components.
The final concentration of methadone in the reaction
mixture was 500 mM, i.e., approximately equal to its
apparent Km value of 424 mM. The concentration used
for each inhibitor was that equal to its reported IC , K , or
5
0
i
2.6. The activity of placental CYP19/aromatase
apparent Km values for a specific CYP isoform [20–22].
The following are the inhibitors used and the name of each
is followed by its concentration and the name of the
isozyme that it is selective for: a-naphthoflavone
The activity of CYP19/aromatase in catalyzing the
conversion of testosterone to17b-estradiol was determined
using placental mitochondrial and microsomal fractions.
The following reaction components were pre-incubated for
5 min at 37 8C: 250 mg of protein (mitochondrial or micro-
somal) and 1.0 mM testosterone in a final volume of 1 mL
of 0.1 M potassium phosphate buffer. The reaction was
initiated by the addition of NADPH-regenerating system,
incubated for 5 min and terminated by the addition of
100 mL of 10% (w/v) TCA. Estrone, 100 mL of a 10 mg/
mL solution, was added to the reaction components as an
internal standard, centrifuged at 12,000 ꢂ g for 10 min and
the pellet discarded. The amount of 17b-estradiol in the
supernatant was determined by HPLC as described below.
(0.1 mM), CYP1A; sulfaphenazole (10 mM), CYP 2C;
quinidine (5 mM) CYP2D6; 4-methylpyrazole (25 mM),
CYP2E1; nifedipine (200 mM), CYP3A4; ketoconazole
(
(
0.5 and 2.5 mM), CYP3A4; 4-hydroxyandrostenedione
1 and 10 mM), CYP19; and aminoglutethimide (1 and
1
0 mM) CYP19. Each inhibitor was simultaneously
added with methadone to the reaction components and
pre-incubated for 5 min at 37 8C. The NADPH-regener-
ating system was added to initiate the reaction, and the
incubation was continued for another 20 min until ter-
minated by the addition of 15% (w/v) TCA. All stock
solutions of the inhibitors were made in methanol. The
maximum final concentration of methanol in the reaction
was 0.5%. The control reaction included all components
except for the inhibitor and in presence of 0.5% methanol
2.7. Western blot analysis of aromatase
Microsomal and mitochondrial fractions were prepared
as described above. The amount of protein loaded for all
(
v/v).

586
T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
samples was 10 mg/well. Resolution of the samples was
achieved using a 12% SDS–PAGE and electro-transferred
to nitrocellulose membranes overnight at 4 8C using
a constant potential of 25 V. The blots were probed, at
room temperature, with a rabbit polyclonal antiserum
against human aromatase (Hauptman–Woodward Medical
research Institute), at a dilution of 1:2000 for 90 min. Goat
anti rabbit IgG conjugated to horseradish peroxidase
Statistical analysis of the data on the effect of inhibitors
on methadone metabolism was carried out using one-way
ANOVA with Tukey’s comparison and deemed significant
if the P value was <0.05.
3. Results
(Upstate) was used as secondary antibody at a dilution
3.1. Methadone metabolite(s)
of 1:5000. Detection was achieved by an enhanced che-
miluminescence system (Pierce), and the resulting signal
was quantitated by spot densitometry using a digital
imaging system (Alpha Innotech Corp.). Microsomes
of cDNA-expressed aromatase (Gentest) were used as
standards.
Separation of methadone and its metabolite EDDP was
achieved by HPLC according to the method described. A
chromatogram showing the retention times of the opioid
and its metabolite is presented in Fig. 2A. Aliquots of
the reaction supernatant containing methadone and the
2
.8. HPLC-UV
The HPLC systems used was made of a Waters 600E
multi-solvent delivery system with a Waters 2487 dual
wavelength absorbance detector and a Waters 717 auto-
sampler and were controlled by a Waters Millennium
chromatography manager (Waters). The stationary
phase was a 250 mm ꢂ 4:6 mm Luna 5 mm C-18 column
(Phenomenex).
2
.8.1. Identification of EDDP
Separation and identification of EDDP was achieved by
the method described by Iribarne et al. [4]. Briefly, the
mobile phase was made of acetonitrile/water 35/65, v/v,
containing 1% (v/v) triethylamine, and the pH was
adjusted to 2.8 with orthophosphoric acid. Elution was
at a flow rate of 1 mL/min and was monitored at a
wavelength of 210 nm. Chromatographic peaks were iden-
tified by their retention times and compared to that of the
standard compounds. The quantity of EDDP formed was
determined using a standard curve made of six concentra-
tions ranging between 0.25 and 2.0 mg/mL.
2
.8.2. Determination of estradiol
The amount of 17b-estradiol formed was determined by
the method described earlier [23]. The mobile phase was
made of 0.1% triethylamine in acetonitrile/water (45:55,
v/v), and the pH was adjusted to 3.5 by orthophosphoric
acid. Isocratic elution was at a flow rate 1.2 mL/min and
the eluent monitored at a wavelength of 280 nm. All
calculations were based on the ratio of estradiol peak area
to that of the internal standard (estrone).
2
.9. Data analysis
All data are represented as mean ꢀ S:D. Values for
Fig. 2. (A) An HPLC chromatogram for the retention times of standards:
(
1) EDDP (14 min); (2) methadone (18 min); (3) LAAM (24.5 min). The
the apparent K and V
m
were calculated from the satura-
max
latter compound is used as an internal standard. A chromatogram of the
reaction components: (B) in absence and (C) in presence of the NADPH-
regenerating system. EDDP is the only metabolite formed in presence of
the co-enzyme (C).
tion curves using Michaelis–Menten equation and non-
linear regression (SPSS 11.0 for windows software, SPSS
Inc.).

T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
587
microsomal fraction in absence (Fig. 2B) and presence
Fig. 2C) of the NADPH-regenerating system are shown.
(
The chromatogram revealed the formation of the metabo-
lite EDDP (14 min) in the presence of NADPH suggesting
that a CYP isozyme might be catalyzing the reaction. The
metabolite EMDP was not detected under this experimen-
tal conditions. Also, EMDP was not detected when EDDP
was used as a substrate instead of methadone.
The catalytic activity of the mitochondrial and cytosolic
fractions was compared to the microsomal. Eight placentas
were used for this comparison and equal protein con-
centration of each fraction was utilized. Data obtained
revealed that the highest rates for N-demethylation of
methadone was in the microsomal fraction (432 ꢀ 176
ꢁ
1
ꢁ1
pmol (mg protein) min ) followed by the mitochondrial
ꢁ
1
ꢁ1
(
(
252 ꢀ 128 pmol (mg protein) min ) and the cytosolic
ꢁ ꢁ1
1
58 ꢀ 20 pmol (mg protein) min ).
3
.2. Methadone metabolism by microsomal preparations
Fig. 4. The variations observed in the reaction velocity for N-demethyla-
tion of methadone by microsomal preparations from 38 term placentas.
Preliminary data on the reaction conditions revealed
that the amount of EDDP formed was linear with protein
concentration and time (up to 3 mg/mL and 20 min,
respectively). The rate of EDDP formation was depen-
dent on methadone concentration and exhibited typical
Michaelis–Menten saturation kinetics (Fig. 3). Analysis
of the hyperbolic saturation curves obtained from seven
microsomal preparations revealed a mean apparent Km
and Vmax values of 424 ꢀ 92 mM and 420 ꢀ 89 pmol
enzyme kinetics in the range of methadone concentrations
tested.
TheN-demethylation activity of microsomal preparations
from 38 placentas ranged between 184 and 839 pmol
ꢁ
1
ꢁ1
(mg protein) min (Fig. 4). However, the distribution
ꢁ
1
ꢁ1
of the enzyme activity, in pmol (mg protein) min , was
as follows: <250, 10% of the preparations; 250–500, 82%;
and >500, 8%. This range of activity could be attributed to
the variations between placentas obtained from different
women (interplacental variations).
ꢁ
1
ꢁ1
(
mg protein) min , respectively.
Eadie–Hofstee plot of the data (Fig. 3, insert) indicated
that N-demethylation of methadone exhibited monophasic
3.3. Inhibition of methadone N-demethylation
The effect of compounds that are selective inhibitors of
CYP isoforms on EDDP formation was investigated in five
placental preparations. In each reaction, the final concen-
trations of methadone was 500 mM and each inhibitor was
1
0ꢂ its reported IC value for its specific CYP isozyme.
5
0
The maximum inhibition of EDDP formation was observed
in presence of 10 mM aminoglutethimide (88% of control)
and 4-hydroxyadrostenedione (70% of control) (Fig. 5).
Both compounds are selective for CYP19 and the inhibi-
tion was statistically significant (P < 0:001). On the other
hand, the following compounds, at their concentrations
shown in Fig. 5, did not affect EDDP formation: quinidine
(
CYP2D6), sulfaphenazole (CYP2C9), a-naphthoflavone
(
CYP1A), 4-methylpyrazole (CYP2E1), ketoconazole, and
nifedipine (CYP3A). These data suggest that CYP19 is the
microsomal enzyme catalyzing the biotransformation of
methadone to EDDP.
Antibodies raised against human CYP isoforms were
utilized to confirm the identity of the microsomal enzyme
catalyzing the biotransformation of methadone to EDDP. A
pool of microsomal fractions prepared from 12 placentas
Fig. 3. A plot of the relation between increasing methadone concentrations
and the rate of methadone metabolism by placental microsomes indicates
m
saturation kinetics. The apparent K and Vmax values calculated from the
ꢁ
1
saturation curve were 424 ꢀ 92 mM and 420 ꢀ 89 pmol (mg protein)
ꢁ1
min . (Inset) Monophasic kinetics was confirmed using Eadie–Hofstee
plots of reaction velocity (v) against v/[S].

588
T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
Fig. 6. The effect of antibodies raised against human CYP450 isozymes
on the N-demethylation of methadone by placental microsomes. The rate
of metabolite formation was expressed as percentage of control (absence of
antibodies). The values are given as mean ꢀ S:D: of three to six
experiments. Antibodies raised against CYP19 caused >80% inhibition
of EDDP formation.
Fig. 5. The effect of chemical inhibitors on methadone N-demethylation
by placental microsomes. The inhibitors are: quinidine, QN; sulfaphena-
zole, SF; a-naphthoflavone, a-NP; 4-methylpyrazole, 4-MP; ketoconazole,
KT; nifedipine, NF; aminoglutethimide, AG; and 4-hydroxyandrostene-
dione, 4-OH. The final concentration of methadone in the reaction was
5
m
00 mM, i.e., equal to its apparent K . The rates of metabolite formation
the following values, 334 ꢀ 91 mM and 6:3 ꢀ 2:3 pmol
are expressed as percent of control (absence of inhibitors) and represent the
mean ꢀ S:D: of three to six experiments. Only AG and 4-OH-A caused a
significant inhibition of methadone N-demethylation.
ꢁ1 ꢁ1
pmol CYP19) min , respectively. This apparent K
m
(
value is similar to that determined for the placental micro-
somal fractions catalyzing the same reaction.
was utilized in these experiments and each antibody was
tested in triplicates. Antibodies to CYP19 were the most
potent and caused an 80% inhibition of EDDP formation.
Antibodies against 2B6, 2C8 and 2C19 caused <20%
inhibition, while the effect of the remaining antibodies
did not reveal any effect on EDDP formation (Fig. 6).
Therefore, compounds that are selective inhibitors of
CYP19 and antibodies raised against the same isozyme
caused the most pronounced inhibition of methadone
metabolism to EDDP. Taken together, these data strongly
suggest that CYP19 is the enzyme catalyzing the metabo-
lism of methadone.
Taken together, three different approaches to the identi-
fication of the enzyme catalyzing the metabolism of
methadone indicated that it is CYP19/aromatase.
3.5. Aromatization of testosterone to estradiol by
placental subcellular fractions
The activity of CYP19/aromatase in catalyzing the
aromatization of testosterone (its natural substrate) to
estradiol in microsomal and mitochondrial fractions pre-
pared from nine placentas was determined. The mean ratio
of mitochondrial to microsomal CYP19 activity was
0
:52 ꢀ 0:2. The N-demethylation of methadone by the
3.4. Kinetic constants for N-demethylation of
methadone by cDNA-expressed CYP19
same placental preparations was determined, and the mean
activity ratio for the same fractions was 0:43 ꢀ 0:16.
CYP19 protein expression in the mitochondrial and
microsomal fractions from nine placentas was compared
to that of the cDNA-expressed isozyme using Western
blots (data for four placentas are shown in Fig. 7). The
The saturation curves for the N-demethylation of metha-
done to EDDP by the cDNA-expressed CYP19 were
used to calculate the apparent Km and Vmax and reveled
Fig. 7. The expression of microsomal and mitochondrial aromatase as revealed by Western blots. In general, the intensity of the bands corresponding to
aromatase in the mitochondrial preparations were approximately half that of the microsomal.

T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
589
including the pregnant. However, information on its bio-
disposition by human placenta is scarce to nonexistent.
This report provides information on the identification of
the major enzyme responsible for the metabolism of
methadone in term placentas obtained from healthy preg-
nancies without obstetrical complications.
Microsomal preparations from human liver catalyzed
the N-demethylation of methadone to EDDP but EMDP
was not detected by the analytical methods used [2–4]. The
concentration of EMDP, when present in human urine, was
reported as <5% of that for EDDP, leading to difficulties in
its determination because of the detection limits for the
methods used [24–26]. The major enzyme responsible for
the metabolism of methadone by human liver microsomal
fractions was identified as CYP3A4 [2,4]. In human intes-
tine, the major enzyme responsible for the metabolism of
methadone to EDDP is also CYP3A4 but small amounts of
EMDP were detected and attributed to the increase in the
detection limits of the method used namely, liquid chro-
matography-mass spectrometry instead of HPLC-UV [27].
CYP3A4 was also responsible for the metabolism of the
opioids buprenorphine and LAAM in the human liver and
intestine [2,5,27–29]. However, in term human placenta,
CYP19 was identified as the major enzyme responsible for
the N-dealkylation of buprenorphine to norbuprenorphine
and the N-demethylation of LAAM to norLAAM [16,17].
In our initial experiments to identify the major enzyme
responsible for the metabolism of methadone in human
placenta, it was assumed that it could be a microsomal
CYP450 isozyme because it required the presence of
NADPH-regenerating system. Moreover, placental
CYP19 metabolized LAAM which is a congener of metha-
done [17]. The activity of microsomal preparations in
metabolizing methadone was determined in 38 term pla-
centas and revealed a range of four- to six-fold (Fig. 4). The
only metabolite detected under our experimental condi-
tions and utilizing the HPLC-UV method described was
EDDP (Fig. 2). EMDP was not detected in any of the
placental preparations, i.e., no sequential demethylation of
methadone was observed. Similar lack of detectable
sequential demethylation was reported for LAAM to nor-
LAAM only (no dinorLAAM) by placental microsomal
CYP19 [17]. It appears that placental metabolism of
methadone and its congener LAAM by CYP19 is different
from that reported for their hepatic metabolism by
CYP3A4. Another similarity between the metabolism of
methadone and that of its congener LAAM as well as the
opioid buprenorphine is the four- to five-fold range of
enzymatic activities between different placentas [16,17].
The effect of increasing concentrations of methadone on
the rate of EDDP formation exhibited hyperbolic Michae-
lis–Menten saturation kinetics. Analysis of the data
revealed an apparent Km of 424 ꢀ 92 mM. An Eadie–
Hofstee plot of the data indicated monophasic kinetics
and suggested that either a single enzyme is catalyzing the
biotransformation of methadone to EDDP (Fig. 3), or
Fig. 8. Correlation between N-demethylation of methadone and aromati-
zation of testosterone in (A) the microsomal and (B) the mitochondrial
preparations.
data revealed that aromatase expression in the mitochon-
drial fraction was half that of the microsomal.
An examination of the correlation between CYP19 activ-
ities in its demethylation of methadone to EDDP and its
conversion of testosterone to estradiol, in nine placental
2
microsomal and mitochondrial preparations revealed R
values of 0.87 and 0.85, respectively (Fig. 8A and B).
Taken together, these data indicate that CYP19/aroma-
tase is involved in the N-demethylation of methadone in
placental microsomal and mitochondrial fractions.
4
. Discussion
Methadone maintenance programs are considered the
standard for pharmacotherapy of the heroin/opioid addict,

590
T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
multiple enzymes with similar affinities to the substrate.
Similar monophasic kinetics for the metabolism of metha-
done by hepatic microsomes was reported [3]. However,
in human intestine, the metabolism of methadone by
microsomal CYP3A4 exhibited biphasic kinetics [27].
The intrinsic clearance of methadone by human placen-
approximately 0.5. Moreover, the expression of aromatase
in the mitochondrial fraction as determined by Western
blots was 50% of that in the microsomal fraction (Fig. 7).
These data indicate that aromatase activity is present in the
placental mitochondrial fractions and is approximately half
of that in the microsomal. In addition, the correlation
between the N-demethylation of methadone in the micro-
somal and mitochondrial fractions with the aromatization
ꢁ4
ꢁ1
tal microsomes was 8:5 ꢂ 10 ꢀ 2:6 mL (mg protein)
ꢁ
1
min and is one order of magnitude less than that reported
ꢁ
3
2
for intestinal microsomes (11:3 ꢂ 10 ꢀ 1:1 mL (mg
of testosterone to estradiol had an R value of 0.87 and
ꢁ1
ꢁ1
protein) min ) [27]. The latter clearance was compar-
able to that for hepatic microsomes and suggests that
placental metabolism of methadone is significantly less
than that by the liver and or intestine.
0.84, respectively (Fig. 8A and B). This correlation is
consistent with CYP19 being the enzyme catalyzing the
aromatization and the N-demethylation reactions.
The data, from our laboratory and as reported by others,
do not provide evidence for the presence of CYP19 in
placental mitochondria and our experimental conditions
cannot rule out contamination of both the microsomal
and mitochondrial fractions with each other. However, the
focus of our investigation was to identify the major placental
enzyme responsible for the metabolism of methadone rather
than its subcellular localization. Our ultimate goal is a better
understanding of the placental role in the bio-disposition of
the opioids used for treatment of the pregnant woman.
Data cited in this report on the metabolism of methadone
by CYP19 indicate its role as xenobiotic-metabolizing
enzyme in human placenta. Recent data indicated that a
concentration of 0.24 mg/L methadone in maternal circu-
lation, or more, is necessary to prevent fetal and maternal
withdrawal and that it can be achieved by a dose of the drug
ranging between 50 and 150 mg with occasional need for
higher doses during the third trimester [31]. Our data
revealed that the activity of term placental preparations
could vary by four- to six-folds in the rates of methadone
metabolism. This variation could be one of the contributing
factors for the increased metabolism of methadone during
pregnancy, especially during the third trimester, and
emphasizes appropriate methadone dosing to prevent
maternal and fetal/neonatal withdrawal. Moreover, it is
unclear whether the metabolism of methadone or BUP by
placental CYP19 could affect its role in placental biosynth-
esis of estrogens.
In this report, three experimental approaches were uti-
lized to identify the major placental enzyme responsible
for N-demethylation of methadone to EDDP namely; the
effect of chemical inhibitors selective for specific CYP
isoforms; the effect of monoclonal antibodies against
human CYP isoforms; and comparing the kinetics of the
reaction in placentas to that catalyzed by a cDNA-
expressed preparation of CYP19. Aminoglutethimide
and 4-hydroxyandrostenedione, at their concentrations that
are most selective for CYP19, were the most potent
inhibitors of EDDP formation (88 and 70% inhibition,
respectively). These data suggests that CYP19/aromatase
is the major placental enzyme responsible for N-demethy-
lation of methadone (Fig. 5).
The effect of monoclonal antibodies on the metabolism
of methadone by placental microsome revealed that those
raised against human placental CYP19 were the most
potent and caused approximately 80% inhibition of EDDP
formation (Fig. 6). Antibodies against CYP2B6, 2C8, and
2
2
C19 caused <20% inhibition while those against 1A2,
A6, 2C9, and 2E1, and 3A4/5 had no effect. The lack
of a total (100%) inhibition of CYP19 demethylation of
methadone to EDDP by either the chemical inhibitors or
the monoclonal antibodies suggests that CYP19 might not
be the only enzyme responsible for the metabolism of
methadone.
The apparent K values for methadone metabolism by
m
placental microsomal preparations and by the cDNA-
expressed CYP19 were similar. Taken together, the inhibi-
tion experiments and the similarity of the kinetics between
of the cDNA-expressed CYP19 and that of the placenta
indicate that aromatase is the major enzyme involved in the
N-demethylation of methadone in human placenta.
The activity of placental mitochondrial fraction in the N-
demethylation of methadone was approximately 50% of
the microsomal. A similar distribution for the activity of
CYP19 between placental microsomal and mitochondrial
fractions was reported earlier for the conversion of andro-
gens to estrogens [30]. The activity of placental mitochon-
drial and microsomal CYP19 in the aromatization of its
natural substrate testosterone to estradiol was determined.
The ratios for the enzymatic activities of the mitochondrial
to microsomal metabolism for both substrates were
In summary, CYP19/aromatase is a key enzyme in
human placental steroidogenesis specifically the aromati-
zation of androstenedione and testosterone to estrogens
[32]. The enzyme was also implicated in the biotransfor-
mation of xenobiotics [15,33]. Our laboratory, reported on
the metabolism of buprenorphine [16] and LAAM [17] by
placental CYP19 and the same enzyme also metabolizes
methadone. Taken together, it can be concluded that
CYP19/aromatase is the major enzyme responsible for
the metabolism of the three opioids in term human placenta.
Acknowledgments
The authors appreciate the support of the National
Institute on Drug Abuse drug supply program for providing

T.N. Nanovskaya et al. / Biochemical Pharmacology 68 (2004) 583–591
591
[
[
[
[
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Metabolic activation of aflatoxin B1 by human placental microsomes.
J Toxicol Sci 1993;18:129–32.
methadone, EDDP and, LAAM. We also thank Dr. Andrew
Parkinson of XenoTech LLC for his generous gift of
monoclonal antibodies. We greatly appreciate the assis-
tance of the medical staff, the Chairman’s Research Group,
and Publication, Grant, & Media Support Office of the
Department of Obstetrics and Gynecology, University
of Texas Medical Branch, Galveston, Texas. Supported
by a grant from the National Institute on Drug Abuse to
M.S.A. (DA-13431).
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enzyme metabolizing buprenorphine in human placenta. J Pharmacol
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17] Deshmukh SV, Nanovskaya TN, Hankins GDV, Ahmed MS.
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