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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (Y.Sh., Y.Su.); Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama, Japan (Y.Sh., Y.Su.); School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan (T.I.); Faculty of Pharmaceutical Sciences, Showa University, Tokyo, Japan (H.S.); and In Vitro Technologies, Inc., Baltimore, Maryland (A.P.L.)
Received July 23, 2002; accepted October 28, 2002.
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Abstract |
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). Cerivastatin (CER) is a potent HMG-CoA reductase inhibitor
(statin) with a high oral bioavailability, which makes it effective at low
doses (Moghadasian, 1999). CER
is extensively taken up into the liver and subsequently metabolized by two
different enzymes, CYP2C8 and CYP3A4
(Mück, 2000). This dual
metabolic pathway is a distinctive feature of CER among statins.
Patients who develop hypercholesterolemia after tissue transplantation are
sometimes treated with combination therapy with statins and cyclosporin A
(CsA). CsA is an inhibitor of CYP3A4, and therefore, this immunosuppressant is
likely to cause a drug-drug interaction (DDI) with simvastatin, lovastatin,
and atorvastatin, which are all substrates of CYP3A4
(Deseger and Horsmans, 1996).
This DDI may also cause an increase in the plasma concentration of statins and
result in myopathy and/or fatal rhabdomyolysis. Since CER can undergo
metabolism via two pathways, the frequency of DDI was believed to be low.
However, Mück et al.
(1999) have reported that the
plasma concentrations of CER are increased in kidney transplant patients
following CsA treatment. That is, the area under plasma concentration-time
curve (AUC) of CER was increased 4-fold by the coadministration of CsA
compared with the control. The plasma concentrations of CER were not affected
by coadministration of erythromycin, a potent mechanism-based inhibitor of
CYP3A4 (Kanamitsu et al.,
2000), suggesting that it is unlikely that the DDI between CER and
CsA is due to CYP3A4-mediated metabolism
(Mück et al., 1998).
Moreover, the AUC of pravastatin, which is not a substrate of CYP3A4, is also
increased approximately 20-fold by CsA
(Regazzi et al., 1993). Until
now, the mechanism of this DDI between CsA and these statins has remained
unknown.
Statins are taken up into the liver before undergoing metabolism. The
hepatic uptake of some statins has already been studied. For example, in rats,
the hepatic uptake of CER (Hirayama et al.,
2000) and pravastatin (Komai et
al., 1992) has been investigated, and their saturable transport
systems have been studied. Pravastatin also exhibits saturable uptake in human
hepatocytes (Nakai et al.,
2001). However, the uptake of CER by human hepatocytes has not yet
been investigated.
Recent studies of drug transport in the liver have provided detailed
information on drug transporters, including substrate and inhibitor profiles.
More recent studies clarifying the mechanism of drug uptake in the liver have
used cloning to identify a number of transporters expressed at the sinusoidal
membrane of hepatocytes. At present, organic anion transporting polypeptide-2
(OATP2/OATP-C; gene symbol, SLC21A6), OATP8 (SLC21A8),
OATP-B (SLC21A9), and organic anion transporter-2 (OAT2;
SLC22A7) are reported to be expressed in the human liver and involved
in the hepatic uptake of a number of important substrates, including
therapeutic drugs (Abe et al.,
1999; Hsiang et al.,
1999; Kok et al.,
2000; König et al.,
2000a,b;
Tamai et al., 2000).
Pravastatin has been shown to be a substrate of OATP2, and this transporter is
at least partly responsible for its hepatic uptake
(Hsiang et al., 1999;
Nakai et al., 2001). As each
of these transporters accepts a number of compounds as substrates, they may
competitively inhibit the transport of other substrates. Moreover, CsA
functions as an inhibitor of rat Oatp1 and Oatp2
(Shitara et al., 2002). It is
therefore possible that CsA affects the plasma concentrations of substrates,
leading to a clinically relevant DDI
(Kusuhara and Sugiyama, 2001).
In the present study, we examined the effect of CsA on the uptake of CER into
human hepatocytes together with its metabolism to clarify the mechanism of
their DDI.
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Materials and Methods |
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Hepatocyte Preparation. The human hepatocytes used in the study were
isolated from human livers donated for transplantation purposes but not used
mainly due to the lack of appropriate recipients. All the donors were free of
known liver diseases. All the livers were stored for less than 24 h in
University of Wisconsin solution. The hepatocytes were isolated by perfusion
using a two-step collagenase digestion procedure
(Li et al., 1992). After
enzymatic dissociation, the hepatocytes were further separated from
nonparenchymal cells by centrifugation through 30% Percoll. The purified
hepatocytes were cryopreserved (Li et al.,
1999) in liquid nitrogen until analysis. Immediately before the
uptake studies, the hepatocytes (1-ml suspension) were thawed at 37°C then
immediately suspended in 10 ml of ice-cold Krebs-Henseleit buffer and
centrifuged (50g) for 2 min at 4°C, followed by removal of the
supernatant. This procedure was repeated to remove cryopreservation buffer,
and then the cells were resuspended in the same buffer at a cell density of
2.0 x 106 viable cells/ml for the uptake studies.
Uptake of [14C]CER into Hepatocytes. Prior to starting the uptake studies with [14C]CER, the cell suspensions were prewarmed in an incubator at 37°C for 3 min. A pilot experiment confirmed that a 3-min preincubation was sufficient to raise the temperature of the cells to 37°C. The uptake studies were initiated by adding an equal volume of [14C]CER solution containing various concentrations of unlabeled CER or CsA to the cell suspension. At 0.5 and 2 min, the reaction was terminated by separating the cells from the substrate solution. For this purpose, an aliquot of 100 µl of incubation mixture was collected and placed in a centrifuge tube (450 µl) containing 50 µl of 2 N NaOH under a layer of 100 µl of oil (density, 1.015; a mixture of silicone oil and mineral oil; Sigma-Aldrich), and subsequently, the sample tube was centrifuged for 10 s using a tabletop centrifuge (10,000g; Beckman Microfuge E; Beckman Coulter, Inc., Fullerton, CA). During this process, the hepatocytes pass through the oil layer into the alkaline solution. After an overnight incubation in alkali to dissolve the hepatocytes, the centrifuge tube was cut, and each compartment was transferred to a scintillation vial. The compartment containing the dissolved cells was neutralized with 50 µlof 2 N HCl, mixed with scintillation cocktail, and the radioactivity was determined in a liquid scintillation counter (LS6000SE; Beckman Coulter, Inc.).
Uptake Study of [14C]CER in OATP2-Expressing Cells. The
construction and culture of OATP2-expressing cells have been described
previously (Sasaki et al.,
2002). For the uptake study of [14C]CER, MDCKII cells
transfected with OATP2 or vector only as a control were seeded on cell culture
inserts (BD Biosciences Discovery Labware, Bedford, MA). After 2 days, the
culture medium was replaced with one containing 10 mM Nax
butyrate for the induction of OATP2. After culturing for a further day, the
culture medium was replaced with ice-cold Krebs-Henseleit buffer and washed
twice with the same buffer, followed by preincubation at 37°C. The uptake
study was initiated by replacing the buffer on the basal side of the cells
with that containing [14C]CER in the presence or absence of
unlabeled CER or CsA. At the designated times, the reaction was terminated by
aspirating the incubation buffer and washing four times with ice-cold buffer.
Subsequently, the cells were dissolved in 0.75 ml of 0.1 N NaOH overnight,
followed by neutralization with 0.75 ml of 0.1 N HCl. Then, 1.3-ml aliquots
were transferred to scintillation vials, and the radioactivity associated with
the cells and that in the medium was determined in a liquid scintillation
counter (LS6000SE). The remaining 0.1-ml aliquots of the cell lysate were used
for protein assay by the Lowry method with bovine serum albumin as a standard
(Lowry et al., 1951).
Metabolism of [14C]CER and Testosterone in Human
Microsomes. To measure the effect of CsA on the metabolism of
[14C]CER and testosterone, its in vitro metabolism was examined.
Prior to the metabolism study, human microsomes (final 0.5 mg of protein/ml;
BD Gentest, Woburn, MA) were incubated at 37°C for 10 min in 100 mM
potassium phosphate buffer, pH 7.4, containing 3.3 mM MgCl2, 3.3 mM
glucose 6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, 1.3 mM NADPH,
and 0.8 mM NADH. A 500-µl volume of incubation mixture was transferred into
a polyethylene tube, and [14C]CER (final 1 µM) or testosterone
(final 30 µM; Wako, Osaka, Japan) were added to initiate the reaction with
or without inhibitors. After incubation for a designated time, the reaction
was terminated by the addition of 500 µl of ice-cold acetonitrile and 200
µl of ice-cold methanol for the metabolism of [14C]CER and
testosterone, respectively, followed by centrifugation. To measure the
metabolic rate of [14C]CER, the supernatant was collected and
concentrated to approximately 20 µl in a centrifugal concentrator, followed
by thin layer chromatography. The analytes were separated on silica gel
60F254 (Merck KGaA, Darmstadt, Germany) using a mobile phase
(toluene/acetone/acetic acid, 70:30:5, v/v/v). The intensity of the bands for
intact [14C]CER separated by thin layer chromatography was
determined by the BAS 2000 system (Fuji Film, Tokyo, Japan). To measure the
metabolic rate of testosterone, 6
-hydroxytestosterone in the incubation
mixture was determined by a high-performance liquid chromatography-UV
detection method. To a 100-µl volume of supernatant, 100 µl of internal
standard (10 µg/ml phenacetin) was added and subjected to a
high-performance liquid chromatography system (VP-5; Shimadzu, Kyoto, Japan).
The analyte was separated by a C18 column (Cosmosil
5C18-AR; 5-mm, 4.6-mm i.d. x 250 mm; Nakalai Tesque, Kyoto,
Japan) at 45°C. The mobile phase comprised solvent A (20% tetrahydrofuran
and 80% water) and solvent B (methanol). A 20-min linear gradient from 20% B
to 30% B was applied at a flow rate of 1.0 ml/min. The product was detected by
its absorbance at 254 nm and quantitated by comparing with the absorbance of a
standard curve for 6-hydroxytestosterone.
Data Analysis. The time courses of the uptake of [14C]CER
into the hepatocytes were expressed as the uptake volume (microliters per
106 viable cells) of radioactivity taken up into the cells
(dpm/106 cells) divided by the concentration of radioactivity in
the incubation buffer (disintegrations per minute per microliter). The initial
uptake velocity of [14C]CER was calculated using the uptake volume
obtained at 0.5 and 2 min and expressed as the uptake clearance
(CLuptake; microliters per minute per 106 cells). The
kinetic parameters for the uptake of [14C]CER were calculated using
the following equation:
 | (1) |
When the substrate concentration is much lower than the
Km value, the data obtained in the inhibition study of the
uptake into isolated hepatocytes regardless of inhibitor type (i.e.,
competitive or noncompetitive inhibitor) can be fitted to the following
equation to calculate the inhibition constant (Ki).
 | (2) |
The data were fitted to these equations by a nonlinear least-squares method
using a computer program, MULTI, to obtain the kinetic parameters or
inhibition constant with computer-calculated S.E. values
(Yamaoka et al., 1981). The
input data were weighted as the reciprocal of the observed values, and the
Damping Gauss-Newton method was used as the fitting algorithm. The uptake of
[14C]CER into OATP2-expressing MDCKII cells was also expressed as
the uptake volume (microliters per milligram of protein) for the radioactivity
in the cell lysate (disintegrations per minute per milligram of protein)
divided by that in the incubation buffer (disintegrations per minute per
milliliter).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Uptake Study in OATP2-Expressing MDCKII Cells. The time courses of uptake of [14C]CER into human OATP2-expressing MDCKII cells and vector-transfected cells are shown in Fig. 3. The uptake of [14C]CER into OATP2-expressing cells was 2.6 times higher at 9 min than that into vector-transfected cells (Fig. 3). In OATP2-expressing cells, the uptake of [14C]CER observed in the presence of excess unlabeled CER (30 µM) was reduced to the same level as that in vector-transfected cells (Fig. 3). OATP2-mediated uptake of [14C]CER was also inhibited by CsA in a concentration-dependent manner (Fig. 4). The Ki value for the OATP2-mediated uptake of [14C]CER was 0.238 ± 0.129 µM (mean ± computer-calculated S.E.) (Fig. 4).
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Metabolic Stability of [14C]CER. The metabolic stability
of [14C]CER in human microsomes was examined. In
Fig. 5, a time profile of the
metabolic stability of [14C]CER in pooled human microsomes is
shown. As a linear metabolic rate in human microsomes was observed for up to
45 min (Fig. 5), the inhibitory
effects of CsA, 10 µM quercetin (a CYP2C8 inhibitor;
Ohyama et al., 2000), and 0.2
µM ketoconazole (a CYP3A4 inhibitor;
Kawahara et al., 2000) on the
metabolism of [14C]CER were followed for 45 min. In
Fig. 6a, the metabolic rates of
[14C]CER when incubated in human microsomes in the absence or
presence of inhibitors are shown. CsA did not alter the metabolic rate of
[14C]CER up to a concentration of 3 µM and reduced it to, at
most, 71% of the control value at 10 to 30 µM, whereas 10 µM quercetin
and 0.2 µM ketoconazole reduced it to 63 and 72% of the control value,
respectively (Fig. 6a). The
effect of CsA on testosterone 6-hydroxylation, which is mediated by
CYP3A4, was also followed for 2 min (Fig.
6b). The metabolic rate of testosterone 6-hydroxylation
measured in the absence of inhibitors was 1560 pmol/min/mg of protein, and it
was reduced to 30 and 5.9% of the control value in the presence of 3 and 30
µM CsA, respectively (Fig.
6b). It was also reduced to 6.5% of the control value by 0.2 µM
ketoconazole and 52% by 10 µM quercetin
(Fig. 6b).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) due
to a DDI between the two drugs. In the present study, we examined the effect
of CsA on the hepatic uptake and metabolism of CER, especially on its hepatic
uptake, to clarify the mechanism underlying this DDI.
In vitro uptake studies in isolated hepatocytes revealed saturable
transport of [14C]CER in human hepatocytes
(Fig. 1), suggesting the
involvement of transporters in the uptake process. In this study, we found
that transporter-mediated uptake accounted for 70 to 80% of the total hepatic
uptake. In clinical situations, the maximum plasma concentration
(Cmax) of CER is approximately 4 nM (after a single oral
dose of 0.2 mg; Mück et al.,
1999), which is much lower than the Km values
(2.6–18 µM) obtained in the present study
(Fig. 1 and
Table 1), suggesting that the
hepatic uptake of CER is largely mediated by transporters over the therapeutic
range.
The present study revealed a concentration-dependent inhibition of
transporter-mediated [14C]CER uptake by CsA in human hepatocytes,
with Ki values of 0.28 to 0.69 µM
(Fig. 2). The obtained data may
at least partly explain the clinically observed DDI
(Mück et al., 1999).
Mück et al. (1999)
reported that the Cmax and the AUC of CER in kidney
transplant patients given CsA was increased 4- and 3-fold, respectively, when
the Cmax of CsA was approximately 1 µM. In the present
study, the saturable component of the uptake of [14C]CER was mostly
inhibited in the presence of 1 µM CsA
(Fig. 2). However, considering
that approximately 90% of the CsA in blood is bound to plasma proteins that
consist of mainly lipoproteins (Lemaire
and Tillement, 1982), the clinically relevant unbound
concentration of CsA is estimated to be 0.1 µM, which may not be enough to
inhibit hepatic uptake of CER. This discrepancy may be explained by a number
of factors. First, in the case of oral administration, the plasma
concentration of CsA in the circulating blood and portal vein are different,
and therefore, the concentration exposed to the liver may be much higher than
that observed in the circulating blood (Ito
et al., 1998; Sugiyama et al.,
2002). Second, the increase in the plasma concentration of CER
reported by Mück et al.
(1999) could be partly due to
the change in the intrinsic hepatic clearance associated with renal failure
and/or kidney transplantation. In the present study, although the increase in
the plasma concentration observed clinically cannot be fully predicted from
the in vitro uptake study, the results suggest that the increase in the plasma
concentration of CER is at least partly due to the interaction between CER and
CsA involving transporter-mediated hepatic uptake.
The range of CLuptake values for [14C]CER observed among human hepatocytes from the three donors (Fig. 1; Table 1) may be due to the interindividual differences in the expression level and/or function of transporters, although it may be caused by other factors, such as the cell integrity being affected during the cryopreservation process. Indeed, the fact that the interindividual differences were greater for the Vmax/Km values, which reflect transporter-mediated uptake and can be affected by the expression level and/or intrinsic function of transporters, than for the Pdif values, which mainly represent passive diffusion, supports our hypothesis (Table 1). If this hypothesis is correct, there must be a wide range of interindividual differences in the hepatic clearance and/or the extent of transporter-mediated DDI. Human hepatocytes may represent an important experimental system in the near future for evaluating the genetic and environmental factors that may be responsible for the interindividual differences in transporter functions.
In the present study, CER was shown to be a substrate of human OATP2
(Fig. 3), like pravastatin
(Hsiang et al., 1999;
Nakai et al., 2001).
OATP2-mediated uptake of [14C]CER was also inhibited by CsA
(Fig. 4), and the obtained
Ki value (0.24 µM) was within the same range as the
values obtained in the inhibition study using human hepatocytes (0.28–
0.69 µM) (Fig. 2). These
results suggest that the inhibition by CsA on the uptake of CER in human
hepatocytes is partly due to OATP2-mediated transport. Since OATP2 accepts a
wide variety of compounds as substrates
(Abe et al., 1999;
Hsiang et al., 1999), these
substrates in addition to CER may possibly exhibit DDI. Indeed, a DDI between
pravastatin, a substrate of OATP2, and CsA was reported, which could also be
due to OATP2-mediated uptake in the liver
(Regazzi et al., 1993). To
avoid this kind of DDI, the characterization of transporters, which are
responsible for the drug uptake, and their contributions to total hepatic
uptake are very important (Kouzuki et al.,
1999a,b;
Kusuhara and Sugiyama, 2002;
Mizuno and Sugiyama, 2002;
Shitara et al., 2002). The
increase in the plasma concentration of drugs associated with a
transporter-mediated DDI may be quantitatively predicted from in vitro studies
that determine the extent of inhibition of transport in hepatocytes and/or in
transporter-expressing cells (Ueda et al.,
2001).
We also examined the effect of CsA on the metabolism of [14C]CER
in human microsomes (Fig. 6a).
CsA did not markedly reduce the metabolic rate of [14C]CER up to a
concentration of 3 µM, and 10 to 30 µM CsA reduced it only to 70% of the
control value (Fig. 6a). On the
other hand, 30 µM CsA markedly reduced testosterone 6-hydroxylation,
which was mediated by CYP3A4, to 30% of the control value
(Fig. 6b). To explain this
different effect of CsA on the metabolisms of CER and testosterone, we
examined the effect of ketoconazole, a potent CYP3A4 inhibitor
(Kawahara et al., 2000). As the
Ki values of ketoconazole for the inhibition of CYP2C8 and
CYP3A4 functions are 2.5 and 0.03 µM, respectively
(Kawahara et al., 2000;
Ong et al., 2000), 0.2 µM
ketoconazole should be enough to inhibit most of the CYP3A4-mediated
metabolism of [14C]CER and have only a slight effect on that
mediated by CYP2C8. Indeed, we have confirmed that 0.2 µM reduced the
CYP3A4-mediated metabolism of testosterone to 7% of the control value.
However, 0.2 µM ketoconazole reduced the metabolism of [14C]CER
only to 72% of the control (Fig.
6a). This study supports that CYP3A4 plays a limited role in the
metabolism of CER as previously reported by Mück
(2000), and CYP3A4 inhibitors,
such as ketoconazole and CsA, reduce the metabolism of CER to only a limited
extent. From the present study, the contribution of CYP3A4 to the total
metabolism of CER is estimated to be approximately 38%
(Fig. 6a). We also examined the
effect of quercetin, a CYP2C8 inhibitor
(Ohyama et al., 2000). As the
Ki value of quercetin for the inhibition of
CYP2C8-mediated metabolism is 1.3 µM
(Rahman et al., 1994), 10
µM quercetin should be enough to inhibit most of the CYP2C8-mediated
metabolism of [14C]CER, although it also reduces the
CYP3A4-mediated metabolism of testosterone to 50% of the control value
(Fig. 6b). In the presence of
10 µM quercetin, the metabolism of [14C]CER was reduced to 63%
of the control value (Fig. 6a).
This result supports a contribution of CYP2C8 to the metabolism of CER is less
than 37%, considering that the CYP3A4-mediated metabolism is partly inhibited
by 10 µM quercetin (Fig.
6b). The present study suggests that at low concentrations (<3
µM), CsA does not inhibit the metabolism of CER
(Fig. 6a), although it does
inhibit its transporter-mediated hepatic uptake with a much lower
concentration (<1 µM) (Fig.
2). This confirms that it is less likely that the DDI between CER
and CsA is due to the metabolism of CER.
Recently, a severe DDI between CER and gemfibrozil was reported, and in the
USA, 31 deaths from severe rhabdomyolysis in patients taking CER were
reported, of whom 12 were taking concomitant gemfibrozil
(Charatan, 2001). This resulted
in the withdrawal of CER from the market. It is still unknown whether this
severe DDI is mainly due to the pharmacokinetic event (i.e., the change of the
plasma concentration of CER caused by gemfibrozil) or due to the
nonpharma-cokinetic event (for example, an increased formation of toxic
metabolites of CER or the effects on the energy of the cell, which may lead to
rhabdomyolysis). In the present study, however, we tried to clarify the
mechanism of the effect of CsA on the pharmacokinetics of CER and found that
the inhibition of the OATP2-mediated hepatic uptake of CER by CsA was the
major mechanism. There is also a report that the AUC of CER was increased
4.2-fold when coadministered with gemfibrozil
(Mueck et al., 2001), which
may be one mechanism of this serious DDI. At the time of this report,
Prueksaritanont et al. (2002)
reported that both oxidation and glucuronidation of CER in human liver
microsomes were inhibited by gemfibrozil with IC50 values of 82 and
87 to 220 µM, respectively, which at least in part explains the mechanism
of DDI between CER and gemfibrozil. However, considering the relatively high
plasma protein binding of gemfibrozil (plasma unbound fraction of
1.4–3%; Todd and Ward,
1988), the unbound therapeutic concentration of gemfibrozil is
estimated to be less than 7.5 µM (total Cmax,upto250
µM; Prueksaritanont et al.,
2002), which is much lower than the recently reported
IC50 values (80–220 µM) for CER metabolism. Therefore, the
increase in the AUC of CER caused by the DDI may not necessarily be accounted
for only by the inhibition of metabolism. Although there has been no report
concerning the effect of gemfibrozil on the transporter-mediated hepatic
uptake of CER, this should also be examined in a future study. In conclusion,
we should pay more attention to DDI that may originate from the inhibition of
transporter-mediated hepatic uptake, since it may occur with a large number of
drug combinations when their elimination (metabolism and/or biliary excretion)
takes place following transporter-mediated hepatic uptake.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; DDI, drug-drug interaction; CER, cerivastatin; CsA, cyclosporin A; OATP, organic anion transporting polypeptide; MDCK, Madin-Darby canine kidney; OAT, organic anion transporter; AUC, area under plasma concentration-time curve; CL, clearance.
1 Present address: Faculty of Pharmaceutical Sciences, Showa University,
Tokyo, Japan. 
2 Present address: Phase 1 Molecular Toxicology, Inc., Santa Fe, NM.
Address correspondence to: Dr. Yuichi Sugiyama, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp
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