Biomedical Research Centre, University of Dundee, Ninewells
Hospital and Medical School, Dundee, United Kingdom (L.B.G., C.R.W.,
T.F.); and Department of Dermatology, University Hospital,
Rheinland-Westfälische Technische Hochschule (RWTH), Aachen,
Germany (J.M.B.)
Some compounds used for phenotyping of cytochrome P450s are substrates
of P-glycoprotein (pgp). It is likely that in these cases, the level of
pgp modulates the metabolism of in vivo probes. To address this
important issue, we have analyzed the effects of pgp on CYP3A4-mediated
reactions in two newly established cell lines (3A4/HR/MDR
and 3A4/HR/MDR+), which express CYP3A4 in the absence and
presence of pgp, respectively. In cultured cells, the presence of pgp
increased the apparent Km for the
6
-hydroxylase activity of CYP3A4 toward testosterone and cortisol by
a factor of 1.7 and 4, respectively. These steroids are poor and good
substrates of pgp, respectively, and cortisol 6-hydroxylase has been
frequently used as an in vivo probe for CYP3A4. Interestingly, we also
found that pgp modulated the inhibition of CYP3A4-mediated metabolism
by several compounds in intact cells. Although quinidine inhibited
testosterone 6-hydroxylase activity in membranes or in intact cells
that expressed recombinant CYP3A4 in the absence of pgp, low
concentrations of this compound increased CYP3A4 activity in intact
cells that expressed pgp. These results imply that pharmacokinetic
drug-drug interactions involving CYP3A4 can be influenced by pgp.
 |
Introduction |
Drug
disposition may be conceptualized as consisting of absorption,
distribution, biotransformation, and excretion (Parkinson, 1996
). The
latter three processes also control the disposition of several
endogenous compounds such as steroids. Phase I of drug metabolism is
mainly catalyzed by cytochrome P450s (P450). The cellular uptake and
export of xenobiotics or their metabolites is mediated by drug transporters.
P450-mediated metabolism displays interindividual variability either
due to genetic polymorphisms, which affect the catalytic properties of
the various P450 isoforms, or due to differences in the level of P450
enzymes, which are determined by endogenous and exogenous factors (Daly
et al., 1993; Kroemer and Eichelbaum, 1995; Smith et al., 1998).
Because interindividual differences of P450-catalyzed drug metabolism
have important implications for drug therapy and also determine disease
susceptibility (Wormhoudt et al., 1999), geno- and phenotyping of these
enzymes have become important issues. Ideally, the metabolism of the in
vivo metabolic probes should be only dependent on the level and the
catalytic properties of the P450 that metabolizes them. Phase II
metabolism of the resulting metabolites would interfere with accurate
phenotyping. Similarly, drug uptake or export is likely to affect the
probe reaching its target P450, thereby influencing these assays. This can occur with compounds that are simultaneously substrates of drug
transporters and of P450s.
Most studies have investigated the interaction of CYP3A4 or orthologous
CYP3A enzymes, and an important drug transporter, namely,
P-glycoprotein (pgp, human MDR1), which share many substrates and/or
inhibitors (Kivisto et al., 1995). For example, cortisol, quinidine,
and erythromycin, which are substrates of in vivo phenotyping assays
for CYP3A4, are also substrates of pgp (Ged et al., 1989; Barnes et
al., 1996; Kim et al., 1999). Several reports have addressed the
important question of how the combined action of CYP3A4 and pgp
influences the pharmacokinetics and the cytotoxicity of therapeutic drugs and how drug-drug interactions impinge on this process (Gan et
al., 1995; Wacher et al., 1995; Lown et al., 1997; Watkins, 1997; Hall
et al., 1999). However, it has been extremely difficult to evaluate the
contribution of each component to these parameters, mostly because
selective inhibitors that discern between both processes have been
until recently unavailable. One attempt to circumvent this problem has
been to correlate the levels of CYP3A and pgp in tissues with
pharmacokinetic data (Lown et al., 1997). Another approach used
transgenic mice in which the mdr1a and the mdr1b
genes have been inactivated by homologous recombination, resulting in changes in bioavailability and the toxicity of several compounds (Schinkel et al., 1995). However, to our knowledge, no data
are available on the effect of pgp on CYP3A-mediated metabolism in this
model. This approach may also be problematic because the catalytic
properties of rodent P450s differ from those of human P450s (Gonzalez,
1992). It has been also demonstrated that human and mouse pgp have
different substrate specificities (Tang-Wai et al., 1995).
Consequently, the interplay between P450 and pgp in rodents is likely
to be different from that in humans.
The development of cell lines that coexpress pgp and CYP3A4 was
hampered by the rapid repression of CYP3A4 in vitro. One study achieved
culture conditions that lead to CYP3A4 expression in the human
intestinal cell line Caco-2 (Schmiedlin-Ren et al., 1997), which
expresses low levels of pgp. Another report described the heterologous
expression of CYP3A4 in Caco-2 cells. These cell lines could be used to
study the influence of pgp on CYP3A4-mediated metabolism, by comparing
metabolism in intact cells with that in membrane fractions. However, no
data are available on this issue. This approach has also the
disadvantage that other factors such as membrane permeability, cellular
milieu, and cofactor availability may contribute to differences in
intact cells compared with lysates.
Some pharmacokinetic drug-drug interactions are often a composite,
resulting from the interplay of pgp and CYP3A4, which unfortunately complicates interpretation of the observed effects. In one study, it
was shown that the antifungal ketoconazole or the immunosuppressant cyclosporin A increased the area under the curve for vinca alkaloids, most likely by inhibiting pgp and CYP3A. However, the contribution of
both pathways to the increased bioavailability of vinca alkaloids is
not clear (Chan, 1998). Similarly simultaneous inhibition of pgp and
CYP3A has been postulated to be responsible for the effects of
ketoconazole on the bioavailability of digoxin or the cysteine protease
inhibitor KO2 (Salphati and Benet, 1998).
Because the impact of pgp on CYP3A4-mediated reactions and
pharmacokinetic drug-drug interactions is difficult to dissect in vivo,
we developed an in vitro model to address this important issue.
| |
Experimental Procedures |
Materials.
Bovine serum albumin, verapamil, cortisol,
testosterone, 
-naphthoflavone, ketoconazole, and quinidine were
obtained from Sigma (Poole, UK). 6-Hydroxytestosterone was kindly
provided by Steraloids Inc. (New Port, RI) and 6-hydroxycortisol was
obtained from Research Plus (Bayonne, NJ). The multidrug resistance
modulator LY335979 was provided by Lilly (Windlesham, UK). Cell culture media and the Lipofectin reagent were purchased from GIBCO (Paisley, Scotland). Calcein acetoxymethyl ester (calcein AM) was purchased from
Molecular Probes (Eugene, Oregon).
[3H]Vinblastine (12.7 Ci/mmol) was obtained
from Amersham (Cardiff, UK). Other reagents were of highest grade
commercially available.
Cell Culture, DNA Transfection, and Isolation of Cell Lines.
1847 human ovarian cancer cells (Louie et al., 1986) were grown under
standard cell culture conditions in 1640 RPMI medium supplemented with
10% fetal bovine serum, penicillin (50 IU/ml), and streptomycin
(50 µg/ml).
This cell line was made resistant to paclitaxel (Taxol, Bristol-Myers
Squibb, Stamford, CT) by initially treating the cells with the
mutagen ethylmethanesulfonate (EMS) for 24 h, allowing the cells
to recover in the absence of EMS for 48 h before treating with 1 nM paclitaxel. The cells were allowed to adapt to this concentration of
paclitaxel for three passages before the paclitaxel concentration was
increased to 2.5 nM. This incremental exposure to drug was continued
until cells became resistant to 10 µM paclitaxel. Expression of pgp
in different clones was analyzed by immunoblotting.
1847 cells overexpressing human pgp (termed MDR+
cells) as well as parental cells (termed MDR
cells) were cotransfected with the plasmids pDHFR/3A4 carrying the
CYP3A4 cDNA under the control of the cytomegalovirus promoter and pCDNA
containing the G-418 resistance gene (Ding et al., 1997) using the
Lipofectin. In brief, 2 × 105 cells were
incubated in 60-mm tissue culture plates in 4 ml of standard growth
medium for 24 h. Plasmid DNA (2 µg) and 20 µl of Lipofectin
reagent were each diluted in 100 µl of Opti-MEM I medium. Both
solutions were allowed to stand at room temperature for 45 min, mixed,
and incubated at room temperature for further 15 min. Opti-MEM I (1.8 ml) was added and the 1847 cells were overlaid with this solution after
being washed with Opti-MEM1 once. After 24 h of incubation at
37°C, the DNA-containing medium was replaced with 4 ml of normal
growth medium and the incubation continued for another 48 h.
Finally, cells were split and selected by G-418 (400 µg/ml). Cells
derived from MDR+ clones were grown in the
presence of additional paclitaxel (1 µM). Expression of CYP3A4 was
detected by immunoblotting.
Lipofection was also used for the cotransfection of cells with the
vectors pcDNAHR and pcDNA/Zeo+ carrying the human P450 reductase cDNA
under the control of the cytomegalovirus promoter (Ding et al., 1997)
and, respectively, the marker conferring resistance to zeocin. Cell
clones were selected using zeocin (100 µg/ml). After isolation of
zeocin-resistant colonies, the concentration of zeocin was changed to
50 µg/ml and 400 µg/ml G-418. Cells derived from
MDR+ clones were grown in the presence of
additional paclitaxel (1 µM).
Immunochemical Detection of pgp, CYP3A4, and P450 Reductase.
For isolation of total cellular protein, cells were harvested by
trypsin treatment and resuspended in a buffer containing 10 mM sodium
phosphate (pH 8.0), 1 mM EDTA, and 2 mM dithiothreitol. Cells were
lysed by two 8-s bursts at an amplitude of 12 µm using an MSE
Soniprep 150 sonicator, leaving the samples frequently on ice. Protein
concentration was determined spectrophotometrically using the Bradford
reagent (Bio-Rad, Hemel Hempstead, UK) and bovine serum albumin as standard.
The expression of pgp, CYP3A4, and P450 reductase was analyzed by
immunoblotting (Towbin et al., 1979) using as primary antibodies the
monoclonal anti-pgp C219 (Signet Lab, Glasgow, UK) and polyclonal antibodies directed against CYP3A4 or human P450 reductase (Li et al.,
1999). Secondary antibodies were coupled to horseradish peroxidase and
were detected by fluorography using the enhanced chemiluminescence
Western blotting kit (Amersham, Cardiff, UK), according to the
manufacturer's instructions. For detection of pgp, samples were
denatured by suspension in loading buffer containing 200 mM
dithiothreitol and subsequent incubation at room temperature for 5 min.
For the detection of CYP3A4 and P450 reductase, samples were boiled in
loading buffer. Generally, 60 µg of protein was loaded per lane.
Determination of the CYP3A4 Activity in Cultured Cells and Cell
Lysates.
For the detection of the testosterone 6-hydroxylase
activity in cultured cells, 5 × 105 cells
were seeded per 60-mm culture dish and incubated in standard growth
medium at 37°C for 24 h (Ding et al., 1997). Hemin was added to
a final concentration of 5 µg/ml and cells cultured for a further
24 h. Medium was replaced with 4 ml of fresh medium and incubated
for 1 h at 37°C. Testosterone (dissolved in methanol) was added
directly into the culture medium to a final concentration of 100 µM
if not stated otherwise. After 3 h of incubation, 3.3 ml of medium
was transferred to a polypropylene tube containing 1.1 ml of ice-cold
methanol and left on ice for about 10 min. Subsequently, the remaining
medium was removed and cells were harvested and lysed by sonication for
determination of the total protein concentration. Before HPLC,
metabolites were concentrated using Isolute C18 columns (IST Ltd,
Midglamorgan, UK) and after washing with 4 ml of 25% methanol,
the samples were eluted with 1 ml of methanol. The solvent was
evaporated and the residue was resuspended in 200 µl of 35%
methanol, and then centrifuged to pellet-insoluble material.
Metabolites were separated by HPLC.
For the detection of testosterone 6-hydroxylase activity in cell
lysate, 5 × 106 cells were cultured for 3 days as described above and harvested by trypsinization, resuspended,
and sonicated (see above). Cell lysate (5 mg) was incubated for 1 h at 37°C in an assay buffer containing 0.05 M Hepes (pH 7.4), 30 mM
MgC12, 200 µM testosterone, and 1 mM NADPH. The
HPLC assay was performed as described above. For determination of
CYP3A4 activity in membranes isolated from recombinant
Escherichia coli (Blake et al., 1996), the cell lysate in
the assay was replaced with membranes containing 100 pmol CYP3A4 and
P450 reductase (approximately 1000 nmol of cytochrome c
reduced per minute per milligram of protein). Incubation was for
20 min at 37°C. The assay conditions were found to be linear with
time and protein.
The determination of the cortisol 6-hydroxylase was conducted as
follows: 1.5 × 106 cells were cultured for
3 days as described above. The medium was replaced by 7 ml of fresh
medium and incubated for 1 h at 37°C. Cortisol (dissolved in
DMSO) was added directly into the culture medium to a final
concentration of 200 µM unless stated otherwise. After 14 h of
incubation, 6.6 ml of medium was transferred to a polypropylene tube
containing 2.2 ml of ice-cold methanol and left on ice for about 10 min. Samples were prepared as described above using Isolute C18 columns
for concentration of the metabolites and the total protein
concentration was determined. Separation of the metabolites by HPLC was
performed on an Inertsil ODS-2 column (5 µm, 4.6 × 250 mm;
Phase Separations, Ltd, Watford, UK) at a flow rate of 1 ml/min using
isocratic elution with 8% ethanol. The effluent was monitored at 240 nm. The assay conditions were found to be linear with time and protein.
Determination of pgp Activity.
Calcein AM ester (Molecular
Probes) was used to study the efflux activity of pgp (Homolya et al.,
1993). Cells were seeded at 5 × 105
cells/ml for 5 days in 10 ml of cell culture medium in an
80-cm2 flask. Briefly, cells were trypsinized and
washed once with HPMI buffer [120 mM NaCl, 5 mM KCl, 0.4 mM
MgCl2, 10 mM HEPES-Na (pH 7.4), 10 mM
NaHCO3, 10 mM glucose, and 5 mM
NaHPO4]. They were then resuspended in the HPMI
buffer and incubated with 0.25 µM calcein AM at 37°C for 10 min
(concentration of solvent in medium, 0.5% DMSO). Incubation was
terminated by centrifugation, followed by washing of the cell pellet
with HPMI buffer. The final pellet was resuspended in 1 ml of lysis
solution [0.25 M sucrose, 10 mM Hepes (pH 7.6), and 1 mM EDTA].
Fluorescence was measured at 493/515 nm. A high level of fluorescence
indicates a high intracellular retention of calcein, reflecting a low
activity of pgp.
Effects of pgp on the accumulation of vinblastine, testosterone, and
cortisol in these cell lines were determined by a modification of the
published procedure using radiolabeled substrate (Stein et al., 1994).
Cells were seeded onto six-well plates at a density of
105 cells/well and grown for 3 to 4 days in 5 ml
of medium. For the accumulation of the steroids, cells were washed
three times with 5 ml of phosphate-buffered saline, leaving them for 10 min at room temperature between each wash. Subsequently, the experiment was carried out using serum-free medium. To measure vinblastine accumulation, cells were washed only once and accumulation was studied
using serum-containing medium. To initiate the experiment, cells were
incubated with 1 ml of fresh medium. After 30 min at 37°C in 5%
CO2, another 1 ml of medium containing 1 µM
tritiated substrate was added to a final concentration of 0.5 µM
(concentration of solvent in medium, 0.5% DMSO). After 30 min (for
vinblastine) or 1 h (for steroids), reactions were terminated by
aspirating the medium and rinsing the cells three times with 5 ml of
cold phosphate-buffered saline. Cells were lysed by adding 0.5 ml of 0.4 M NaOH and neutralized with 0.5 ml of 0.5 M
NH4OAc, pH 6.6. Lysate (400 µl) was counted in
a scintillant. Separate wells were seeded to determine the number of
cells per well. Results were normalized against cell number.
| |
Results |
Establishment and Characterization of Cell Lines.
The cell
lines that were used for the coexpression of CYP3A4 and P450 reductase
were the human ovarian cancer cell line 1847 (Louie et al., 1986) and a
derivative cell line that had been previously selected to overexpress
endogenous pgp. The latter cell line was isolated by treating the
parental pgp-negative 1847 cells (termed MDR
cells) with the mutagen EMS followed by selection on increasing concentrations of paclitaxel (up to 10 µM). One of the resistant clones (termed MDR+ cells) and
MDR cells were subsequently transfected with an
expression vector containing the CYP3A4 cDNA and selected on G-418. In
the case of MDR+-derived cells, the medium also
contained paclitaxel to prevent loss of pgp. The resulting clones were
screened by immunoblotting for the expression of CYP3A4. One of the
positive clones derived either from MDR or
MDR+ cells was transfected with an expression
vector containing the human P450 reductase. Subsequent selection was on
zeocin and G-418. Transfectants were screened for expression of the
recombinant proteins and endogenous pgp.
Figure 1 shows the immunoblot analysis of
the cell lines that were used in the present work. Pgp was undetectable
in cells that had been grown in the absence of paclitaxel (Fig. 1,
lanes 1 and 3). The expression of pgp in the
3A4/HR/MDR+ cells was weaker than in
MDR+ cells (Fig. 1, cf. lanes 4 and 2). The
antibody against CYP3A4 recognized two proteins in total cellular
lysates, however the protein with the slower mobility was only detected
in cells that had been transfected with the CYP3A4 cDNA. The level of
this protein was rather similar in 3A4/HR/MDR
and 3A4/HR/MDR+ cells (Fig. 1, cf. lanes 3 and
4). These two cell lines also displayed similar expression levels of
P450 reductase as determined by immunoblotting. This was confirmed by
assaying the cytochrome c reductase activity (Table
1), which was more than 10 times higher
than the activity in cells that did not express recombinant P450
reductase. Importantly, cell lysates isolated from
3A4/HR/MDR and
3A4/HR/MDR+ cells displayed rather similar
CYP3A4-mediated testosterone 6-hydroxylase activities (Table 1).
This activity was not detected in cells that did not express the
recombinant proteins, namely, MDR+ and
MDR cells.
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Fig. 1.
Immunoblot analysis of pgp, CYP3A4, and P450
reductase proteins. Samples of total cellular protein (60 µg)
prepared from different cell lines were electrophoresed on a 7% (pgp)
or 10% (CYP3A4 and P450 reductase) SDS-polyacrylamide gel and
transferred to a nitrocellulose membrane. pgp (A) was detected using a
mouse antibody to human pgp (C219), and CYP3A4 (B) and P450 reductase
(C) were detected by antibodies to human CYP3A4 and P450 reductase.
Lane 1, MDR cells; lane 2, MDR+ cells; lane
3, 3A4/HR/MDR cells; and lane 4, 3A4/HR/MDR+
cells.
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TABLE 1
Enzyme activities in total cellular protein isolated from various
cell lines
For isolation of total protein, cells were trypsinized and sonicated.
Protein concentrations were determined as described under
Experimental Procedures. The cytochrome c
reductase and the testosterone 6-hydroxylase assay was performed at
37°C. Incubation time for the testosterone 6-hydroxylase assay was
20 min. Assays were done in triplicate.
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The activity of pgp in 3A4/HR/MDR and
3A4/HR/MDR+ cells was determined by measuring the
accumulation of calcein AM ester, vinblastine, testosterone, and
cortisol (Fig. 2). Compared with
3A4/HR/MDR cells, accumulation of calcein,
vinblastine, and cortisol was strongly reduced in
3A4/HR/MDR+ cells. This effect was not as strong
for testosterone. These experiments were also performed at a
concentration of testosterone and cortisol of 25 instead of 0.5 µM,
yielding similar results (data not shown).
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Fig. 2.
Measurement of pgp activity. The net accumulation of
calcein AM ester (A), vinblastine (B), testosterone (C), and cortisol
(D) was determined as described under Experimental
Procedures either fluorimetrically (calcein AM ester) or
radiometrically in 3A4/HR/MDR cells ( ) or in
3A4/HR/MDR+ cells ( ). Accumulation of calcein AM ester,
of vinblastine, and of steroids was allowed to proceed for 10, 30, and
60 min, respectively, and was performed either in duplicate (A) or
triplicate (B) or quadruplicate (C and D), respectively. Single values
obtained in A for each cell line differed by less than 20% from the
means. Values obtained for the two cell lines differed significantly
(*p  0.05, **p 0.01, ***p 0.001).
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Effect of pgp on CYP3A4-Dependent Metabolism.
Testosterone has
been shown to be a poor substrate of pgp, whereas cortisol has been
reported to be efficiently transported by pgp (Barnes et al., 1996).
The effects of pgp on CYP3A4-mediated metabolism were investigated by
determining the kinetic parameters of the testosterone 6-hydroxylase
and the cortisol 6-hydroxylase in cultured
3A4/HR/MDR+ and
3A4/HR/MDR cells without prior
trypsinization (Fig. 3; Table
2), under conditions that were linear
with time and cell number (data not shown). The two reactions followed
linear Lineweaver-Burk kinetics irrespective of whether pgp was
expressed (Fig. 3). The apparent Km of
the testosterone 6-hydroxylase was almost 2-fold higher in
3A4/HR/MDR+ cells compared with their
counterparts that did not express pgp (Table 2). Differences in the
apparent Km were even more pronounced for the cortisol 6-hydroxylase activity, with
3A4/HR/MDR+ cells displaying a 4-fold higher
Km compared with
3A4/HR/MDR cells. The
Vmax of testosterone 6-hydroxylase
activity in the cell line that expressed pgp was 3-fold lower than in
cells that did not express this transporter (cf.
3A4/HR/MDR versus
3A4/HR/MDR+ cells). Interestingly, the
Vmax of the cortisol 6-hydroxylase was similar in cells that expressed the transporter compared with those
that were deficient in pgp.
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Fig. 3.
Determination of an apparent
Km and Vmax for
cells expressing CYP3A4 and 450 reductase with and without pgp. Kinetic
assays were performed by determining the testosterone 6-hydroxylase
(A and B) and cortisol 6-hydroxylase (C and D) activity in intact
cells. The following cell lines were investigated:
3A4/HR/MDR cells (A and C) and 3A4/HR/MDR+
cells (B and D). Two determinations were performed per concentration.
The data were plotted on a reciprocal plot according to
Lineweaver-Burk. Km and
Vmax values derived from this experiment
(experiment a) are presented in Table 2 together with data from a
separate experiment.
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TABLE 2
Kinetic analysis of CYP3A4-mediated steroid hydroxylations in intact
cells
The Km and Vmax values were
derived from Lineweaver-Burk analysis. Two independent experiments
(footnoted a and b) were performed for each
substrate on intact cells. The Lineweaver-Burk plot for experiment a is
displayed in Fig. 3.
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Effects of CYP3A4 Inhibitors on P450 Activity Are Modulated by
pgp.
The following experiments were performed to investigate the
impact of pgp on CYP3A4-mediated metabolism in the presence of weak and
strong inhibitors of this P450.
The effects of quinidine, LY335979, which had been originally developed
as a reversal agent, verapamil, ketoconazole, and -naphthoflavone on
the testosterone 6-hydroxylase activity of CYP3A4 were analyzed in
membrane fractions containing CYP3A4 as well as in intact
3A4/HR/MDR cells and
3A4/HR/MDR+ cells at a concentration of 40 µM
testosterone. In these experiments, membranes isolated from E. coli, which coexpressed CYP3A4 and human P450 reductase, were used
(Fig. 4). LY335979 and ketoconazole were
the most potent inhibitors of CYP3A4, with the metabolic IC50 of both compounds being less than 1 µM.
The IC50 of verapamil and -naphthoflavone was
higher than 5 µM. Quinidine was a very weak inhibitor of testosterone
6-hydroxylase activity, with the IC50 being
higher than 100 µM. Compared with the results obtained with membrane
fractions, qualitatively similar effects of the inhibitors on the
activity of CYP3A4 were noted in cultured cells that expressed CYP3A4
in the absence of pgp (3A4/HR/MDR cells; Fig.
5, striped columns). As expected from the
experiments using bacterially expressed CYP3A4, increasing
concentrations of the compounds lead to increased inhibition of CYP3A4
activity. Again, ketoconazole and LY335979 were the most potent
inhibitors of testosterone 6-hydroxylase activity. Interestingly,
-naphthoflavone affected the testosterone 6-hydroxylase activity
of intact cells only slightly. Importantly, in the presence of pgp
(3A4/HR/MDR+ cells; Fig. 5, striped columns) the
effects of quinidine and LY335979 on testosterone 6-hydroxylase
activity were different than those seen in membranes or in intact
3A4/HR/MDR cells. Quinidine at a concentration
of 5 and 20 µM stimulated CYP3A4 activity and LY335979 failed to
inhibit this enzyme.
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Fig. 4.
Effects of compounds on the testosterone
6-hydroxylase activity in membranes. Membranes derived from
E. coli that coexpressed CYP3A4 and P450 reductase were
used. One hundred picomoles of CYP3A4 was used per assay and the final
testosterone concentration was 40 µM. Inhibitors were added together
with testosterone and the assay was run for 20 min. Data [mean ± range (bars) for duplicate determinations] are expressed as percentage
of control. Activities in the absence of inhibitors (solvent control)
were set at 100%.
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Fig. 5.
Effects of compounds on testosterone 6-hydroxylase
activity in intact cells. Testosterone (40 µM) was added together
with the indicated compounds to cultured cells. Incubation proceeded
for 4 h. Metabolites were separated by HPLC. The following cell
lines were investigated: 3A4/HR/MDR cells ( ) and
3A4/HR/MDR+ (). Data [mean ± range (bars) for
duplicate determinations] are expressed as percentage of control.
Activities in the absence of inhibitors (solvent control) were set at
100%.
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Discussion |
Cell lines were developed that allow investigations into some
pharmacokinetic consequences resulting from P450-catalyzed metabolism in the presence of pgp. Pgp is absent in
3A4/HR/MDR cells as revealed by immunoblotting
(Fig. 1) and by PCR (data not shown) but is clearly present in
3A4/HR/MDR+ cells. This is also reflected by the
decreased accumulation of calcein, vinblastine, testosterone, and
cortisol in 3A4/HR/MDR+ compared with
3A4/HR/MDR cells (Fig. 2). As expected from
published data on the activity of pgp toward cortisol and testosterone
(Barnes et al., 1996), the presence of this transporter in the former
cell line reduced the accumulation of cortisol more strongly than that
of testosterone. Even though anti-CYP3A4 antibodies reacted with a
protein in cells that had not been transfected with the CYP3A4 cDNA
(Fig. 1) this protein is unlikely to represent a CYP3A isoform since
untransfected cells did not display any testosterone 6-hydroxylase activity.
Steroids were used for the characterization of CYP3A4-mediated
metabolism in pgp-positive and -negative cell lines, because the
activities of both proteins toward these entities were previously well
characterized. The kinetics of testosterone 6- and cortisol 6-hydroxylase activity in both cell lines did not deviate from linearity when analyzed according to Lineweaver-Burk (Fig. 3). Also
analyzing the data according to Eadie-Hofstee failed to detect any
cooperativity in these reactions. A small cooperative effect (Hill
coefficient of 1.3) had been previously noted for the testosterone 6-hydroxylase activity of CYP3A4 in reconstituted membrane vesicles (Ueng et al., 1997). The Km of CYP3A4
toward cortisol and testosterone (as determined by the 6-hydroxylase
activity of CYP3A4) had been found by others to be 10 and 56 µM,
respectively (Ged et al., 1989; Ding et al., 1997). These values are
similar to those found by us in intact
3A4/HR/MDR cells. However, in the presence of
pgp, the apparent Km for the 6-hydroxylase activity of CYP3A4 toward testosterone and cortisol was increased by a factor of 1.7 and 4, respectively (Table 2, 3A4/HR/MDR+ cells). This difference can be
predicted from the higher activity of pgp toward cortisol compared with
testosterone (Barnes et al., 1996; our data), resulting in a stronger
intracellular depletion of the former compared with the latter steroid.
It is unlikely that pgp affected the apparent
Km of the P450 by somehow altering the
intrinsic affinity of CYP3A4 for the substrate. It is furthermore unlikely that differences in the apparent
Km resulted from the slightly lower
level of CYP3A4 in 3A4/HR/MDR+ compared with
3A4/HR/MDR cells (Table 1).
In accordance with literature (Ged et al., 1989; Ding et al., 1997),
the Vmax of CYP3A4 toward cortisol was
much lower than that toward testosterone and was not significantly
modulated by the expression of pgp (Table 2). However, the expression
of pgp lead to a 3-fold reduction of the
Vmax for the testosterone
6-hydroxylase activity. This strong reduction cannot be explained by
the slightly lower level of CYP3A4 in cells that overexpress pgp (Table
1). One may speculate that the net accumulation of substrate, which is
determined by uptake and pgp-mediated efflux, became rate limiting for
the rapid metabolism of testosterone but not for the slow metabolism of cortisol.
The ratio of cortisol to 6-hydroxycortisol has been used as an in
vivo marker activity for CYP3A4 (Ged et al., 1989; Nakamura et al.,
1997). Our finding that pgp strongly affected the apparent Km of CYP3A4 toward cortisol could
have relevance for the use of this marker, provided that the
intracellular level of cortisol is below the
Km of the cortisol 6-hydroxylase
and that the level of hepatic pgp influences the concentration of this
steroid in hepatocytes. The plasma level of free cortisol, which unlike
protein-bound cortisol is responsible for the biological effects of
this steroid, has been reported to be maximally 0.5 µM and its entry
into cells is mainly by diffusion (Milgrom, 1990). Modulation of
cortisol 6-hydroxylase activity by pgp in the liver could explain
the observation that the correlation between this enzyme activity and
CYP3A4 protein level was only moderate when applied separately to
either patients that had been untreated or to individuals that had been
treated with rifampicin (Ged et al., 1989). This correlation became
only strong when applied to both groups together, thus showing, as
discussed previously (Ged et al., 1989), that cortisol 6-hydroxylase
activity can be used as a marker of CYP3A induction but not to assess
interindividual variations in CYP3A protein levels. In this respect, it
is important to note that levels of pgp in the liver showed marked
interindividual differences (Schuetz et al., 1995) and that rifampicin
is a potent inducer of pgp (Schuetz et al., 1996). It remains to be
seen whether intraindividual differences in the level of pgp may also
be partially responsible for the lack of correlation between marker
activities of CYP3A4 such as erythromycin N-demethylase,
dapsone N-hydroxylase, cortisol 6-hydroxylase, and
midazolam 1'-hydroxylase (Kinirons et al., 1999), with the added
complication that some of these metabolic probes will be more sensitive
to intestinal pgp, whereas others may be more influenced by hepatic
pgp.
Metabolic in vivo probes have been also used to predict pharmacokinetic
drug-drug interactions relating to CYP3A4. One study investigated the
effects of human immunodeficiency virus protease inhibitors on dapsone
N-hydroxylation and cortisol 6-hydroxylation in vivo
(Gass et al., 1998). Interestingly these compounds, some of which have
been shown to interact with pgp, failed to affect both activities of
CYP3A4 in vivo as would have been expected from in vitro experiments.
Another group investigated the effects of stiripentol on CYP3A4
activities in vivo and found that this compound inhibited
CYP3A4-catalyzed carbamazepine metabolism but that the
cortisol/6 hydroxycortisol ratio did not prove to be a reliable
indicator of this inhibition (Tran et al., 1997). Here, we wanted to
explore the possibility that pgp may modulate drug-drug interactions
acting via CYP3A4. We have investigated the effects of some pgp
inhibitors on CYP3A4 mediated reactions in intact 3A4/HR/MDR and
3A4/HR/MDR+ cells and in membrane fractions
containing recombinant CYP3A4 (Figs. 4 and 5). Verapamil, LY335979, and
quinidine have been previously tested as reversal agents. LY335979 was
found to be more than 1000-fold as potent as verapamil in the
inhibition of pgp-dependent accumulation of daunomycin (Dantzig et al.,
1996). Moreover, this study showed that LY335979 at a concentration of 100 nM lowered the cytotoxic
CIC50 of vinblastine in
pgp-overexpressing cells 400-fold, whereas verapamil at a concentration
of 5 µM lowered the cytotoxic
CIC50 only 50-fold.
However, no data are available on the interaction of LY335979 with
CYP3A4. Recently, the selectivity of several reversal agents toward
CYP3A4 and pgp was published (Wandel et al., 1999). This study showed
that quinidine was more selective than verapamil to inhibit
preferentially pgp over CYP3A4, which is in agreement with our data
that quinidine, but not verapamil, stimulated the testosterone
6-hydroxylase activity in intact 3A4/HR/MDR+
cells due to preferential inhibition of pgp compared with CYP3A4 (Fig.
5), thereby making more substrate available to the P450. A similar but
weaker effect was also noted with LY335979. This demonstrates that
compounds that are stronger inhibitors of pgp than of CYP3A4 can
stimulate the activity of this P450 in intact cells. Stimulation of
testosterone 6-hydroxylase activity by quinidine (5 µM) was 2-fold
in cells that expressed pgp (Fig. 5), whereas the presence of pgp
decreased the concentration only by 1.5-fold. This difference is not
entirely unexpected because Michaelis-Menten kinetics predicts that
Va/Vb = (Vmax × Sa/Km + Sa)/(Vmax × Sb/Km + Sb), with
Sa and
Sb being the concentrations of
testosterone in the presence and absence, respectively, of pgp and
Va and
Vb being the velocities of the
testosterone 6-hydroxylation under these two conditions. Note that
the concentration of testosterone in the experiments displayed in Fig.
5 was 40 µM and the Km for the
testosterone hydroxylase is 60 µM.
The inhibitory effects of verapamil on CYP3A4 activity seem to plateau
in 3A4/HR/MDR+ but not in
3A4/HR/MDR cells. This most likely results from
inhibition of both CYP3A4 and pgp with both inhibitions having an
opposing impact on CYP3A4 activity, albeit only in pgp-positive cells.
However, the resulting net effect on CYP3A4 activity is difficult to
predict accurately.
The data presented demonstrate that the expression of pgp, as predicted
by theoretical considerations, impinges on CYP3A4-mediated metabolism
by influencing the apparent Km values
of the enzyme in intact cells. In cases such as quinidine where a
compound is oxidized by CYP3A4 at two different positions with
different Km values (Sanwald et al.,
1996; Nielsen et al., 1999), product ratios can be expected to be
influenced by the activity of pgp. Even though our observations are
mainly pertinent to reactions catalyzed by CYP3A4, it should be noted
that debrisoquine, which has been used as a metabolic in vivo probe for
CYP2D6, is a substrate of pgp (Kim et al., 1999). In addition, we show
that the influence of pharmacokinetic drug-drug interactions on CYP3A4
is strongly modulated by the activity of pgp.
P450, cytochrome P450;
pgp, human
P-glycoprotein;
calcein AM, calcein acetoxymethylester;
EMS, ethylmethanesulfonate;
HPLC, high performance liquid chromatography;
DMSO, dimethyl sulfoxide;
IC50, concentration of inhibitor
that reduces metabolism of substrate by 50%;
CIC50, concentration of cytotoxic compound that
reduces cell survival by 50%.
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Abstract
Introduction
