Involvement of cytochrome P450 3A4 enzyme in the N-demethylation of methadone in human liver microsomes

Article abstract of DOI:10.1021/tx950116m

Methadone has become one of the most widely used drugs for opiate dependency treatment. This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man, yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethy

Full text of DOI:10.1021/tx950116m

Chem. Res. Toxicol. 1996, 9, 365-373
365
In volvem en t of Cytoch r om e P 450 3A4 En zym e in th e
N-Dem eth yla tion of Meth a d on e in Hu m a n Liver
Micr osom es
Christelle Iribarne,† Franc¸ois Berthou,*,† Susan Baird,‡ Yvonne Dre´ano,†
Daniel Picart,† J ean Pierre Bail,§ Philippe Beaune,‡ and J ean Franc¸ois Me´nez†
Laboratoires de Biochimie-Nutrition, EAD 948, Faculte´ de Me´decine, BP 815-29285,
Brest Cedex, France, and INSERM U-75, Universite´ Rene´ Descartes, F 73730,
Paris Cedex 15, France, and Service de Chirurgie Digestive, CHU Morvan, 29285 Brest, France
Received J une 27, 1995X
Methadone has become one of the most widely used drugs for opiate dependency treatment.
This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man,
yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethylidene-1,5-
dimethyl-3,3-diphenylpyrrolidine. The specific forms of cytochrome P450 involved in this
oxidative N-demethylation were examined in a panel of 20 human liver microsomal preparations
previously characterized with respect to their P450 enzyme contents. Methadone was
demethylated with an apparent Km of 545 ( 258 µM (n ) 3). The metabolic rates were 745 (
574 pmol/(min‚mg of protein). This metabolic pathway was strongly correlated with estradiol
2-hydroxylation, testosterone 6â-hydroxylation, nifedipine oxidation, erythromycin N-de-
methylation, and toremifene N-demethylation, all of these monooxygenase activities being
supported by P450 3A4. Furthermore, the total P450 3A content of liver microsomal samples,
determined by immuno-quantification using a monoclonal anti-human P450 3A4 antibody, was
correlated with methadone demethylation (r ) 0.72; p <0.003). Methadone metabolism was
60-72% inhibited either by three mechanism-based inhibitors of P450 3A4 (gestodene, TAO,
and erythralosamine) or by four reversible inhibitors of P450 3A (ketoconazole, dihydroer-
gotamine, quercetin, and diazepam with an apparent Ki of 50 µM) and by two nonspecific
inhibitors (metyrapone and SKF-525A). Conversely, quinidine (inhibitor of P450 2D6), 7,8-
benzoflavone (inhibitor of P450 1A), or sulfaphenazole (inhibitor of P450 2C) did not significantly
inhibit, and may even have activated, methadone metabolism. Four heterologously expressed
P450 proteins were able to catalyze the N-demethylation of methadone, namely, P450 2C8,
P450 2C18, P450 2D6, and P450 3A4. However, referring to their relative liver content, it
can be asserted that P450 3A4 is the major enzyme involved in the N-demethylation of
methadone on average. Accordingly, caution should be advised in the clinical use of methadone
when other drugs are also administered that induce or inhibit P450 3A4, such as rifampicin
or diazepam, respectively.
named metabolite M1. The urinary ratio of methadone
to its pyrrolidine metabolite is between 1/1 and 1/5,
demonstrating an extensive metabolism (5). Despite its
widespread use, however, there are no direct studies on
the nature of P450 involved in the major metabolic
pathway of methadone. Only drug interactions have
been reported between methadone and other drugs, such
as dextromethorphan (6) and P450 2D6 substrates (7) or
fluvoxamine (8). More interesting is the increase in
methadone metabolism by rifampicin (9, 10), known to
be a powerful inducer of P450 3A4 in humans (11).
In tr od u ction
Since the discovery of the effectiveness of 6-(dimethy-
lamino)-4,4-diphenyl-3-heptanone (methadone) as a sub-
stitute treatment for opiate abuse (1), methadone main-
tenance has become one of the most widely used drugs
for opiate dependency treatment. Weaning patients off
opiates using methadone is now the most common
treatment for opioid addicts in Western countries (2).
Optimum treatment of these patients requires a knowl-
edge of the drug’s metabolic pathways and in particular
the nature of the enzyme(s) involved in its biotransfor-
mation.
Methadone is mainly N-demethylated in vitro by
cytochrome P450 (P450)1 enzymes, a conclusion based
upon the fact that incubation with rat and rabbit liver
microsomal preparations liberates formaldehyde (3).
This N-demethyl metabolite spontaneously cyclizes into
2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (4),
1 Abbreviations: P450, cytochrome P450 (EC 1.14.14.1); methadone,
6-(dimethylamino)-4,4-diphenyl-3-heptanone; M1, primary metabolite
of methadone: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; M2,
secondary metabolite of methadone: 2-ethyl-5-methyl-3,3-diphenylpyr-
rolidine; R-NF, R-naphthoflavone: 7,8-benzoflavone; TAO, troleando-
mycin; Tor, toremifene N-demethylation; E2, estradiol 2-hydroxylation;
TST, testosterone 6â-hydroxylation; NIF, nifedipine oxidation; Erythr,
erythromycin N-demethylation; DOR, dextromethorphan O-demethy-
lation; MEM, dextromethorphan N-demethylation; CHZ, chlorzoxazone
6-hydroxylation; NP, 4-nitrophenol hydroxylation; POD, phenacetin
O-deethylase; MROD, methoxyresorufin O-demethylase; Coh, coumarin
7-hydroxylase; SKF-525A, 2-(diethylamino)ethyl 2,2-diphenylvalerate;
S-MP, (S)-mephenytoin 4′-hydroxylation; TB, tolbutamide hydroxyla-
tion.
* To whom correspondence should be addressed. Fax (33) 98 01 66
03.
† Laboratoires de Biochimie-Nutrition, Faculte´ de Me´decine, Brest.
‡ INSERM U-75, Universite´ Rene´ Descartes.
§ Service de Chirurgie Digestive.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0893-228x/96/2709-0365$12.00/0 © 1996 American Chemical Society
+
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366 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Iribarne et al.
above except that microsomal proteins or NADPH was omitted.
For the determination of kinetic parameters, methadone was
added to the reaction mixture in the range 10-5000 µM.
In vitro techniques using liver microsomes and heter-
ologously expressed enzymes have shown their potenti-
alities in determining the identity of P450(s) involved in
the drug metabolism and predict potential drug-drug
interactions in humans (12). The aim of this in vitro
study, by means of such methods, was to identify the
hepatic P450 enzyme(s) involved in the major metabolic
pathway of methadone. Such information may prove
useful in explaining the variable drug concentrations
observed in patients given uniform dosages and may be
used to avoid inefficiency brought about the simultaneous
administration of other drugs. To this end, correlation
studies were carried out between methadone metabolism
and selective monooxygenase activities in 20 human liver
microsomes, the effect of P450-selective chemical inhibi-
tors was tested, and heterologously expressed P450
proteins were used for methadone metabolism.
The HPLC analysis was performed on a Carbosorb ODS-3
analytical column, particle diameter 5 µm, 150 × 4.6 mm
(Metachem Tech., Torrance, CA). The mobile phase consisted
of acetonitrile/water (35/65 v/v) containing 1% (v/v) triethy-
lamine, pH adjusted to 2.8 with orthophosphoric acid, with a
flow rate of 1 mL/min. The analytes were detected at 210 nm
(Spectromonitor II, LDC, Riviera Beach, FL) at a sensitivity of
0.01 absorbance. Peaks were identified by their retention times
and UV characteristics in comparison with standard compounds.
Peaks were quantified by the Chromato-integrator D2000
(Merck, Germany). Overall biotransformation was calculated
by calibrating with known amounts of metabolites M1 and M2
added to the reaction mixture and extracted as described above
or by means of the internal standard, clomipramine. Linearity
of M1 formation was checked by varying protein concentration
(0.5-2 mg) and time (15-45 min) with methadone concentra-
tions varying from 0.5 to 2.5 mM.
Ma ter ia ls a n d Meth od s
For the methadone metabolism inhibition study, two types
of inhibitors were used: (i) reversible inhibitors such as R-naph-
thoflavone (activator of P450 3A (22)), metyrapone, SKF-525A,
dihydroergotamine, quinidine (inhibitor of P450 2D6 and sub-
strate of P450 3A4 (12, 23)), sulfaphenazole (inhibitor of P450
2C), ketoconazole, quercetin, and diazepam; (ii) mechanism-
based inhibitors such as gestodene, TAO, and erythralosamine.
As these mechanism-based inhibitors require NADPH-depend-
ent complexation for inactivation, 0.25 mM TAO, 0.1 mM
gestodene, or 0.1 mM erythralosamine was preincubated in the
presence of 1 mM NADPH and 1 mg of microsomal protein at
37 °C for 20 min in a volume of 0.5 mL. The incubation medium
was then diluted 2-fold in 0.1 mM potassium phosphate buffer
containing 0.5 mM methadone and 1 mM NADPH. After 20
min incubation at 37 °C, metabolites were analyzed by HPLC
as described above. Inhibitors sparingly soluble in water were
added to the reaction medium by means of solvents as vehicle,
either dimethyl sulfoxide for quinidine, dihydroergotamine,
erythralosamine, and quercetin, or methanol for ketoconazole,
metyrapone, R-NF, TAO, and gestodene. Control reactions were
performed by adding to the reaction medium the same amount
of solvent that was needed for the addition of inhibitor. The
volume of solvent never exceeded 0.2% (v/v). Sulfaphenazole
was added in a phosphate buffer/KOH (60 mM) mixture (50/50
v/v).
Ch em ica ls. Methadone HCl, as a racemate mixture, was
purchased from Sigma-Chimie (St. Quentin Fallavier, France),
while its two metabolites, primary metabolite M1 or 2-eth-
ylidene-1,5-dimethyl-3,3-diphenylpyrrolidine HClO4 (dl-EDPP)
and secondary metabolite M2 or 2-ethyl-5-methyl-3,3-diphe-
nylpyrrolidine HCl (dl-EMDP) were obtained from Alltech (State
College, PA). Clomipramine, diazepam, troleandomycin (TAO),
ketoconazole, metyrapone, dihydroergotamine, 7,8-benzoflavone
(R-naphthoflavone; R-NF), quercetin, and quinidine were pro-
vided by Sigma (St. Louis, MO). Sulfaphenazole, (S)-(+)-
mephenytoin, 4′-hydroxymephenytoin, and hydroxytolbutamide
were from Ultrafine Chemicals (Salford, Manchester, U.K.).
Gestodene was given by Schering SA (Lys-Lez-Lannoy, France).
Erythralosamine was a gift from Dr. M. Delaforge (URA-400-
CNRS, Universite´ R. Descartes, Paris) and 2-(diethylamino)-
ethyl 2,2-diphenylvalerate (SKF-525A) was from Smith-Kline
and French laboratories (Paris, France). Solvents were of the
highest quality available from Merck (Darmstadt, Germany).
Hu m a n Liver Sa m p les, Micr osom e P r ep a r a tion s, a n d
Mon ooxygen a se Activities. Human liver samples were ob-
tained as previously described (13). The specific forms of
cytochrome P450 involved in different monooxygenation reac-
tions have been previously characterized according to methods
reported elsewhere (13-15). These reactions include five
catalytic reactions specific to P450 3A4, namely, toremifene
N-demethylation (13), estradiol 2-hydroxylation (14), testoster-
one 6â-hydroxylation, erythromycin N-demethylation (15), and
nifedipine oxidation measured according to ref 16. Other
monooxygenase activities known to be specific to different P450
families were determined: chlorzoxazone 6-hydroxylation and
4-nitrophenol hydroxylation, both specific to P450 2E1 (17);
methoxyresorufin O-demethylation and phenacetin O-deethy-
lation, both specific to P450 1A (18). Dextromethorphan N-
demethylation to 3-methoxymorphinan (MEM) and O-demeth-
ylation to dextrorphan (DOR), specific to P450 3A and P450 2D6
(19), respectively, were measured as previously described (13).
Coumarin 7-hydroxylation, specific to P450 2A6, was performed
according to (20). (S)-Mephenytoin 4-hydroxylation and tolb-
utamide hydroxylation, specific to P450 2C, were determined
according to ref 21. The mean intra-individual variations of
catalytic activities were less than 10%.
In a ctiva tion of P 450 by Meth a d on e Meta bolism . Total
2+-CO versus Fe2+
P450 concentrations were measured by Fe
difference spectrophotometry as described by Omura and Sato
(24) with a molecular extinction coefficient of 91 mM-1‚cm-1
.
For the study of P450 inactivation, 1 mg of microsomal proteins
was incubated with 1 mM NADPH and 2.5 mM methadone for
a fixed time, and the reaction was quenched by the addition of
an equal volume of cold 0.1 M potassium phosphate buffer (pH
7.4), containing 1 mM EDTA, 40% glycerol (v/v), 0.5% sodium
cholate (w/v), and 0.4% Emulgen 911 (w/v). The P450 spectrum,
Fe2+-CO versus Fe2+, was then recorded according to Omura
and Sato (24). The formed M1 metabolite was then extracted
as described above. Control reactions were performed either
without cofactor or without substrate as described above.
Im m u n o-In h ibition of Meth a d on e Meta bolism . Microso-
mal preparations from Br022 liver containing 350 pmol of total
P450 in 50 mM potassium phosphate buffer (pH 7.4) in the
absence of methadone and NADPH were incubated at room
temperature for 20 min in the presence of increasing amounts
(1-5 mg of IgG/nmol of P450) of rabbit serum polyclonal
antibody raised against rat P450 3A1 (Human Biologics, Phoe-
nix, AZ) or nonimmune rabbit IgG. Reaction was started by
2.5 mM methadone and 1 mM NADPH additions and processed
as described above. This antibody did not cross-react with other
P450 families other than the 3A subfamily, but cross-reacted
with the P450 3A enzymes in all mammalian species, including
humans (P450 3A4, 3A5, and 3A7) according to the manufac-
turer.
Deter m in a tion of Meth a d on e Meta bolism . The final
standard 1 mL incubation mixture contained 1 mg of microsomal
protein, 50 mM potassium phosphate buffer (pH 7.4), and 2.5
mM methadone HCl. After 5 min of preincubation at 37 °C,
the reaction was started by addition of 1 mM NADPH. After
20 min shaking at 37 °C, the reaction was stopped by addition
of 100 µL of 2 N sodium hydroxide and 5 µg of clomipramine as
an internal standard. Extraction was performed by 2 × 5 mL
of diethyl ether. The organic layer was evaporated at 40 °C
under nitrogen stream. The dried residue was dissolved in 1
mL of 0.005 N HCl. Control incubations were run as described
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Methadone N-Demethylation by Human Liver P450 3A4
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 367
Im m u n ob lot An a lysis. Protein samples (20 µg) were
separated by electrophoresis and transferred to nitrocellulose
sheets according to the usual procedures (25, 26). A human
monoclonal anti-P450-NIF (27) antibody was used as previously
described (13-15). The anti-human P450 3A4 did not distin-
guish between the four members of the 3A family. So, the blots
quantified by means of image processing scan analysis (Biosoft,
Cambridge, U.K.) indicated the amount of total P450 3A in
microsomal preparations.
Het er ologou sly E xp r essed P 450 P r ot ein s. Different
P450 enzymes (1A1, 1A2, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4, and
3A5) were expressed separately in yeast using an adaptation
of the procedure described elsewhere (28-30). P450 1A1, 1A2,
2C8, 2C9, 2C18, 3A4, 3A5, and 2D6 coding sequences were
cloned by polymerase chain reaction either from human liver
cDNA or from plasmids, as described elsewhere (31). All these
sequences, checked by DNA sequencing, were inserted into the
yeast expression vector YeDP60 (32) using classical cloning
procedures or by gap repair (29). They were expressed into the
engineered Saccharomyces cerevisiae strain W(R) which coex-
presses yeast NADPH-P450 reductase when grown with ga-
lactose as a carbon source (28-32). Preparation of microsomal
fractions was performed as previously described (28). All
preparations were checked regarding their respective catalytic
activities toward typical substrates (28-31). For example,
nifedipine was oxidized by P450 3A4 and 3A5 with a turnover
number of 0.6 and 1 min-1, respectively, without P450 reductase
addition; these values were in the range previously reported
(28).
Human P450 2E1 was obtained from Gentest (Gentest Corp.,
Woburn, MA). Human B-lymphoblastoid cell lines, transfected
with human P450 2E1 cDNA, expressed high P450 2E1 activity,
characterized by turnover numbers of 3.9 and 10.5 min-1 for
chlorzoxazone 6-hydroxylation and 4-nitrophenol hydroxylation
(33), respectively.
Sta tistica l An a lysis. Correlation coefficients were calcu-
lated using an ANOVA table by the least squares regression
analysis from the raw data. As a quite normal Gaussian
distribution in the population was observed (skewness ) 0.66),
correlation coefficients were calculated by including all the
samples. Correlation coefficients, r, were considered to be
statistically significant when p was <0.05.
F igu r e 1. HPLC chromatograms of methadone metabolism.
(A) Pure compounds; (B) chromatogram of Br032 sample (1 mg
of microsomal protein) incubated with or without 1 mM NADPH
(C) or bubbled with CO (D). The CO gas was bubbled through
the reaction medium not containing substrate for 3 min, and
then the reaction was started by substrate addition and tubes
were stoppered during incubation. M1: Primary methadone
metabolite; M2: secondary metabolite; IS: internal standard
(clomipramine). Analysis was carried out on a Carbosorb ODS-3
column (15 × 0.4 cm); mobile phase was acetonitrile/water (35/
65 v/v) containing 1% triethylamine (pH adjusted to 2.8 by H3-
PO4) at a flow rate of 1 mL/min. Eluates were detected at 210
nm with 0.01 absorbance.
Resu lts
(1) Met a bolic P r ofile of Met h a d on e. By using
optimal HPLC conditions and in particular by adjusting
the pH to 2.8, the two metabolites, M1 and M2, were
separated from the substrate within 15 min (Figure 1A).
Furthermore, the internal standard clomipramine was
resolved from the M2 metabolite. One main metabolite
of methadone, namely, M1, was detected when incubated
with human liver microsomes (Figure 1B). Preliminary
experiments suggested the involvement of P450 in metha-
done metabolism. This evidence included localization of
activity in the microsomal subcellular fraction and
absolute dependence of catalytic activity on the presence
of NADPH (Figure 1C). Morever, M1 formation was
inhibited when the medium reaction was bubbled with
a CO stream prior to incubation (Figure 1D).
(2) Kin etic P a r a m eter s. Figure 2 shows the double
reciprocal plot of M1 formation from methadone by
microsomal preparations from the human liver Br032
characterized by a high P450 3A4 activity. Km was
determined to be 0.36 mM with a Vm of 2.8 nmol/(min‚mg
of protein). The use of a concentration of methadone as
low as 10 µM did not allow us to detect biphasic steady-
state kinetics. Km were determined on three samples
choosen on the basis of their very different metabolic
rates (FH1, FH3, and Br032) as 545 ( 258 µM (mean (
SD). In order to establish the metabolic rates of metha-
F igu r e 2. Reciprocal plot of v (nmol/(min‚mg of protein))
against methadone concentration (mM-1) according to Lin-
eweaver-Burk based on microsomal preparations from Br032
liver. The graph was plotted by the linear regression method.
done N-demethylation in the panel of 20 liver mi-
crosomes, the drug was therefore incubated at the
saturating concentration of 2.5 mM, so that the intra-
individual rate differences reflect the level of P450
enzyme(s) involved.
(3) Dest r u ct ion of P 450 R ela t ed t o Met h a d on e
Oxid a tion . When the human Br022 liver microsomal
preparation, high in P450 3A4, was incubated with
methadone in the presence of NADPH, P450 enzyme
content, measured spectrophotometrically according to
ref 34, was found to decrease from 3.5 to 2.1 µM during
20 min incubation (Figure 3) while the formation of the
M1 metabolite decreased by 10%. Any significant de-
crease of spectral P450 content was observed in the
absence of methadone or NADPH incubated under the
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368 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Iribarne et al.
F igu r e 3. (A) Variation of total P450 spectrophotometrically measured. (() Incubation with methadone and NADPH; ()) incubation
in the absence of NADPH; (4) incubation in the absence of methadone; and (+) M1 metabolite formation (nmol/mg of protein) vs
incubation time. (B) Spectra showing the destruction of human P450 following incubation with 2.5 mM methadone and 1 mM NADPH
for 10-20 min. Human liver microsomes (1 mg of protein, Br022) were incubated with 1 mM NADPH and 2.5 mM methadone, and
the reaction was quenched by the addition of an equal volume of cold buffer containing detergents. P450 spectra (Fe2+-CO versus
Fe2+) were measured as described by Omura and Sato (24). (C) Inhibition of human liver microsomal nifedipine oxidation vs methadone
concentration. Human liver microsomes (1 mg of protein, Br022) were preincubated with 1 mM NADPH and 2.5 mM methadone for
20 min at 37 °C. Nifedipine oxidation was subsequently performed using the same sample diluted 2-fold into 0.1 M potassium phosphate
buffer (pH 7.4) containing 200 µM nifedipine and 1 mM NADPH. After 10 min of incubation, the oxidized product was measured by
using HPLC. Nifedipine oxidation was 5.77 nmol/(min‚mg of protein) in Br022 control sample. The points shown are for individual
data.
same conditions. This result suggests that methadone
was metabolized into an active metabolite which binds
to heme.
Methadone was effective in the inactivation of nife-
dipine oxidation in human liver microsomes (Figure 3C).
This inactivation is dependent upon cofactor, time, and
inhibitor concentration.
(4) Cor r ela tion betw een Meth a d on e Meta bolism
a n d Differ en t Mon ooxygen a se Activities in Hu m a n
Liver Micr osom es. If two reactions are catalyzed by
the same enzyme, then the rates should be correlated to
each other when compared in a panel of microsomal
preparations containing varying levels of the enzyme.
Table 1 reports the metabolic rates of six P450 catalytic
activities in 20 human liver microsomal samples. The
N-demethylation of methadone, leading to M1 formation,
presented a 20-fold inter-individual variation ranging
from 122 to 2447 pmol/(min‚mg) with a mean of 745 (
574 pmol/(min‚mg).
by the P450 3A4 enzyme, namely, nifedipine oxidation
(r ) 0.89; Figure 4), testosterone 6â-hydroxylation (r )
0.66), erythromycin N-demethylation (r ) 0.60), estradiol
2-hydroxylation (r ) 0.59), toremifene N-demethylation
(r ) 0.78), and N-demethylation of dextromethorphan
(MEM) (r ) 0.60) (Table 2). Conversely, no r values
>0.40 were found for substrate markers of P450 1A2
(phenacetin O-deethylation, methoxyresorufin O-de-
methylation), P450 2E1 (chlorzoxazone 6-hydroxylation
or 4-nitrophenol hydroxylation), P450 2D6 (dextrometho-
rphan O-demethylation), or P450 2A6 (coumarin 7-hy-
droxylation). It is of interest to note that dextrometho-
rphan O-demethylation to dextrorphan, supported by
P450 2D6, was not significantly correlated with metha-
done metabolism, while the minor metabolic pathway of
dextromethorphan, namely, N-demethylation to 3-meth-
oxymorphinan supported by P450 3A, was correlated (r
) 0.60; p <0.01). Tolbutamide hydroxylation and (S)-
mephenytoin 4-hydroxylation, specific to P450 2C9 and
C19 activities, repectively, were not correlated with P450
3A4 catalytic activities (Table 2).
The N-demethylation of methadone correlated signifi-
cantly with six catalytic activities known to be mediated
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Methadone N-Demethylation by Human Liver P450 3A4
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 369
Ta ble 1. P 450-Dep en d en t Activities in Micr osom a l Sa m p les fr om 20 Hu m a n Liver s
subjects
sex, age
total P450a
P450 3Ab
TSTc
NIFd
Erythrd
Torc
E2c
M1c
FH1
FH3
M, 18 y
M, 47 y
M, 43 y
M, 44 y
M, 26 y
F, 45 y
M, 45 y
M, 23 y
F, 15 y
M, 2 y
M, 27 y
M, 23 y
M, 36 y
M, 44 y
M, 56 y
M, 56 y
M, 2 d
M, 60 y
M, 40 y
M, 40 y
34
269
470
414
369
593
334
241
350
505
110
165
77
154
286
252
153
264
143
198
433
289
142
1.2
3.6
2.8
2.9
2.6
1.8
1.1
3.3
4.5
1.2
1.9
0.7
0.4
4.9
2.4
1.1
1.2
3.2
2.4
2.2
2.27
1.23
815
3420
985
1850
1795
950
0.60
3.37
0.97
1.67
1.78
0.61
1.17
5.26
4.14
0.22
1.55
0.41
0.22
5.98
3.68
1.67
0.81
2.87
2.38
2.11
2.07
1.67
1.14
1.86
0.85
1.15
1.42
1.00
0.82
1.90
2.12
0.98
0.50
0.35
1.17
1.66
1.57
0.64
0.36
2.18
1.18
1.06
1.19
0.55
31
139
30
40
70
30
30
88
146
27
30
8
21
192
102
56
25
96
26
213
25
85
83
302
1212
268
1029
793
793
383
1661
774
122
157
468
407
2447
1243
717
392
862
539
911
745
574
Br015
Br016
Br017
Br018
Br019A
Br022
Br024
Br025
Br028
Br029
Br031
Br032
Br035
Br036
Br040
Br042
Br043
Br046
mean
32
52
740
3650
3935
1085
845
165
430
2445
2120
1310
30.3
ND
236
385
76
250
49
96
459
565
194
41
367
370
530
207
180
3.5
ND
1476
1219
122
75
68
(SD
17
51
a
b
pmol/mg of microsomal protein (measured by spectrophotometry). Arbitrary relative units in µg of protein. c pmol/(min‚mg of protein).
d
nmol/(min‚mg of protein). TST, testosterone 6â-hydroxylation; E2, estradiol 2-hydroxylation; NIF, nifedipine oxidation; Erythr,
erythromycin N-demethylation; Tor, toremifene N-demethylation; M1, methadone N-demethylation.
done by as much as 84%. Figure 7 illustrates the
competitive inhibition of diazepam on methadone N-
demethylation with an apparent Ki of 50 µM. Quinidine,
a potent inhibitor of P450 2D6, did not significantly
inhibit methadone metabolism in the four samples
studied (102 ( 30% residual activity, range 70-127%).
Sulfaphenazole, a potent inhibitor of the P450 2C family,
was not able to inhibit methadone N-demethylation more
than 10% (90.1 ( 16% residual activity, n ) 6, range 78-
113%).
(6) Im m u n o-In h ibition of Meth a d on e Meta bolism .
In order to confirm that P450 3A enzymes are involved
in methadone metabolism, immuno-inhibition studies
with anti-rat P450 3A1 were carried out. This study
showed that the methadone N-demethylation was 50%
inhibited (vs control) with 5 mg of IgG/nmol of P450
(Figure 8). The anti-rat P450 3A1 was shown to cross-
react with P450 3A enzymes but not with other P450
families. This antibody was able to inhibit nifedipine
oxidation, specific to P450 3A4, up to 86% under the same
conditions. The lack of immuno-inhibition stronger than
50% by antibody raised against rat P450 3A1 suggests
the partial involvement of other P450s in methadone
N-demethylation in the Br022 sample. However, by
using an anti-rat-P450 3A2 (Gentest, MA), the immuno-
inhibition reached 62% for the same sample (data not
shown). Furthermore, under the same conditions (5 mg
of IgG/nmol of P450), the immuno-inhibition was greater
than 72% (vs control) for FH1 sample, characterized by
low P450 3A activities.
F igu r e 4. Correlation between N-demethylation of methadone
and nifedipine oxidation in microsomal samples from 20 human
livers. Correlation coefficient, r, was calculated by the least-
squares regression method.
The N-demethylation of methadone correlated signifi-
cantly with the amount of total P450 3A immunodetected
by a monoclonal anti-human P450 3A4 antibody (r )
0.72; p <0.003) by means of Western blot technique. The
regression line failed to intersect the y axis at zero,
suggesting the possible involvement of other P450 isozyme-
(s) in M1 formation (Figure 5).
(5) In h ibition of Meth a d on e Meta bolism by Dif-
fer en t Com p ou n d s. To assess further that the main
metabolic pathway of methadone is catalyzed by the P450
3A enzymes, as is suggested by the correlation studies,
the inhibitory effect of different substrates or inactivators
of P450 was tested on liver microsomal samples. Figure
6 reports the percentage of methadone N-demethylation
inhibition by three mechanism-based inhibitors, namely,
TAO, gestodene, and erythralosamine. Inhibition was
between 60% and 72% with respect to controls. Con-
versely, R-naphthoflavone, known to enhance some P450
3A4 reactions, stimulated methadone metabolism in the
sample Br036. Other chemical inhibitors were tested,
including P450 3A selective inhibitors such as ketocona-
zole, quercetin, and dihydroergotamine as well as non-
specific inhibitors such as metyrapone and SKF-525A.
These substances inhibited the N-demethylation of metha-
(7) Meta bolism of Meth a d on e by Heter ologou sly
Exp r essed P 450 P r otein s. Microsomal preparations
of cells, genetically engineered for stable expression of
nine human P450s, catalyzed the N-demethylation of
methadone at different rates (Table 3). P450 2C8, P450
2C18, P450 2D6, and P450 3A4 preferentially demethy-
lated methadone with a turnover number of 0.97-2.52
min-1 for P450 3A4 and P450 2D6, respectively. By
taking into account the mean content of immunodetect-
able P450 isoforms in human liver microsomes (35, 36),
however, Table 3 clearly demonstrates that the N-
demethylation of methadone is mainly mediated by P450
+
+

370 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Iribarne et al.
Ta ble 2. Cor r ela tion Coefficien ts,c r , betw een Ca ta lytic Activities a n d N-Dem eth yla tion of Meth a d on e (M1) in 20 Hu m a n
Liver Micr osom a l Sa m p les
P450 act.
(substrate)
M1
NIF
Erythr
E2
Tor
TST
MEM
POD
MROD
CHZ
NP
DORb
Coh
S-MP
M1
NIF
Erythr
E2
0.89
0.60
0.59
0.78
0.66
0.60
0.07
0.05
0.04
0.20
0.12
0.36
0.29
0.30
0.74
0.64
0.86
0.82
0.42
0.19
0.18
0.06
0.27
0.04
0.34
0.25
0.41
0.62
0.80
0.88
0.55
0.30
0.13
0.05
0.21
0.11
0.50
0.42
0.42
Tor
0.75
0.57
0.52
0.14
0.45
0.15
0.30
0.13
0.49
0.15
0.49
TST
MEMa
POD
MROD
CHZ
NP
0.78
0.63
0.26
0.27
0.16
0.34
0.09
0.55
0.40
0.61
0.37
0.11
0.03
0.02
0.09
0.17
0.25
0.24
0.14
0.26
0.50
0.24
0.15
0.14
0.72
0.30
0.71
0.63
0.08
0.28
0.08
0.33
0.13
0.44
0.01
0.38
0.03
0.06
0.07
0.58
0.77
0.61
0.31
0.01
0.08
DORb
Coh
S-MP
Tolb
0.54
0.67
0.29
0.53
0.20
0.51
0.64
0.54
0.78
0.59
a
b
MEM ) 3-methoxymorphinan ) N-demethylation of dextromethorphan (P450 3A). DOR ) O-demethylation of dextromethorphan
) dextrorphan (P450 2D6). c Correlation coefficients, r, were calculated by the least-squares regression method. For r >0.56, p <0.01 (n
) 20). M1, methadone; NIF, nifedipine; Erythr, erythromycin; E2, estradiol; Tor, toremifen; TST, testosterone; POD, phenacetine; MROD,
methoxyresorufin; CHZ, chlorzoxazone; NP, 4-nitrophenol; Coh, coumarin 7-hydroxylation; S-MP, (S)-mephenytoin 4-hydroxylation; Tolb,
tolbutamide hydroxylation.
F igu r e 5. Correlation between N-demethylation of methadone
and total P450 3A enzymes immunodetected in human liver
microsomes. Intensity units of total P450 3A are relative
arbitrary units determined by image processing quantification.
3A4 in human liver. Values of N-demethylation reported
in Table 3 must be regarded as rough estimates of mean
activity in human liver on average.
Discu ssion
(1) Meta bolism . Methadone has previously been
F igu r e 6. Percent residual activity (vs controls) of methadone
shown to be extensively metabolized in man (5) by the
N-demethylation in microsomal human liver samples (FH1,
N-demethylation reaction. Indeed, after extended bio-
transformation in the liver, the major metabolite M1 and
the unmetabolized drug remaining are excreted in the
urine and bile with an elimination rate of ca. 20-90% of
the dose within 24 h. The present study demonstrates
that methadone is efficiently N-demethylated by P450
enzymes, leading to a pyrrolidine derivative through
spontaneous cyclization. This primary metabolite, M1,
was not subsequently demethylated by liver microsomal
preparations (data not shown), which is in agreement
with the results of a previous study (5) in which only
traces of this N-didemethylated pyrrolidine metabolite
were detected in the urine of patients. Methadone
interacts with P450 with a good affinity, the association
constant Ks being around 1 µM (37). The methadone
plasma level was about 1 µM during chronic treatment
with oral doses of 40 mg/day (5). The present study,
however, demonstrates that a wide range of inter-
individual metabolic capacities can be observed, which
FH3, Br022, Br024, Br032, Br035, Br036) by different chemical
inhibitors. (A) Mechanism-based inhibitors (erythralosamine,
gestodene, and TAO) and inhibitors specific to P450 2D6
(quinidine), P450 1A (R-NF), and P450 2C (sulfaphenazole,
sulpha...). (B) Three P450 3A-specific inhibitors (dihydroergota-
mine, ketoconazole, and quercetin) and two nonspecific inhibi-
tors (SKF-525A and metyrapone). Control values were per-
formed by adding to the reaction medium the same amount of
solvent that is needed for addition of inhibitor. Inhibitors were
used at different concentrations: TAO at 0.25 mM; gestodene,
erythralosamine, R-NF, dihydroergotamine, metyrapone, quer-
cetin, and SKF-525A at 0.1 mM; quinidine at 0.01 mM;
ketoconazole at 0.002 mM; and sulfaphenazole at 0.005 mM.
The values are given as mean ( SD of three to six liver samples.
suggested that the monitoring of methadone levels during
treatment should be carried out.
Methadone exhibited monophasic steady-state kinetics
with a Km of 0.545 mM in man, while in rat, biphasic
kinetics with low- and high-affinity Km of 0.95 and 0.08
mM were found (data not shown). Accordingly, it can
+
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Methadone N-Demethylation by Human Liver P450 3A4
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 371
of NADPH, methadone gave metabolite P450 complexes
absorbing at 450 nm, stable in the presence of NADPH
and dithionite. This complex was dissociated by addition
of ferricyanide (37). These results suggest the formation
of a nitroso metabolite-P450 complex.
(2) In volvem en t of P 450 3A4. The work presented
here demonstrates that human liver P450 3A4 has a
major role in the N-demethylation of methadone. Such
a conclusion is based on four kinds of results: correlation
studies, P450 3A4-selective chemical inhibitors, immuno-
inhibition by antibody raised against rat P450 3A1, and
metabolism by heterologously expressed P450 proteins.
Indeed, the N-demethylation of methadone, which varied
20-fold between individuals, was shown to be signifi-
cantly correlated with six P450 3A-specific monooxyge-
nase activities in a large panel of 20 human liver
microsomes. As the methadone N-demethylation was
measured at a saturating substrate concentration of 2.5
mM, the differences in reaction rates reflect the level of
P450.
F igu r e 7. Competitive inhibition of methadone N-demethyla-
tion by diazepam. Liver microsomal preparation from Br032 was
assayed for methadone (O 206 µM; 2 404 µM; 9 777 µM). The
data were analyzed according to the Dixon plot.
The use of various P450 3A selective chemical inhibi-
tors similarily points to the involvement of P450 3A4 in
methadone metabolism. Gestodene (34), erythralosamine
(38), and TAO (39), well-known to be mechanism-based
inhibitors of P450 3A4, inhibited the N-demethylation
of methadone by up to 72%. Furthermore, four specific
inhibitors of P450 3A, namely, dihydroergotamine (40),
ketoconazole (41), quercetin (42), and diazepam (43), were
found to strongly inhibit methadone biotransformation.
Quinidine, a well documented drug known to be a P450
3A substrate but also a potent inhibitor of P450 2D6 (23),
did not significantly modify the methadone metabolism,
suggesting that P450 2D6 is not significantly involved
in the metabolism. Similarly, the lack of inhibition by
7,8-benzoflavone on methadone metabolism precluded
the involvement of the P450 1A enzyme family. Sul-
faphenazole, known to be a potent inhibitor of the P450
2C family (12), did not significantly inhibit the metha-
done N-demethylation, suggesting that P450 2C enzymes
are not the main enzymes of this reaction. The contribu-
tion of P450 2D6 and P450 2C, however, cannot be totally
excluded in some circumstances, as judged by 25%
inhibition by 10 µM quinidine in FH3 sample and by 30%
inhibition by 5 µM sulfaphenazole in Br022 sample. The
variable response of immuno-inhibition experiments (50%
in Br022 and 72% in FH3 samples) allows us to suggest
that P450 isoforms other P450 3A could be involved, but
a low level of contribution in the methadone metabolism
in some samples.
F igu r e 8. Immuno-inhibition of methadone N-demethylation
by rabbit antibody raised against rat P450 3A1. One milligram
of microsomal proteins from Br022 sample (350 pmol of total
P450) was incubated with 2.5 mM methadone and 1 mM
NADPH for 20 min at 37 °C after preincubation for 20 min at
room temperature with anti-P450 3A1 IgG. Microsomes were
preincubated with nonimmune IgG (2) or anti-P450 3A1 (0).
The points shown are for individual data.
be suggested that a single P450 form is involved in the
metabolism of methadone in man and that the P450
involved catalyzes reactions at nonsaturating substrate
concentrations.
Spectrally detectable P450 was destroyed in mi-
crosomes when incubated with methadone in the pres-
ence of NADPH, suggesting that the primary metabolite
of methadone is a mechanism-based inactivator of P450
3A4. Such a mechanism was shown to be NADPH
dependent, as previously reported (37). In the presence
Finally, the use of nine heterologously expressed P450
enzymes allowed the determination of which isoforms of
Ta ble 3. Meth a d on e N-Dem eth yla tion Activity of Hu m a n Heter ologou sly Exp r essed P 450s
yeast
(cell) expressing
methadone
P450 content
methadone
total P450a
N-demethylationb
in liver microsomesc
N-demethylationd
P450 1A1
P450 1A2
P450 2C8
P450 2C9
P450 2C18
P450 2D6
P450 3A4
P450 3A5
P450 2E1*
74.6
166.7
87.7
139.7
44.1
0.50
0.02
1.33
0.14
2.24
2.52
0.97
0.04
0.05
2
25
10
125
20
25
250
50
22
0.99 (<1)
0.60 (<1)
13.34 (3)
17.00 (4)
44.90 (12)
62.95 (16)
242.00 (63)
1.83 (<1)
1.15 (<1)
14.3
391.5
191.2
109.0
a
b
pmol/mg of microsomal preparation. Activity expressed as turnover number, min-1 or pmol/(min‚pmol of P450). c pmol/mg of
d
immunodetectable protein; data from refs 35 and 36. Hepatic activity calculated from the turnover number × P450 isoform content
(pmol/(min‚mg of protein)), in parentheses is given the percentage (%) (estimated on average by extrapolation) of catalytic activity supported
by each P450 isoform. (*) Human P450 2E1 supplied from a commercial source.
+
+

372 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Iribarne et al.
P450 are involved in methadone N-demethylation. Five
P450s can be considered as candidates in the catalysis
of this reaction: P450 1A1, P450 2C8, P450 2C18, P450
2D6, and P450 3A4, with a kcat (or turnover number) of
0.5, 1.33, 2.24, 2.52, and 0.97 min-1, respectively. On
the basis of the relative levels of these P450s in human
liver microsomes, it can be concluded that P450 3A4,
which contributes 30-40% of the total P450 pool in
human liver microsomes (35, 36), is the major enzyme
involved in methadone N-demethylation. Such an as-
sumption, however, is limited by some caveats. First,
estimates of P450 form content in human liver are based
on immunodetectable proteins and not on active P450
enzyme content (35, 36). Second, it must be borne in
mind that the rough estimates of methadone N-demethy-
lation activity calculated in Table 3 represent an average.
So P450 3A4 will be the major catalyst of this N-
demethylation on average. Two other P450 isoforms
could be involved in livers depleted in P450 3A4, namely,
P450 2D6 (25% inhibition by 10 µM quinidine in FH3
sample) and P450 2C (30% inhibition by 5 µM sul-
faphenazole in Br022 sample). Until now, P450 3A4 and
P450 3A5 could not be differentiated based on their
catalytic activities (44). Results of Table 3 suggest that
methadone could be a good substrate for discriminating
between P450 3A4 and P450 3A5 since the activity of
P450 3A4 is about 25 times higher.
assuming that a human liver contains about 67 g of
microsomal proteins (50). Intrinsic clearance was esti-
mated to be around 350-400 mL/min. From pharmaco-
kinetic data of methadone (50, 51), i.e., t1/2 ) 40 h, Vd )
300 L, and unbound methadone of 0.2, the intrinsic
clearance from in vivo data was calculated as 400-450
mL/min. Such a result is in agreement with data
reported in this study.
Finally, taken together, all the data reported in this
study suggest that the N-demethylation of methadone
is mainly mediated by P450 3A4 enzyme in human liver
microsomes.
Ack n ow led gm en t. This work was financially sup-
ported by the Ministe`re de l’Enseignement Supe´rieur et
de le Recherche (Equipe d’accueil EAD-948). The authors
wish to thank to Dr. M. Delaforge (URA-400 CNRS,
Universite´ Rene´ Descartes, Paris) and Dr. I. Castel
(Schering SA, Lys-Lez-Lannoy, France) for their gifts of
erythralosamine and gestodene, respectively. The yeast
expression was made possible by Rhoˆne-Poulenc-Rorer
and Ministe`re de l’Enseignement Supe´rieur et de la
Recherche through the Bioavenir programme (S.B., P.B.).
The authors thank Miss M. J ohnson for her editorial
help.
Refer en ces
Furthermore, methadone N-demethylation increased
2-fold vs control rats by phenobarbital or pregnenolone
16R-carbonitrile treatment of rats (data not shown). As
these two drugs are known to induce P450 3A1 in rat
(45), these data support the conclusion of this study; i.e.,
P450 3A enzyme family is the main enzyme involved in
methadone N-demethylation.
(1) Dole, V. P., and Nyswander, M. (1965) A medical treatment for
diacethylmorphine (heroine) addiction. J . Ann. Med. Assoc. 193,
646-650.
(2) Kreck, M. J . (1994) Biological correlates of methadone mainte-
nance pharmacotherapy. Ann. Med. Intern. 145, 9-14.
(3) Axelrod, J . (1956) The enzymatic N-demethylation of narcotic
drugs. J . Pharmacol. Exp. Ther. 117, 322-330.
(4) Beckett, A. H., Taylor, J . F., Casy, A. F., and Hassan, M. M. A.
(1968) The biotransformation of methadone in man: synthesis
and identification of a major metabolite. J . Pharm. Pharmacol.
20, 754-762.
(5) Verebely, K., Volavka, J ., Mule, S., and Reswick, R. (1975)
Methadone in man: pharmacokinetic and excretion studies in
acute and chronic treatment. Clin. Pharmacol. Ther. 18, 180-
190.
(6) Wu, D., Otton, S. V., Sproule, B. A., Busto, U., Inaba, T., Kalow,
W., and Sellers, E. M. (1993) Inhibition of cytochrome P450 2D6
(P450 2D6) by methadone. Br. J . Clin. Pharmac. 35, 30-34.
(7) Kerry, N. L., Somogyi, A. A., Mikus, G., and Bochner, F. (1993)
Primary and secondary oxidative metabolism of dextromethor-
phan. Biochem. Pharmacol. 45, 833-839.
(8) Perucca, E., Gatti, G., and Spina, E. (1994) Clinical pharmaco-
kinetics of fluvoxamine. Clin. Pharmacokinet. 27, 175-190.
(9) Baciewicz, A. M., and Self, T. H. (1984) Rifampin drug interac-
tions. Arch. Intern. Med. 144, 1667-1671.
(10) Brockmeyer, N. H., Mertins, X., and Goos, M. (1991) Pharmaco-
kinetic interaction of antimicrobial agents with levomethadone
in drug-addicted AIDS patients. Klin. Wochenschr. 69, 16-18.
(11) Combalbert, J ., Fabre, I., Fabre, G., Dalet, I., Derancourt, J ., Cano,
J . P., and Maurel, P. (1989) Purification and identification of the
rifampicin-inducible human liver cytochrome P-450 (cyclosporin
A oxidase) as a product of P450 IIIA gene subfamily. Drug Metab.
Dispos. 17, 197-207.
(3) Dr u g In ter a ction s. The drugs which have been
characterized as being specific substrates or inducers of
human P450 3A4 enzymes include erythromycin, nife-
dipine, testosterone, diltiazem, cyclosporin, diazepam,
propafenone, lidocaine, imipramine, rifampicin, and dex-
amethasone (for a complete list, see ref 46). It can
therefore be anticipated that any of these drugs might
lead to interaction with methadone when given in as-
sociation. Two major drug interactions merit further
discussion. It has been reported that addicts treated for
tuberculosis by rifampicin develop methadone with-
drawal symptoms (9, 10), requiring increased methadone
dosage. Based on the results of this study, such observa-
tions are easily explained by the fact that rifampicin,
known to be a potent inducer of P450 3A enzymes (11),
increases the P450 3A4 liver content which thereby also
increases methadone biotransformation, resulting in
withdrawal symptoms (9).
Patients subject to long-term methadone treatment
often simultaneously use benzodiazepines, such as diaz-
epam (47). Data reported in this study suggest that
diazepam and methadone share a similar affinity for the
same P450 enzyme and, accordingly, could lead to a
competitive inhibition of the hydroxylation metabolic
pathway to temazepam supported by P450 3A (43). Such
an interaction has been previously reported (48). The
acute administration of diazepam with daily methadone
maintenance doses enhances methadone effects, demon-
strating a decrease of methadone biotransformation due
to an effect of competition inhibition by diazepam.
(4) In Vitr o a n d In Vivo Cor r ela tion s. Intrinsic
clearance was calculated as Vm/Km (49). Vm was given
for the amount of enzyme present in the whole liver
(12) Rodriguez, A. D. (1994) Use of in-vitro human metabolism studies
in drug development. An industrial perspective. Biochem. Phar-
macol. 48, 2147-2156.
(13) Berthou, F., Dre´ano, Y., Belloc, C., Kangas, L., Gautier, J . C.,
and Beaune, P. H. (1994) Involvement of cytochrome P450 3A
enzyme family in the major metabolic pathways of toremifene in
human liver microsomes. Biochem. Pharmacol. 47, 1883-1895.
(14) Kerlan, V., Dre´ano, Y., Bercovici, J . P., Beaune, P. H., Floch, H.
H., and Berthou, F. (1992) Nature of cytochromes P450 involved
in the 2/4-hydroxylation of estradiol in human liver microsomes.
Biochem. Pharmacol. 44, 1745-1756.
(15) J acolot, F., Simon, I., Dre´ano, Y., Beaune, P. H., Riche´, C., and
Berthou, F. (1991) Identification of the cytochrome P450 IIIA
family as the enzyme involved in the N-demethylation of tamox-
ifen in human liver microsomes. Biochem. Pharmacol. 41, 1911-
1919.
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Methadone N-Demethylation by Human Liver P450 3A4
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 373
(35) Guengerich, F. P., and Turvy, C. G. (1991) Comparison of levels
of several human microsomal cytochrome P450 enzymes and
epoxide hydrolase in normal and diseases state using immu-
nochemical analysis of surgical liver samples. J . Pharmacol. Exp.
Ther. 256, 1189-1194.
(36) Shimada, T., Yamazaki, H., Minura, M., Inui, Y., and Guengerich,
F. P. (1994) Interindividual variations in human liver cytochrome
P-450 enzymes involved in the oxidation of drugs, carcinogens
and toxic chemicals: studies with liver microsomes of 30 J apanese
and 30 Caucasians. J . Pharmacol. Exp. Ther. 270, 414-423.
(37) Delaforge, M., Hoellinger, H., Sonnier, M., J aouen, M., and Do-
Cao-Thang. (1991) Cytochrome P-450 complex and covalent
binding to liver microsomal proteins of methadone and L R-acetyl-
methadol. Prog. Pharmacol. Clin. Pharmacol. 8, 167-176.
(38) Sartori, E., Delaforge, M., Mansuy, D., and Beaune, P. (1985)
Some erythromycin derivatives are strong inducers in rats of a
cytochrome P450 very similar to that induced by 16R-preg-
nenolone carbonitrile. Biochem. Biophys. Res. Commun. 128,
1431-1439.
(39) Chang, T. H. K., Gonzalez, F. J ., and Waxman, D. J . (1994)
Evaluation of triacetyloleandomycin, R-naphthoflavone and di-
ethyldithiocarbamate as selective chemical probes of human
cytochromes P450. Arch. Biochem. Biophys. 311, 437-442.
(40) Delaforge, M., Rivie`re, R., Sartori, E., Doignon, J . L., and Grognet,
J . M. (1989) Metabolism of dihydroergotamine by a cytochrome
P450 similar to that invoved in the metabolism of macrolide
antibiotics. Xenobiotica 19, 1285-1295.
(41) Maurice, M., Pichard, L., Daujat, M., Fabre., I., J oyeux, H.,
Domergue, J ., and Maurel, P. (1992) Effects of imidazole deriva-
tives on cytochrome P450 from human hepatocytes in primary
cultures. FASEB J . 6, 752-758.
(42) Rashid, J ., McKinstry, C., Dirnhuber, M., Waller, D. G., and
George, C. F. (1993) Quercetin, an in-vitro inhibitor of P450 3A4
does not contribute to the ineraction between nifedipine and
grapefruit juice. Br. J . Clin. Pharmacol. 36, 460-463.
(16) Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P.,
Wolff, T., and Wasma, D. J . (1986) Characterization of rat and
human liver microsomal cytochrome P450 forms involved in
nifedipine oxidation, a prototype for genetic polymorphism in
oxidative drug metabolism. J . Biol. Chem. 261, 5051-5060.
(17) Carrie`re, V., Goasduff, T., Ratanasavanh, D., Morel, F., Gautier,
J . C., Guillouzo, A., Beaune, P., and Berthou, F. (1993) Both
cytochrome P450 2E1 and 1A1 are involved in the metabolism of
chlorzoxazone. Chem. Res. Toxicol. 6, 852-857.
(18) Berthou, F., Flinois, J . P., Ratanasavanh, D., Beaune, P., Riche´,
C., and Guillouzo, A. (1991) Evidence for the involvement of
several cytochrome P-450 in the first steps of caffeine metabolism
by human liver microsomes. Drug Metab. Dispos. 19, 561-567.
(19) J acqz-Aigrain, C., Funck-Brentano, C., and Cresteil, T. (1993)
P450 2D6 and P450 3A dependent metabolism of dextrometho-
rphan in humans. Pharmacogenetics. 3, 197-204.
(20) Maurice, M., Emiliani, S., Dalet-Beluche, I., Deancourt, J ., and
Lange, R. (1991) Isolation and characterization of a cytochrome
P450 of the IIA subfamily from human liver microsomes. Eur. J .
Biochem. 200, 511-517.
(21) Relling, M. V., Aoyama, T., Gonzalez, F. J ., and Meyer, U. A.
(1990) Tolbutamide and mephenytoin hydroxylation by human
cytochrome P450s in the CYP2C subfamilly. J . Pharmacol. Exp.
Ther. 252, 442-447.
(22) Brian, R., Sari, M. A., Iwasaki, M., Shimada, T., Kaminsky, L.
S., and Guengerich, F. P. (1990) Catalytic activities of human liver
cytochrome P450 3A4 expressed in Saccharomyces cerevisiae.
Biochemistry 293, 11280-11292.
(23) Guengerich, F. P., Muller-Enoch, D., and Blair, I. A. (1986)
Oxidation of quinidine by human liver cytochrome P-450. Mol.
Pharmacol. 30, 287-295.
(24) Omura, T., and Sato, R. (1964) The carbon monoxide-binding
pigment of liver microsomes. Evidence for its hemoprotein nature.
J . Biol. Chem. 239, 2370-2378.
(25) Laemmli, U. K. (1970) Change of structural protein during the
assembly of the head of bacteriophage T4. Nature 227, 680-685.
(26) Towbin, H., Staehelin, T., and Gordon, J . (1970) Electrophoretic
tranfer of protein from polyacrylamide gel to nitrocellulose
sheets: procedures and some applications. Proc. Natl. Acad. Sci.
U.S.A. 76, 4350-4354.
(27) Beaune, P., Kremers, P., Letawe-Goujon, F., and Gielen, J . (1985)
Monoclonal antibodies against human liver cytochrome P450.
Biochem. Pharmacol. 34, 3547-3552.
(28) Renaud, J . P., Cullin, D., Pompon, D., Beaune, P., and Mansuy,
D. (1990) Expression of human cytochrome P450 IIIA4 in yeast.
A functional model for the hepatic enzyme. Eur. J . Biochem. 194,
889-896.
(29) Peyronneau, M., Renaud, J . P., Truan, G., Urban, P., Pompon,
D., and Mansuy, D. (1992) Optimization of yeast-expressed
human liver cytochrome P450 3A4 catalytic activities by coex-
pressing NADPH-P450 reductase and cytochrome b5. Eur. J .
Biochem. 207, 109-116.
(30) Gautier, J . C., Urban, P., Beaune, P., and Pompon, D. (1993)
Engineered yeast cells as model to study coupling between
humans xenobiotic metabolizing enzymes. Eur. J . Biochem. 211,
63-72.
(31) Lemoine, A., Gautier, J . C., Azoulay, D., Kiffel, C., Belloc, C.,
Guengerich, F. P., Maurel, P., Beaune, P., and Leroux, J . P. (1993)
Major pathway of imipramine metabolism is catalyzed by cyto-
chrome P-450 1A2 and 3A4 in human liver. Mol. Pharmacol. 43,
827-832.
(32) Urban, P., Cullin, C., and Pompon, D. (1990) Maximizing the
expression of mammalian cytochrome P450 monooxygenase ac-
tivities in yeast cells. Biochimie 72, 463-472.
(33) Amet, Y., Berthou, F., Baird, S., Dre´ano, Y., Bail, J . P., and Me´nez,
J . F. (1995) Validation of the (ω-1) hydroxylation of lauric acid
as an in-vitro substrate probe for human liver CYP2E1. Biochem.
Pharmacol. 10 (in press).
(34) Guengerich, F. P. (1990) Mechanism-based inactivation of human
liver microsomal cytochrome P450 IIIA4 by gestodene. Chem. Res.
Toxicol. 3, 363-371.
(43) Andersson, T., Miners, J . O., Veronese, M. E., and Birkett, D. J .
(1994) Diazepam metabolism by human liver microsomes in
mediated by both S-mephenytoin hydroxylase and P450 3A
isoforms. Br. J . Clin. Pharmacol. 38, 131-137.
(44) Aoyama, T., Yamano, S., Waxman, D. J ., Lapenson, D. P., Meyer,
U. A., Fischer, V., Tyndale, R., Inaba, T., Kalow, W., Gelboin, H.
V., and Gonzalez, F. J . (1989) Cytochrome P450 hPCN3, a novel
cytochrome P450 IIIA gene product that is differentially expressed
in adult human liver. J . Biol. Chem. 264, 10388-10395.
(45) Okey, A. B. (1990) Enzyme induction in the cytochrome P-450
system. Pharmac. Ther. 45, 241-298.
(46) Guengerich, F. P. (1991) Mechanisms of catalysis and substrate
specificity of human cytochrome P450 enzymes. In Hepatic
metabolism and disposition of endo- and xenobiotics (Bock, K. W.,
Matern, S., Gerok, W., and Schmid, R., Eds.) pp 85-96, Kluwer
Academic Publishers, Dordrecht.
(47) Igushi, M. Y., Handelsman, L., Bickel, W. K., and Griffiths, R. R.
(1993) Benzodiazepine and selective use/abuse by methadone
maintenance clients. Drug Alcohol Depend. 32, 257-266.
(48) Preston, K. L., Griffitts, R. R., Stitzer, M. L., Bigelow, G. E., and
Liebson, I. A. (1984) Diazepam and methadone interaction in
methadone maintenance. Clin. Pharmacol. Ther. 36, 534-541.
(49) Houston, J . B. (1994) Utility of in-vitro drug metabolism data in
predicting in-vivo metabolic clearance. Biochem. Pharmacol. 47,
1469-1479.
(50) Inturrisi, C. E., Colburn, W. A., Kaiko, R. F., Honde, R. W., and
Foley, K. (1987) M. Pharmacokinetics and pharmacodynamics of
methadone in patients with chronic pain. Clin. Pharmacol. Ther.
41, 392-401.
(51) Wolff, K., Hay, A. W., Raistrick, D., and Calvert, D. (1993) Steady-
state pharmacokinetics of methadone in opioid addicts. Eur. J .
Pharmacol. 44, 189-194.
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