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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Obstetrics & Gynecology, University of Texas Medical Branch, Galveston, Texas
Received April 18, 2003; accepted June 5, 2003.
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Abstract |
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;
Fischer et al., 2000;
Eder et al., 2001), suggesting
that BUP might be an alternative to methadone for maintenance of this patient
population.
BUP levels in the fetal circulation can have a direct effect on its normal
growth and development. On the other hand, maternal circulating levels of the
opiate could affect placental physiology and, consequently, the fetus. The
kinetics for transplacental transfer of BUP, its concentration in the fetal
circuit, and effects on placental functions were determined in our laboratory
utilizing the technique of dual perfusion of placental lobule
(Nanovskaya et al., 2002). Our
data indicated that the tissue accumulated 13 times the amount of BUP present
in the maternal circuit, and less than 5% of it was metabolized to norBUP.
However, in the perfusion system mentioned, BUP was delivered in the
intervillous space, thus bypassing the maternal myometrium and endometrium,
and its access to the metabolizing enzymes was less. Therefore, it is likely
that in vivo metabolism of BUP in the placenta is higher.
In humans, BUP is biotransformed to norBUP by hepatic enzymes during first
pass metabolism. The major enzyme responsible for N-dealkylation of
BUP to norBUP in the liver is cytochrome P450 3A4 (CYP 3A4)
(Iribarne et al., 1997;
Kobayashi et al., 1998). The
formation of the glucuronide conjugates of BUP and norBUP was also reported
(Cone et al., 1984). During
pregnancy, the human placenta plays an important role in the metabolism of
endogenous compounds, xenobiotics, and environmental pollutants
(Juchau 1980;
Contractor, 1983;
Nandakumaran et al., 1983,
Blanck et al., 1983;
Roe et al., 1990;
Pienimaki et al., 1997). The
expression and activity of various P450 isoforms depends on placental
gestation age and tissue maturity but is lower than that in the liver (Hakkola
et al.,
1996a,b).
Aromatase/CYP 19 is one of the cytochrome P450 isozymes with a well
established role in the biosynthesis of steroids in human placenta
(Thompson and Siiteri, 1974).
More recently, the role of aromatase in the oxidative metabolism of
xenobiotics by preparations of crude and purified enzymes has been reported
(Toma et al., 1996;
Osawa et al., 1997). These
data were further substantiated by the availability of high specific activity
cDNA-expressed CYP 19 that allowed testing the metabolism of several compounds
and determining their kinetic parameters
(McNamara et al., 1999).
The above underscores the importance of identifying the enzyme responsible for BUP metabolism in human placenta and the effects of prolonged administration of the opiate on its activity. The information obtained is necessary to avoid drug interactions between other therapeutics that may be administered during utilization of BUP for treatment of the pregnant opiate addict. Therefore, the focus of this investigation is to identify and characterize the enzyme responsible for BUP metabolism in term human placenta.
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Materials and Methods |
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TopAbstractMaterials and MethodsOtherResultsDiscussionReferences |
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Clinical Material
All placentas were obtained from term healthy pregnancies after delivery
according to a protocol approved by the Institutional Review Board. Every
effort was made to exclude placentas of women who abused drugs during
pregnancy.
Villous tissue was dissected, rinsed with ice-cold saline, and homogenized in 0.1 M potassium phosphate buffer pH 7.4 (Ultra Turrax, Staufen, Germany). The homogenate was used to prepare subcellular fractions by differential centrifugation; namely, 10,000g pellet for the mitochondrial, 104,000g pellet for the microsomal, and the supernatant for the cytosolic fraction. The mitochondrial and microsomal pellets were resuspended in 0.1 M potassium phosphate buffer (pH 7.4), and their protein content was determined (Bio-Rad kit, Hercules, CA) using bovine serum albumin as a standard. Aliquots from the subcellular fractions were stored at -80°C until used.
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). The
cDNA-expressed CYP 19 system is commercially available as Supersomes from
Gentest (Woburn, MA). The characterization of Supersomes was described in an
earlier report (McNamara et al.,
1999).
Enzyme Reactions
Monooxygenases. The activity of these enzymes was determined by the
O-deethylation of the substrate 7-ethoxy-4-trifluoromethylcoumarin
(EFC) and formation of the fluorescent product
7-hydroxy-4-trifluoromethylcoumarin (HFC). The reaction volume of 1 ml was
made of the placental subcellular fraction (1 mg of protein), the substrate
EFC (50 µM), and NADPH (250 µM) in 0.1 M KPO4 buffer (pH
7.4), followed by incubation for 10 min at 37°C. The reaction was
terminated by the addition of 2 ml of ice-cold acetone followed by
centrifugation at 7500g for 10 min, and the precipitate was
discarded. The inhibition of HFC formation by BUP was determined by two
procedures. In the first, the reaction components and BUP were incubated for
10 min before the substrate EFC was added, and in the second, all compounds
were added simultaneously.
N-Dealkylation of BUP. The activity of placental mitochondrial, microsomal, and cytosolic fractions in catalyzing the N-dealkylation of BUP to norBUP was determined. The reaction volume was made of 1 ml of 0.1 M potassium phosphate buffer (pH 7.4) and contained 1 mg protein of the subcellular fraction, 50 µM BUP. The components were preincubated for 5 min at 37°C followed by addition of the NADPH-regenerating system that started the reaction period of 30 min. The latter was made of: 0.4 mM NADP, 4 mM glucose 6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, and 2 mM MgCl2. The incubation period was terminated by the addition of 100 µl of a 35% (w/v) trichloroacetic acid containing 1 µg/ml terfenadine (internal standard) and centrifuged at 18,000g for 10 min. The amount of norBUP formed in the supernatant was determined by liquid chromatography/mass spectrometry as described below. The control was identical, but the placental fraction was denatured. The dependence of the activity of the microsomal fraction (1 mg/ml) and preparations of human cDNA expressing CYP 19 (40-100 pmol of CYP 19/ml) (Gentest) on the amount of BUP (saturation curve) was determined. The reaction conditions were as described above but with an incubation period of 15 min. The data obtained was used to calculate the apparent Km and Vmax values.
Aromatase (CYP 19) Activity. The activity of aromatase in the
mitochondrial and microsomal fraction was determined by its conversion of
testosterone to 17
-estradiol. The following was preincubated for 5 min
at 37°C: 250 µl of protein and 1.0 µM 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, and the incubation continued for
another 5 min. It was then terminated by the addition of 100 µl of 10%
(w/v) trichloroacetic acid followed by 100 µl of 10 µg/ml estrone as an
internal standard. The reaction solution was centrifuged at 12,000g
for 10 min, the pellet was discarded, and estradiol formed was determined in
the supernatant.
Inhibition of norBUP Formation
Identification of the P450 enzyme catalyzing the metabolism of BUP to
norBUP was achieved by utilizing the following inhibitors: chemicals, i.e.,
compounds selective for specific P450 isoforms; and monoclonal antibodies
against purified human liver P450 isoforms.
Chemical Inhibitors. The concentration range used for each inhibitor
was based on its IC50, Ki, or
Km values for a specific P450 isoform. The following are
the inhibitors, the concentration used, and their corresponding P450 isoforms:
-naphthoflavone, 0. 1 µM (CYP 1A); sulfaphenazole, 10 µM (CYP
2C); quinidine, 5 µM (CYP 2D6); 4-methylpyrazole, 50 µM (CYP 2E1);
ketoconazole, 2.5 µM (CYP 3A4/CYP 19); troleandomycin, 50 µM (CYP 3A4)
(Newton et al., 1995;
Bourrie et al., 1996;
Pelkonen et al., 1998);
4-hydroxyandrostenedione, 1 µM; and aminoglutethimide 10 µM (CYP 19)
(Stresser et al., 2000).
Concentrated stock solutions of the inhibitors in methanol were prepared, and
an aliquot of each was used to attain the final concentration required for
each P450 isoform as specified above. Each inhibitor, BUP at a final
concentration of 12 µM(
Km), and the microsomal
preparation in potassium phosphate buffer (1 mg of protein/ml) were
preincubated for 5 min at 37°C. The reaction was initiated by the addition
of the NADPH-regenerating system and incubated for a period of 30 min. The
control reaction contained all the above-mentioned components but with 0.5%
(v/v) methanol substituting for the inhibitor solution. The effect of 2.5
µM ketoconazole was investigated in commercially available systems
expressing either CYP 3A4 or CYP 19 under identical conditions.
Monoclonal Antibodies. Monoclonal antibodies against human liver CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4/5 and rabbit antiserum to human placental aromatase were utilized to identify/confirm the P450 enzyme responsible for BUP metabolism by placental tissue. A pool of microsomal preparations obtained from 12 placentas was utilized.
In each enzyme assay, 0.1 mg of protein of the microsomal pool was incubated at room temperature with an antibody at its concentration causing 80% inhibition of the P450 isoform it was raised against. The microsomal pool and the antibodies were incubated for 15 min before BUP was added to attain a final concentration of 50 µM, and the reaction was allowed to continue for another 120 min at 37°C before it was terminated as described above. Mouse IgG replaced the monoclonal antibodies in the control reaction.
Quantitative Determination of the Reaction Products
7-Hydroxy-4-trifluromethylcoumarin (HFC). The concentration of HFC
was determined by fluorescence spectroscopy (Cyto-Fluor, Series 4000
Fluorescence; Applied Biosystems, Foster City, CA) according to the method of
Buters et al., (1993). A
standard curve for fluorescence intensity of 20 to 2000 pmol/ml HFC, with
excitation and emission wavelengths of 450 and 530 nm, respectively, was
plotted. The fluorescence intensity at zero time of the incubation period
served as a blank.
BUP and norBUP. BUP and norBUP were identified by HPLC according to
the method of Iribarne et al.
(1997) and as described
earlier in detail (Nanovskaya et al.,
2002).
Liquid Chromatography (LC) and Mass Spectrometry (MS) Analysis
The amounts of norBUP formed in the reaction mixture were determined
utilizing LC/MS. A Spectra system consisting of an AS 3000 autosampler, P 4000
pump (Spectra Physics, San Jose, CA) and a 2 x 30 mm C-18 3-µm Luna
column (Phenomenex, Torrance, CA) was used. The mobile phase was made of a
linear gradient of acetonitrile/water starting with 10% of the former and
ending with 90% at a flow rate of 500 µl/min for a period of 5 min. The
ratio was then reverted to 10% acetonitrile for 1 min before the end of the
run. The volume of the injected sample was 100 µl. The eluant of the column
was coupled to AQA single-quadrupole Navigator LC/MS (Thermo Quest, San Jose,
CA). Mass spectral analyses were performed using electrospray in the positive
mode with an ionizing voltage of 4.0 kV, and the capillary probe temperature
was set at 400°C. Other conditions were as follows: source voltage, 10 V;
lens voltage, 1.0 V; ion energy, 5 eV; detector voltage, 750 V; low mass
resolution, 25; high mass resolution, 0.
For quantitative determinations, the mass spectrometer was operated in the selected ion monitor mode. Under the above conditions, norBUP (m/z = 413.7) formed a potassium adduct (m/z = 454.7) of its positively charged molecular ion [MH+] (m/z = 414.7) that had a greater relative abundance than its molecular ion [MH+] and was used to monitor norBUP.
For quantitative studies, ThermoFinnigan's Xcalibur data processing software (Thermo Finnigan, San Jose, CA) was utilized. The ratios of norBUP potassium adduct peak area (m/z = 454.7) to that of terfenadine (m/z = 471.7) was plotted against the amount of norBUP. All analyses were carried out in the linear range of the instrument's sensitivity. Calibration curves were prepared using known amounts of norBUP added to blank samples of the reaction mixture.
HPLC/UV Determination of Estradiol
The method used for determination of 17-estradiol was as described
earlier (Taniguchi et al.,
1989) with slight modification. The HPLC system used consisted of
a Waters 600E multisolvent delivery system, a Waters 2487 dual wavelength
absorbance detector, and a Waters 717 autosampler controlled by Waters
Millennium32 chromatography manager (Waters, Milford, MA). The
volume of the sample injected was 200 µl and the C-18 column was a 250
x 4.6 mm Luna 5 µm (Phenomenex). 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 of 1.2
ml/min monitored at a wavelength of 280 nm. The ratio of estradiol peak area
to that of the internal standard was used for all calculations of its
quantity.
Data Analysis
Data are represented as mean ± S.D. throughout the text. Kinetic
parameters were determined by use of nonlinear regression analysis with SPSS
Version 11 for Windows (SPSS Science, Chicago, IL). The data were fit to the
Michaelis-Menten equation: v = (Vmax x
S)/(Km + S). Statistical analysis of
data on the effect of inhibitors on BUP metabolism was carried out using
one-way ANOVA with Tukey's comparison and deemed significant if the p
value was <0.05.
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Results |
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TopAbstractMaterials and MethodsOtherResultsDiscussionReferences |
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The activities of placental mitochondrial and microsomal fractions in
catalyzing the dealkylation of BUP and the marker substrate EFC were compared.
The ratio of mitochondrial to microsomal EFC O-deethylation was equal
to 1, and that for N-dealkylation of BUP was 0.5
(Table.1). These data indicate
that the dealkylation of BUP by placental microsomal fractions was more active
than that by the mitochondrial fractions. Minor contamination of the
microsomal fraction with mitochondrial enzymes cannot be ruled out, but a
similar observation has been reported earlier
(Pasanen et al., 1985) and is
addressed further in determination of aromatase activity utilizing its natural
substrate, testosterone.
The effect of BUP (10-200 µM) on EFC deethylation was investigated and its IC50 was determined (Fig. 1). The inhibition was concentration-dependent and was only observed in the placental preparations in the high activity group. The IC50 values for BUP in the mitochondrial and microsomal fractions were 129.2 ± 61.1 and 129.4 ± 75.9 µM, respectively. The inhibitory effect of BUP was observed whether the opiate was added before or simultaneously with EFC to the reaction mixture.
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Kinetic Parameters for BUP Dealkylation. Our data suggest that the enzyme catalyzing the dealkylation of BUP to norBUP may be present in the three subcellular fractions of placental tissue, with the microsomal fraction having the highest activity. Therefore, a pool of microsomal fractions prepared from six placentas (1-6, Table 2) was utilized to determine the apparent Km and Vmax for the dealkylation of BUP. The enzyme catalyzing the reaction required NADPH. Analysis of the saturation curves for the biotransformation of BUP to norBUP revealed an apparent Km of 12 ± 4.0 µM, a Vmax of 2.9 ± 0.7 pmol/mg protein · min, and an intrinsic clearance (Vmax/Km) value of 0.3 ± 0.1 µl/mg protein · min (Table 2; Fig. 2).
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The Effect of Inhibitors Selective for P450 Isoforms on BUP
Metabolism. Inhibitors selective for P450 isoforms were utilized to
identify the enzyme catalyzing the dealkylation of BUP to norBUP. Ketoconazole
(2.5 µM), 4-hydroxyandrostenedione (1.0 µM), and aminoglutethimide (10.0
µM) caused the highest inhibition (70% of control). -Naphthoflavone
and 4-methylpyrazole caused 20% and 40% inhibition at their respective
concentrations of 0.1 and 50 µM (Fig.
3). The least amount of inhibition (20%) was caused by 5 µM
quinidine, whereas sulfaphenazole and troleandomycin did not show any
inhibition of BUP metabolism in the concentration range tested.
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Ketoconazole is considered an inhibitor of hepatic CYP 3A but is also known
to inhibit human placental aromatase (Ayub
and Levell, 1988). The effect of ketoconazole on norBUP formation
by cDNA preparations expressing CYP 3A4 and CYP 19 was investigated.
Ketoconazole (2.5 µM) caused a 90% inhibition of BUP metabolism in both
preparations; i.e., it was as effective an inhibitor of CYP 19 as it was of
CYP 3A4. Taken together, our data suggest that the human placental microsomal
enzyme catalyzing N-dealkylation of BUP to norBUP could be CYP
19.
The Effect of Monoclonal Antibodies on BUP Metabolism. Monoclonal antibodies raised against specific human liver P450 isoforms were used to confirm the identification of the enzyme catalyzing BUP metabolism. A pool of microsomal preparations from 12 placentas was utilized in these experiments. Each monoclonal antibody raised against a specific P450 isoform was tested at its concentration causing 80% inhibition. The highest inhibition observed for BUP metabolism (70%) was observed in the presence of the antibodies raised against CYP 19. Monoclonal antibodies against CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and CYP 3A4/5 did not cause any significant inhibition of norBUP formation (Fig. 4). Data obtained on the effects of the selective inhibitors and monoclonal antibodies on the enzyme-catalyzed dealkylation of BUP indicate that it is CYP 19/aromatase.
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Kinetic Parameters for norBUP Formation by cDNA-Expressed CYP 19. The cDNA-expressed CYP 19 isozyme (Supersomes) catalyzed the dealkylation of BUP to norBUP. Analyses of the saturation curves obtained revealed apparent
Km and Vmax values of 14 ± 8 µM and 0.14 ± 0.1 pmol of norBUP/pmol CYP 19 · min, respectively (Fig. 5); i.e., the apparent Km value obtained is similar to that for the placental microsomal preparation.
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Aromatase Activity in the Subcellular Fractions. The activity of
aromatase/CYP 19 in placental tissue microsomal and mitochondrial fractions
was determined utilizing its natural substrate testosterone conversion to
estradiol. The ratio of the enzymatic activity in the mitochondrial to
microsomal fractions ranged between 0.32 and 0.56 with a mean value of 0.43. A
similar distribution for aromatase activity, as determined by the conversion
of androstenedione to estrogens between placental mitochondrial and microsomal
fractions, was reported earlier (Pasanen
et al., 1985). Taken together, our data indicate that the major
enzyme responsible for human placental tissue metabolism of BUP is CYP
19/aromatase.
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Discussion |
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TopAbstractMaterials and MethodsOtherResultsDiscussionReferences |
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;
Marquet et al., 1997;
Fischer et al., 2000;
Lejeune et al., 2001). A
recent report from our laboratory indicated that BUP transfer to the fetal
circuit is low when administered into the maternal circuit of the dually
perfused placental lobule; it accumulates in the tissue and is metabolized to
norBUP during the experimental period
(Nanovskaya et al., 2002).
This report provides data on identification and characterization of the human
placental enzyme responsible for the metabolism of BUP to norBUP.
Microsomal CYP 3A4 is the enzyme responsible for the biotransformation of
75% of BUP to norBUP during first-pass metabolism. The remaining 25% is
metabolized by CYP 3A5, 3A7, and 2C8, but other minor products may also be
formed by CYP 2C18, 2C19, 2D6, and 2E1
(Iribarne et al., 1997;
Kobayashi et al., 1998,
Moody et al., 2002).
Our working hypothesis was that the enzyme responsible for BUP metabolism
in the placenta was an oxygenase. Accordingly, the oxygenase activity of
placental subcellular fractions was determined by utilizing the EFC
O-deethylation to the fluorescent product HFC
(DeLuca et al., 1988;
Buters et al., 1993). Two
distinct forms of monooxygenases catalyze this reaction in placental tissue;
namely, a constitutive form that includes CYP 19/aromatase
(Meigs, 1987) and an inducible
CYP 1A1 (Pasanen et al.,
1990). The determined EFC O-deethylase activity in
placental subcellular fractions revealed a wide range of values for
Vmax (Table
1) and was arbitrarily divided into two groups: low activity, <
40pmol/mg protein · min, and the remainder as high activity. A similar
observation for O-deethylase activity was reported earlier; the high
activity group of placentas was associated with maternal smoking during
pregnancy and has been attributed to induction of CYP 1A1
(Pasanen et al., 1990). On the
other hand, the activity of our placental preparations in the
N-dealkylation of BUP did not reflect the wide range of variability
observed for the O-deethylation of EFC
(Table 1). Since the term
placentas utilized in this investigation were obtained from women who may have
been smokers, the involvement of CYP 1A1 in BUP metabolism was excluded.
The highest activity for the enzyme catalyzing the metabolism of BUP to
norBUP was in the microsomal fraction
(Table 1). The reaction
required NADPH and exhibited saturation kinetics with an apparent
Km of 12 µM and Vmax of 2.9 pmol/mg
protein · min (Fig. 2;
Table 2). An Eadie-Hofstee plot
of the data reveled monophasic kinetics indicating that either a single enzyme
or multiple enzymes with similar affinities catalyze the reaction. The values
obtained for intrinsic clearance (Table
2) ranged between 0.18 and 0.42 µl/mg protein · min
(mean = 0.27) and is considerably lower than that reported for human liver, 17
µl/mg protein · min (Kobayashi
et al., 1998). These data suggest that human liver is the major
site for BUP metabolism, but the placenta is also involved in
biotransformation of the opiate during pregnancy.
Two types of inhibitors were used to identify the placental microsomal
enzyme catalyzing BUP metabolism: the first type was chemicals selective for
P450 isoforms, and the second was monoclonal antibodies raised against
purified human liver enzymes. The most potent inhibitors of norBUP formation
were ketoconazole, 4-hydroxyandrostenedione, and aminoglutethimide
(Fig. 3), suggesting that CYP
3A4 and CYP 19 could be the enzymes catalyzing the reaction. In our
investigations, ketoconazole caused a 70% inhibition of norBUP formation
(Fig. 3) at its concentration
of 2.5 µM that is selective for its effect on CYP 3A4
(Fabre et al., 1993;
Baldwin et al., 1995;
Pelkonen et al., 1998).
Ketoconazole also inhibited the metabolism of BUP by two cDNA preparations
expressing CYP 3A4 and CYP 19. Therefore, our data confirmed earlier reports
that ketoconazole is an inhibitor of CYP 19
(Ayub and Levell, 1988), with
Ki values similar to those of hepatic microsomal CYP 3A4
(Stresser et al., 2000). A
role for CYP 3A4 as the enzyme catalyzing the metabolism of BUP by placental
microsomal preparations was excluded because troleandomycin, a selective
inhibitor of the isozyme (Newton et al.,
1995), had no effect on norBUP formation. Taken together, our data
on the effects of chemical inhibitors of the activity of placental microsomes
on BUP metabolism suggest that CYP 19 is the enzyme catalyzing the
reaction.
Monoclonal antibodies raised against CYP 19 were the most potent inhibitor of norBUP formation, confirming the profile of the chemical inhibitors and the conclusion drawn (Fig. 4). Moreover, analysis of the kinetics for BUP metabolism by a cDNA-expressed CYP 19 revealed a Km value similar to that obtained for the placental enzyme (Fig. 5).
The activity exhibited by the mitochondrial fraction in metabolizing BUP
and our direct determination of testosterone aromatization by CYP 19 in both
subcellular fractions are in agreement with earlier reports on the presence of
aromatase activity in the mitochodrial fractions
(Finkelstein et al., 1985;
Pasanen et al., 1985).
Our finding that CYP 19 is the enzyme responsible for the metabolism of BUP
in human placenta raises the issue of drug interactions during pregnancy.
Aromatase/CYP 19 is the enzyme responsible for the conversion of androgens to
estrogens and the metabolism of certain xenobiotics in human placenta
(Meigs, 1987;
Toma et al., 1996;
McNamara et al., 1999). It is
unclear whether BUP metabolism by placental microsomes would interfere with
the biosynthesis of these hormones or the detoxification of other drugs.
In summary, data in this report provided evidence that aromatase/CYP 19 is the enzyme catalyzing the metabolism of BUP to norBUP in trophoblast tissue obtained from term human placentas.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: BUP, buprenorphine; norBUP, norbuprenorphine; P450, cytochrome P450; EFC, 7-ethoxy-4-trifluoromethylcoumarin; HFC, 7-hydroxy-4-trifluoromethylcoumarin; LC/MS, liquid chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; ANOVA, analysis of variance.
Address correspondence to: Dr. Mahmoud S. Ahmed, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0587. E-mail: maahmed{at}utmb.edu
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Km) for 30 min at 37°C except troleandomycin, which
was preincubated with the microsomes for 15 min before adding BUP. Data are
represented as percentage of the control (in the absence of inhibitors). Each
concentration of the inhibitor was tested in three placental microsomal
preparations. * indicates statistical significance of p
< 0.05 as determined by one-way ANOVA with Tukey's comparison.
