Vol. 30, Issue 11, 1149-1152, November 2002
SHORT COMMUNICATION
Docetaxel (Taxotere) Is Not Metabolized by Recombinant Human
CYP1B1 in Vitro, but Acts as an Effector of This Isozyme.
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Abstract |
The objective of this study was to determine whether recombinant
human cytochrome P450 1B1 (rhCYP1B1) metabolizes the anticancer agent
docetaxel (Taxotere) in vitro. First, the catalytic activities of Supersomes-expressed rhCYP1B1 toward 17
-estradiol and of rhCYP3A4 toward docetaxel in our conditions were determined. Second,
[14C]docetaxel at 0.1 and 1 µM was incubated with
rhCYP1B1 in the presence of NADPH up to 60 min. No metabolism of
docetaxel was detected. Third, several activators of P450 isoenzymes
were added to docetaxel incubations with rhCYP1B1, such as 2-chloro
3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine 1-carboxamide,
-naphthoflavone, and organic solvents. Again, no metabolism of
docetaxel was detected. As a forth step, 10 incubation factors were
tested at two levels each in 16 different combinations, using a
fractional factorial statistical experimental design. Docetaxel was not
metabolized by rhCYP1B1 under any of the combinations. As a final step,
the effect of docetaxel on the rhCYP1B1-mediated 7-ethoxyresorufin O-deethylase (EROD) activity was studied, to evaluate if
docetaxel can bind to CYP1B1. -Naphthoflavone (1 µM), a CYP1B1
inhibitor, totally inhibited the EROD activity. Docetaxel at 3, 10, and
30 µM did not show major effects on EROD activity. At 100 µM,
docetaxel increased EROD activity by 3.8-fold. Additionally, it was
shown that 7-epidocetaxel, which is in equilibrium with docetaxel as a
minor compound in solutions, was a potent activator of rhCYP1B1, with a
>7-fold increase of EROD activity at 10 µM. In conclusion, docetaxel
was not metabolized by recombinant human CYP1B1 in vitro, under any of
the conditions tested. Docetaxel was shown to bind to recombinant human
CYP1B1 and to act as an effector of this enzyme.
| |
Introduction |
Docetaxel
(Taxotere) is a major chemotherapeutic molecule with particular potency
against breast cancers. Its mechanism of cytotoxicity is thought to be
related to its ability to alter tubulin processing within the cells;
docetaxel increases the rate of tubulin polymerization and inhibits the
depolymerization of microtubules. Docetaxel is metabolized by CYP3A4-
and CYP3A5-mediated oxidations (Shou et al., 1998
). Recently, a
decrease of the in vitro cytotoxic potency of docetaxel was observed in
Chinese hamster ovary cells transfected with human cytochrome P450 1B1
(hCYP1B11). In addition, this effect was reversed
in the presence of -naphthoflavone, an inhibitor of hCYP1B1
(McFadyen et al., 2001). The authors concluded that hCYP1B1 expression
could be a mechanism of tumor resistance to docetaxel, and they
referred to preliminary studies showing that docetaxel was metabolized
by hCYP1B1. However, this was not exactly the case. In fact, these
earlier studies only showed that docetaxel was a competitive inhibitor
of the hCYP1B1-mediated ethoxyresorufin O-deethylation, with
a Ki of 28 µM (Rochat et al., 2001).
Since CYP1B1 is expressed in a wide variety of human tumors (Murray et
al., 1997), it was considered necessary to have direct evidence of its
possible catalytic activity on docetaxel.
| |
Materials and Methods |
Chemicals.
-Naphthoflavone, 17-estradiol, ethoxyresorufin, NADPH, EDTA,
ascorbic acid, and reduced glutathione were purchased from Sigma-Aldrich (St. Louis, MO).
[14C]docetaxel was purchased from Amersham
Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). 2-Chloro
3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine 1-carboxamide (CMV423;
Bournique et al., 2001) was synthesized at the Centre de Recherche de
Vitry-Alfortville, Aventis Pharma, (Vitry-Alfortville, France).
Enzymes.
Recombinant human cytochrome P450 1B1 (rhCYP1B1) and 3A4 (rhCYP3A4)
expressed in insect cells (Supersomes) were purchased from BD Gentest
Corporation (Woburn, MA). The specific contents were 138.9 and
208.3 pmol P450/mg of microsomal protein, respectively. The cytochrome
c reductase activity of the coexpressed human NADPH P450
reductase was 260 and 3500 nmol/min/mg, respectively. Human cytochrome
b5 was also coexpressed with rhCYP3A4, at
a specific content of 1200 pmol/mg. The activities of rhCYP1B1 and 3A4
are shown in Table 1. Purification of
rabbit liver cytochrome b5 was performed
as described by Strittmatter et al., 1978.
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TABLE 1
Catalytic activities of recombinant human CYP1B1 and 3A4
7-ethoxyresorufin O-deethylase activity (EROD) was measured
in 0.1 M phosphate buffer, pH 7.4, containing 10 pmol rhCYP1B1/ml and
1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL
glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride.
17-estradiol biotransformation was measured in 0.1 M phosphate
buffer, pH 7.4, containing 25 pmol CYP1B1/ml, 2 mM ascorbic acid, and 1 mM NADPH. Testosterone 6-hydroxylase activity was measured in the
same conditions as EROD, using 20 pmol CYP3A4/ml. Docetaxel
biotransformation was measured in the same conditions as
17-estradiol, except that ascorbic acid was omitted in the case of
CYP3A4.
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Incubations.
17-Estradiol in methanol was incubated at 1 µM, final
concentration, in the presence of 25 pmol/ml of rhCYP1B1 Supersomes with 1 mM NADPH and 2 mM ascorbic acid, in 0.1 M phosphate buffer, pH
7.4, at 37°C in a shaking water bath, up to 60 min. The concentration of methanol in the incubations was 0.5%. The reaction was stopped by
the addition of 1 volume of methanol/acetonitrile 60:40 (v/v) with 6%
perchloric acid. Supernatants were analyzed by HPLC with fluorescence detection.
[14C]docetaxel, in methanol, was incubated at
0.1, 1, or 30 µM, final concentrations, in the presence of 25 pmol/ml
of rhCYP1B1 and with 1 mM NADPH, in 0.1 M phosphate buffer, pH 7.4, at
37°C in a shaking water bath. The incubation lasted 30 min in the
statistical design and 60 min in the other experiments. The
concentration of methanol in the incubations was 0.5%. Reactions were
terminated by the addition of 1 volume of methanol. After
centrifugation, supernatants were analyzed by HPLC. Alternatively,
[14C]docetaxel was incubated as described above
in the presence of potential P450 activators, namely CMV423 (Bournique
et al., 2001) at 10 and 100 µM, final concentrations,
-naphthoflavone at 3, 10, 30, and 100 µM, final concentrations,
acetonitrile, ethanol, methanol, and DMSO at 2%, final concentration.
These compounds were added with [14C]docetaxel
before the start of the reaction with NADPH. In the statistical
experimental design, [14C]docetaxel in the
presence of 25 pmol/ml of rhCYP1B1 was incubated in 16 different
combinations of 10 incubation factors, using a fractional factorial
design. The incubation factors were the docetaxel (1 and 30 µM), the
buffer (25 and 100 mM), and the NADPH (0.6 and 2 mM) concentration, the
buffer type (phosphate and Tris), the starting mode of the reaction
(NADPH and cold microsomes), and the presence of magnesium (0; 10 mM),
cytochrome b5 (0; 50 pmol/ml), EDTA
(0; 1 mM), ascorbic acid (0; 2 mM), and reduced glutathione (0; 5 mM).
Inhibitions of 7-ethoxyresorufin O-deethylase activity
(EROD) were measured as follows. rhCYP1B1 (5 pmol) in 1 ml 0.1 M
phosphate buffer, pH 7.4, containing 1 mM NADPH were preincubated under agitation at 37°C during 1 min in glass tubes, in the presence of 1 µM -naphthoflavone or in the presence of 0, 3, 10, 30, and 100 µM docetaxel or 7-epidocetaxel. The reactions were started by the
addition of 1 µM 7-ethoxyresorufin and allowed to proceed during 2 min. Stock solutions of substrates and inhibitors were in DMSO. The
total final concentration of DMSO was 1% in all incubations. Reactions
were stopped by the addition of 1 ml of cold phosphate buffer 0.1 M, pH
7.4, containing 1% DMSO, vortex and kept on ice. After centrifugation
at 4°C, supernatants were assayed for resorufin. Resorufin was
measured by fluorescence on a SFM KONTRON (Saint Quentin en Yvelines,
France), using a calibration of 100 mFAU for 0.25 µM
resorufin. The fluorescence of resorufine was not modified in the
presence of docetaxel and 7-epidocetaxel, whatever their concentration.
Preliminary measurements by on-line fluorescence kinetics indicated
that all the reactions were linear within the two first minutes.
Surprisingly, when 7-ethoxyresorufin was preincubated in the presence
of docetaxel, and the reactions started by the addition of NADPH,
although the kinetics were still linear during 2 min, highly variable
results were repeatedly obtained within each triplicate, showing either
activation or inhibition of the rhCYP1B1-mediated EROD activity.
HPLC Analysis.
Supernatants of 17-estradiol incubations were analyzed on a
C18 Hypersil BDS column (Hypersil, Runcorn,
Cheshire, UK). Chromatography was effected using a mobile phase
of water/methanol/acetonitrile (40:40:20 with 0.05% trifluoroacetic
acid) at a flow rate of 0.6 ml/min. The retention times of
4-hydroxyestradiol, 2-hydroxyestradiol, and estradiol were 6, 6.7, and
8.6 min, respectively.
Supernatants of [14C]docetaxel incubations were
analyzed by reverse phase gradient HPLC with radiodetection (limit of
quantification, 5 nM) as previously described (Marre et al., 1996).
Data Analysis.
Rates of metabolism were calculated with GraFit software,
Sigma-Aldrich. The statistical experimental design was built, analyzed, and validated with JMP (Cary, NC) and Modde (Umea, Sweden) software packages, as previously described (Bournique et al., 1999). Analyses of
the results were performed by multilinear regression.
| |
Results and Discussion |
In the first instance, the activity of the preparation of rhCYP1B1
was measured on a prototypic substrate, 17-estradiol. As shown in
Fig. 1 and Table 1, rhCYP1B1 metabolized
17-estradiol (1 µM) in our incubation conditions with an initial
rate of 2.3 min
1 and a
4-hydroxyestradiol/2-hydroxyestradiol ratio of 5, in agreement with
previous studies (Hanna et al., 2000; Li et al., 2000). In the second
instance, the metabolism of docetaxel by Supersomes-expressed P450s in
our incubation conditions was studied. As shown in Fig. 2 and Table 1, docetaxel was
significantly metabolized by rhCYP3A4 Supersomes, as expected. The
metabolism of docetaxel by rhCYP1B1 was then studied. When
[14C]docetaxel at 0.1 and 1 µM was repeatedly
incubated up to 60 min with rhCYP1B1, no metabolism of docetaxel was
detected. It was then decided to investigate other rhCYP1B1/docetaxel
incubation conditions. The effect of some P450 isoenzymes activators,
namely CMV423 (Bournique et al., 1999), -naphthoflavone, and organic solvents (see Materials and Methods) was tested. Recombinant
hCYP1B1 did not metabolize docetaxel in the presence of any of these
potential activators. Therefore, the effect of 10 incubation factors on the activity of rhCYP1B1 on docetaxel was studied. These factors were
the docetaxel concentration, the buffer type, the buffer concentration, the presence of magnesium, the presence of rabbit liver
purified cytochrome b5, the starting
mode of the reaction, the NADPH concentration, and the presence of
EDTA, ascorbic acid, and reduced glutathione. Two levels were selected
for each factor, as indicated under Materials and Methods.
Studying all the factor combinations would have needed 1024 (210) assays. A fractional factorial design of
only 16 factor combinations was used to rapidly and efficiently detect
the factors that could be important for an activity of rhCYP1B1 toward
docetaxel. When these experiments were performed, again, no metabolism
of docetaxel was detected.
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Fig. 1.
Metabolism of estradiol by recombinant
hCYP1B1.
Estradiol concentration was 1 µM. Recombinant hCYP1B1 (Supersomes)
concentration was 25 pmol/ml. Incubation was performed during 60 min in
0.1 M phosphate buffer, pH 7.4, at 37°C, with 1 mM NADPH and 2 mM
ascorbic acid. HPLC analysis is described under Materials and
Methods. E2, 17-estradiol; 4-OH, 4-hydroxyestradiol; 2-OH,
2-hydroxyestradiol. The arrow indicates a presteady state period of 5 min.
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Fig. 2.
Metabolism of docetaxel by recombinant
hCYP3A4.
Docetaxel concentration was 1 µM. Recombinant hCYP3A4 (Supersomes)
concentration was 50 pmol/ml. Incubation was performed during 60 min in
0.1 M phosphate buffer, pH 7.4, at 37°C, with 1 mM NADPH. HPLC
analysis is described under Materials and Methods. Peaks
1, 2, 3, and 4, oxidative metabolites of docetaxel (Marre F. et al.,
1996).
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However, 7-epidocetaxel was detected in some experiments.
7-Epidocetaxel is an epimer of docetacel that is formed from docetaxel when docetaxel is in solution. The concentrations of 7-epidocetaxel are
usually very low as compared with those of docetaxel. In the present
experiments, 7-epidocetaxel was detected when the concentration of
docetaxel was high, and it was already present at the start of the
reaction (t0). After 30 min of
incubation, the concentration of 7-epidocetaxel either increased,
decreased, or maintained relatively to the experiments. The analysis of
the effects of the factors on the concentration of 7-epidocetaxel was
performed by multilinear regression. No significant effects were found
to explain the variation of 7-epidocetaxel from 0 to 30 min of
incubation, but significant effects (p < 0.05)
were detected relatively to the concentration of 7-epidocetaxel at
t0 (Fig.
3). The significantly influential factors
were docetaxel concentration, buffer type, and glutathione. More
precisely, the docetaxel concentration of 30 µM, the phosphate buffer, and the absence of glutathione increased the concentration of
7-epidocetaxel at t0. None of the
other tested factors were influential. As a next step, 1 µM
7-epidocetaxel was incubated with rhCYP1B1 in the standard conditions
described above for docetaxel, to test if docetaxel could be
metabolized by rhCYP1B1 via 7-epidocetaxel. No metabolism of
7-epidocetaxel was detected either. As a final step, the effect of
docetaxel and 7-epidocetaxel on the rhCYP1B1-mediated EROD activity was
studied, to evaluate if these molecules can bind to rhCYP1B1 in our
conditions. Results are shown in Table 2.
-Napthoflavone (1 µM), used as a control inhibitor, totally abolished the rhCYP1B1-mediated EROD activity, as expected. Docetaxel at 3 µM had no significant effect on the rhCYP1B1-mediated EROD activity. At the concentrations of 10 and 30 µM, docetaxel increased and decreased the activity by 30 and 22%, respectively. At 100 µM,
docetaxel increased by 3.8-fold the rhCYP1B1-mediated EROD activity,
showing a marked activatory effect. 7-Epidocetaxel at 3 µM had no
significant effect on the rhCYP1B1-mediated EROD activity. At 10, 30, and 100 µM, 7-epidocetaxel increased the activity by >7-, 4.8-, and
4.7-fold, respectively, showing 7-epidocetaxel as a potent CYP1B1
activator. These results are in complete contradiction to those of
Rochat et al. (2001) who found that docetaxel competitively inhibited
rhCYP1B1-mediated EROD activity with a
Ki of 28 µM. Possibly, the differing
incubation conditions could explain this discrepancy. For example,
Rochat et al. used 10 mM MgCl2 in their incubation medium, whereas we did not add any
MgCl2 in our own incubations. It has been shown
that the concentration of divalent cations has dramatic influences on
the kinetics of cytochrome P450s, particularly on CYP3A4 (Peters and
Fouts, 1970; Gillam et al., 1995; Yamazaki et al., 1995; Bournique et
al., 1999). In addition, the design of the interaction experiment
influenced the effect of docetaxel on the rhCYP1B1-mediated EROD
activity (see Methods and Materials), which may also explain
the discrepancy between the studies. On the whole, this last experiment
demonstrated 1- that docetaxel and 7-epidocetaxel can bind to rhCYP1B1
and 2- that they are effectors of rhCYP1B1 in vitro.
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Fig. 3.
Effects of 10 factors on the concentration
of 7-epidocetaxel at t0 of the incubation.
The effects were calculated by multilinear regression. N.S., not
significant at p = 0.05.
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TABLE 2
Effect of -naphthoflavone, docetaxel and 7-epidocetaxel on
rhCYP1B1-mediated EROD activity in vitro
Effects are given in % of controls without inhibitors (vehicle only)
as means (S.D.) of triplicate. 7-ethoxyresorufin
O-deethylase activity (EROD) was measured in 0.1 M phosphate
buffer pH 7.4, containing 5 pmol rhCYP1B1/mL, 1 mM NADPH, and the
molecule to bested. After 1-min preincubation at 37°C, reactions were
started by the addition of ethoxyresorufin, 1 µM. Substrate and
inhibitors were added in DMSO (1%, total final concentration).
Reactions were stopped after 2-min incubation at 37° by the addition
of 1 ml cold phosphate buffer 0.1 M, pH 7.4, containing 1% DMSO,
vortex and kept on ice. After centrifugation at 4°C, the
concentration of resorufin in the supernatants was measured with a
spectrofluorimeter, using a calibration with reference resorufin.
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In conclusion, docetaxel was not metabolized in vitro by recombinant
human CYP1B1 expressed in Supersomes. Several hypothesis can be
proposed, therefore, to explain the protective effect of transfected
hCYP1B1 on docetaxel-exposed Chinese hamster ovary cells (McFadyen et
al., 2001). If docetaxel was effectively metabolized by CYP1B1 in these
cells, this may suggest an intracellular activator of CYP1B1. If
docetaxel was not metabolized by CYP1B1 in the Chinese hamster ovary
cells, in agreement with the in vitro results, then the demonstrated
binding and effector effect of docetaxel on CYP1B1 might either
decrease the intracellular active concentration of docetaxel or trigger
an intracellular protective signaling pathway. Another possibility is
that CYP1B1 is involved in the synthesis of an important signaling
molecule which may reinforce the tubulin processing within the cell,
rendering the effect of docetaxel less efficient. In that case, the
effect is not due to the binding of docetaxel to CYP1B1. Further
investigations are needed to explain how CYP1B1 may decrease the
cytotoxic potency of docetaxel.
Bruno Bournique
Audrey Lemarié
Biochemistry of Drug Metabolism,
Drug Metabolism and
Pharmacokinetics,
Aventis Pharma, Vitry-Alfortville, France
| |
Footnotes |
Received March 20, 2002; accepted July 22, 2002.
Address correspondence to: Bruno Bournique, ProSkelia
Pharmaceuticals, DMPK, 102 route de Noisy, 93230 Romainville, France.
E-mail: bruno.bournique{at}proskelia.com
| |
Abbreviations |
Abbreviations used are:
hCYPx, human cytochrome
P450 1B1, 3A4;
rhCYPx, recombinant human cytochrome P450 1B1, 3A4;
HPLC, high-performance liquid chromotography;
EROD, 7-ethoxyresorufin
O-deethylase activity;
DMSO, dimethyl sulfoxide;
CMV423, 2-chloro 3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine 1-carboxamide;
mFAV, milli fluorescence absorbance unit.
| |
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DMD, 30:1149-1152, 2002
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
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Abstract
Introduction
