involvement of up-regulation of hepatic breast cancer resistance...
TRANSCRIPT
DMD 15164
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Involvement of up-regulation of hepatic breast cancer resistance protein in decreased plasma
concentration of 7-ethyl-10-hydroxycamptothecin (SN-38) by coadministration of S-1 in rats
Koji Yokoo, Akinobu Hamada, Hiroshi Watanabe, Takanobu Matsuzaki, Tomoyuki Imai, Hiromi
Fujimoto, Kengo Masa, Teruko Imai and Hideyuki Saito
Department of Pharmacy, Kumamoto University Hospital, 1-1-1 Honjo, Kumamoto 860-8556,
Japan (K.Y., A.H., T.M., T.I., H.F., K.M., H.S.)
Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi,
Kumamoto 862-0973, Japan (H.W., T.I.)
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Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title Page
Running Title: Drug interaction between CPT-11 and S-1 via BCRP in rats
Address correspondence to:
Professor Hideyuki Saito, Ph.D.
Department of Pharmacy,
Kumamoto University Hospital,
1-1-1 Honjo, Kumamoto 860-8556,
Japan
Phone & Fax: 81-96-373-5820
E-mail: [email protected]
Text pages:
Number of text page: 22
Number of tables: 1
Number of figures: 6
Number of reference: 29
Number of words (counting references) in:
Abstract: 249 words
Introduction: 624 words
Discussion: 1063 words
List of abbreviations:
AUC, area under the plasma concentration curve; BCRP, breast cancer resistance protein; CDHP,
5-chloro-2,4-dihydroxypyridine; CES, carboxylesterase; Cmax, maximum plasma concentration;
CMC, carboxyl methyl cellulose; CPT-11, 7-ethyl-10-[4-[1-piperidino]-1-piperidino]
carbonyloxycamptothecin; CYP, cytochrome P450; FT, tegafur; 5-FU, 5-fluorouracil; Km,
Michaelis-Menten constant; MRP2, multidrug resistance-associated protein 2; Oxo, potassium
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oxonate; P-gp, P-glycoprotein; p-NP, p-nitrophenol; p-NPA, p-nitrophenylacetate; SN-38,
7-ethyl-10-hydroxycampothecin; SN-38G, SN-38 glucuronide; Vmax, maximum hydrolysis rate;
UGT, uridine diphosphate glucuronosyltransferase
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Abstract
The safety and efficacy of combination therapy with irinotecan (CPT-11,
7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin) and S-1 composed of tegafur,
a prodrug of 5-fluorouracil, gimeracil and potassium oxonate, have been confirmed in patients with
colorectal cancer. Previously, we showed that oral coadministration of S-1 decreased the plasma
concentration of both CPT-11 and its metabolites in an advanced colorectal cancer patient. The aim
of this study was to clarify the mechanism of drug interaction using both in vivo and in vitro
methods. Rats were orally administered S-1 (10 mg/kg) once a day for 7 consecutive days. On day
7, CPT-11 (10 mg/kg) was administered by i.v. injection. Coadministration of S-1 affected the
pharmacokinetic behavior of CPT-11 and its metabolites, with a decrease in the Cmax and AUC of
the active metabolite, SN-38 (7-ethyl-10-hydroxycampothecin) lactone form. Furthermore, the rate
of biliary excretion of SN-38 carboxylate form increased upon coadministration of S-1. In the liver,
the level of breast cancer resistance protein (BCRP), but not P-glycoprotein and multidrug
resistance-associated protein 2, was elevated after administration of S-1. Enzymatic conversion of
CPT-11 to SN-38 by carboxylesterase (CES) was unaffected by the liver microsomes of rats treated
with S-1. In addition, components of S-1 did not inhibit the hydrolysis of p-nitrophenylacetate, a
substrate of CES, in the S9 fraction of HepG2 cells. Therefore, the mechanism of drug interaction
between CPT-11 and S-1 appears to involve up-regulation of BCRP in the liver, resulting in an
increased rate of biliary excretion of SN-38 accompanied by a decrease in the Cmax and AUC of
SN-38.
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Introduction
Irinotecan (CPT-11, 7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin),
an inhibitor of DNA topoisomerase Ι, is widely used in the treatment of several types of solid
tumors, including colorectal cancer (Wiseman and Markham, 1996). CPT-11 exerts its antitumor
activity after enzymatic transformation to its more active metabolite, SN-38
(7-ethyl-10-hydroxycampothecin) by hepatic carboxylesterases (CES) (Fig.1) (Humerickhouse et
al., 2000). In vitro studies suggested that antitumor effects of SN-38 is 100 to 1,000 times more
potent than CPT-11 (Kawato et al., 1991). SN-38 is subsequently conjugated in the liver by uridine
diphosphate glucuronosyltransferase (UGT) 1A, thereby forming the inactive metabolite, SN-38
glucuronide (SN-38G) (Fig.1) (Rivory and Robert, 1995). The biliary excreted SN-38G is
deconjugated by bacterial β-glucuronidases in the intestinal lumen (Takasuna et al., 1996).
Furthermore, a portion of CPT-11, SN-38 and SN-38G in bile is reabsorbed from the intestinal
lumen to enter an entero-hepatic recirculation loop. Biliary excretion is the major elimination route
for CPT-11 and its metabolites, with urinary excretion being a less important pathway.
P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2) and
breast cancer resistance protein (BCRP/ABCG2), expressed at the bile canalicular membrane,
appear to be responsible for the biliary excretion of CPT-11 and its metabolites (Sugiyama et al.,
1998; Nakatomi et al., 2001). On the other hand, CPT-11, SN-38 and SN-38G have a labile
α-hydroxy-3-lactone ring, which can undergo reversible pH-dependent hydrolysis in aqueous
solution (Fig. 1) (Rivory et al., 1994). Under acidic conditions the lactone form is the predominant
species, whereas under physiological conditions hydrolysis of the lactone yields the carboxylate
form. The carboxylate form of SN-38 has much weaker antitumor activity than the lactone form of
SN-38 (Rivory et al., 1994). The complex metabolic profile of CPT-11 explains the observed
interindividual variability in the pharmacokinetics and toxicity of this compound.
S-1 is an oral anticancer agent comprised of tegafur (FT), 5-chloro-2,4-dihydroxypyridine
(CDHP) and potassium oxonate (Oxo) at a molar ratio of 1:0.4:1 (Shirasaka et al., 1996). FT, a
prodrug of 5-fluorouracil (5-FU), is converted to 5-FU mainly in liver and tumor cells (Ikeda et al.,
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2000). CDHP enhances the concentration of 5-FU in the plasma and tumor cells by competitive
inhibition of dihydropyrimidine dehydrogenase, which is responsible for the catabolism of 5-FU
(Takechi et al., 2002). A side effect of 5-FU is diarrhea, which is caused by its phosphorylation in
the intestine by orotate phosphoribosyltransferase. Oxo, a specific inhibitor of orotate
phosphoribosyltransferase, is a protective agent against 5-FU-induced diarrhea (Shirasaka et al.,
1993). In phase II studies, it was demonstrated that single treatment with S-1 had a high response
rate, ranging from 19% to 39%, and good compliance with advanced colorectal cancer patients
(Ohtsu et al., 2000; Van den Brande et al., 2003; Shirao et al., 2004). The safety and efficacy of
combination therapy with CPT-11 and S-1 were investigated in a phase ΙΙ study of patients with
colorectal cancer, and its usefulness was confirmed (Goto et al., 2006). Overall response rate of
combination therapy with CPT-11 and S-1 was 62.5%, and median progression-free survival was
8.0 months.
Previous studies reported that the plasma concentration and area under the plasma
concentration curve (AUC) of CPT-11 was elevated, and those of SN-38 reduced, in combined
therapy with CPT-11 and 5-FU compared to single treatment with CPT-11 in colorectal cancer
patients (Sasaki et al., 1994). Recently, we found that oral coadministration of S-1 appeared to
affect the pharmacokinetic behavior of CPT-11, SN-38 and SN-38G in an advanced colorectal
cancer patient (Yokoo et al., 2006). In particular, coadministration of S-1 resulted in a significant
decrease in the plasma concentration of SN-38 compared to single treatment with CPT-11.
However, the mechanisms of this drug interaction have not been elucidated. Therefore, we
explored the effect of coadministration of S-1 on the pharmacokinetics of CPT-11 and its
metabolites in rats.
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Materials and Methods
Materials. CPT-11, SN-38 and SN-38G were kindly provided from Daiichi
Pharmaceutical Co.Ltd (Tokyo, Japan). S-1 was kindly provided from Taiho Pharmaceutical Co.
Ltd (Tokyo, Japan). p-nitrophenylacetate (p-NPA) and p-nitrophenol (p-NP) were purchased from
the Sigma Chemical Co (St Louis, MO). All other chemicals were commercially available products
and of analytical grade.
Experimental animals. Male Sprague-Dawley (SD) rats (7 weeks-old) were purchased
from Kyudo Co. Ltd. (Kumamoto, Japan). The rats were housed in a standard animal maintenance
facility at constant temperature (21 ºC-23 ºC), humidity (50%-70%) and a 12 h:12 h light/dark
cycle for at least 1 week before the day of the experiment. All animal experiments were conducted
according to the guidelines of Kumamoto University for the care and use of laboratory animals.
Pharmacokinetic and biliary excretion analyses. Rats were orally administered the
carboxyl methyl cellulose (CMC) (0.5 w/v%) (without S-1) or S-1 (10 mg/kg) (with S-1) once a
day for 7 consecutive days. On day 6, the rats were fasted overnight with free access to drinking
water before commencement of the experiments. On day 7, CPT-11 was intravenously (i.v.)
administered to rats at 10 mg/kg via the left jugular vein about 1 min after oral administration of
CMC or S-1. Blood samples were collected from the right jugular vein at 5, 15, 30 and 45 min and
1, 1.5, 2, 3 and 4 hours after injection of CPT-11. Bile was collected from a cannula implanted in
the bile duct. Samples were collected at various intervals from the injection of CPT-11 to 5 min, 5
to 15 min, 15 to 30 min, 30 to 45 min, 45 min to 1 hour or over 1 hour. Thereafter, bile was collected
over 30 min intervals. The blood samples were centrifuged at 3,000 g for 5 min, and the plasma and
bile samples were stored at -80°C until analysis. The concentration of the carboxylate and lactone
forms of CPT-11, SN-38 and SN-38G were measured by high-performance liquid chromatography
as described previously (Hamada et al., 2005). Pharmacokinetic parameters for CPT-11, SN-38 and
SN-38G were estimated by non-compartmental model methods with the use of WinNonlin version
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3.1 software (Pharsight, Cary, NC).
Inhibition studies on the hydrolysis of p-NPA by 9,000 g supernatant (S9) of HepG2
cells. HepG2 cells cultured for 7 days in 75-cm2 culture flasks were washed with ice-cold
phosphate-buffered saline (PBS, without CaCl2 and MgCl2) and then removed with a cell scraper.
The cells were suspended in 50 mM HEPES buffer (pH 7.4). The sonicated cells were then
homogenized under ice-cold conditions. After centrifugation of the cell homogenate at 9,000 g for
20 min at 4°C, the supernatant (S9) was obtained. Protein content was determined by the method
described by Bradford with bovine serum albumin as the standard.
The hydrolysis reaction was initiated by the addition of p-NPA after preincubation (37°C)
of S9 fraction for 5 min, and the spontaneous rate of hydrolysis was recorded. The enzymatic
hydrolysis of p-NPA was followed spectrophotometrically at 400 nm as described previously
(Yoshigae et al., 1998). In experiments involving inhibition by CPT-11, FT, 5-FU, CDHP and Oxo,
the enzyme solution of S9 fraction was preincubated (37°C) for 5 min with various concentrations
of them dissolved in HEPES buffer or without them in controls.
Hydrolysis of CPT-11 by rat liver microsomes. Livers were harvested from male SD rats
that had been administrated CMC or S-1 (10 mg/kg). The livers were stored at -80°C until use.
Hepatic microsomes were prepared by differential centrifugation. The rat liver homogenates were
centrifuged at 9000 g for 20 min at 4°C. The supernatant was then centrifuged at 105,000 g for 1
hour at 4°C. The microsomes were stored at -80°C in PBS (10 mg/mL) until use. Incubations of
microsomes with CPT-11 were carried out in PBS at 37°C in 0.5 mL Eppendorf tubes. A stock
solution of CPT-11 was prepared directly in PBS, and the pH was corrected to pH 7.4. An aliquot of
the enzyme stock (25 µL) was added to 125 µL of PBS. Following 5 min of equilibration at 37°C,
the reaction was initiated by the addition of the required dilution of the CPT-11 stock (100 µL) to
yield concentrations of CPT-11 ranging from 1 to 1000 µM in a total of 250 µL. The tubes were
agitated in a shaking water bath maintained at 37°C. Aliquots (50 µL) were withdrawn at regular
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intervals and immediately mixed with 100 µL of ice-cold methanol to quench the reaction. Samples
were then assayed for total forms (carboxylate form plus lactone form) of SN-38 as detailed earlier.
Western blot analysis. Livers were homogenized in a homogenization buffer comprised of
230 mM sucrose, 5 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, 1
mg/ml leupeptin and 1 mg/ml pepstatin A. The homogenate was centrifuged twice at 3,000 g for 15
min, and the supernatant was further centrifuged at 105,000 g for 30 min. The resultant pellet is
referred to as the crude membrane fraction. After measurement of the protein content using a BCA
protein assay reagent (Pierce, Rockford, IL), each samples was mixed in a loading buffer (2% SDS,
125 mM Tris-HCl (pH 6.4), 20% glycerol, 5% 2-mercaptoethanol) and heated at 100°C for 2 min.
The sample were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford,
MA) by semi-dry electroblotting. The blots were blocked overnight at 4°C with 2% ECL advance
blocking agents (GE Healthcare Bio-sciences Corp., Piscataway, NJ) in Tris-buffered saline
containing 0.3% Tween 20 (TBS-T), and incubated for 1 hour at room temperature with primary
antibody specific for P-gp (C219 monoclonal antibody, Signet Laboratories, Inc., Dedham, MA),
MRP2 (MRP2 monoclonal antibody, CHEMICON International, Inc., Temecula, CA) and BCRP
(ABCG2 polyclonal antibody, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The blots were
washed with TBS-T and incubated with the secondary antibody (horseradish peroxidase-linked
antirabbit immunoglobulin F (ab)2 or horseradish peroxidase-linked antimouse immunoglobulin
F(ab)2; both from GE Healthcare Bio-sciences Corp.) for 1 hour at room temperature. Immunoblots
were visualized with an ECL system (ECL Advance Western Blotting Detection Kit; GE
Healthcare Bio-sciences Corp.). The relative amount of each band was determined
densitometrically using Densitograph Imaging Software (ATTO Corporation, Tokyo, Japan).
Densitometric ratios were normalized to the value obtained without S-1.
Statistical Analysis. The differences were analyzed statistically using Student’s unpaired t
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test. A value of P < 0.05 was considered significant.
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Results
Effect of S-1 on the pharmacokinetics of CPT-11 and its metabolites. Figure 2 shows the
plasma concentration-time profiles of the carboxylate form, lactone form and total form (i.e.
carboxylate form plus lactone form) of CPT-11, active metabolite SN-38 and inactive metabolite
SN-38G after i.v. administration of CPT-11 at a dose of 10 mg/kg with or without S-1 (10
mg/kg/day) in rats. Oral coadministration of S-1 appeared to affect the pharmacokinetic parameters
of CPT-11, SN-38 and SN-38G (Table 1). In particular, coadministration of S-1 resulted in a
significant decrease in the plasma concentration of the SN-38 lactone form compared to single
treatment with CPT-11. The maximum plasma concentration (Cmax) of SN-38 lactone form
decreased by 30.1% from 144.1 ± 15.8 ng/mL (without S-1) to 100.8 ± 8.9 ng/mL (with S-1).
Furthermore, the AUC of SN-38 lactone form decreased by 33.8% from 235.8 ± 43.6 ng·h/mL
(without S-1) to 156.2 ± 15.0 ng·h/mL (with S-1). However, Cmax and AUC of the SN-38
carboxylate form were unchanged by coadministration of S-1. Whereas, Cmax and AUC of both
CPT-11 and SN-38G showed a slight decrease in concentration upon coadministration with S-1.
Effect of S-1 on the biliary excretion of CPT-11 and its metabolites. Figure 3 shows the
cumulative biliary excretion curves of the carboxylate form, lactone form and total forms of
CPT-11, SN-38 and SN-38G after i.v. administration of CPT-11 with or without S-1 in rats. After i.v.
treatment of CPT-11, excretion of CPT-11 carboxylate and lactone forms into bile was unaltered by
coadministration of S-1. SN-38 was excreted into bile mostly as the SN-38 carboxylate form,
whereas SN-38G was excreted into bile mostly as the SN-38G lactone form. The biliary excretion
of both forms of CPT-11 and SN-38G or SN-38 lactone form was unaltered by administration of
S-1. However, the biliary excretion of SN-38 carboxylate form was significantly increased by
coadministration of S-1. The amount of SN-38 carboxylate form excreted via bile significantly
increased from 9.7 ± 3.3% (without S-1) to 14.9 ± 3.7% (with S-1) 4 hours after coadministration
of S-1. In addition, the total amount of total forms of SN-38 also significantly increased after
coadministration of S-1 from 12.4 ± 2.8% (without S-1) to 17.6 ± 1.0% (with S-1).
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Effect of each component of S-1 and 5-FU on the relative inhibition of p-NPA
hydrolysis. Kinetic analysis of p-NPA hydrolysis at a range of concentrations (0.1 to 5.0 mM) was
performed (Fig. 4A). The hydrolysis of p-NPA was best fitted by a single Michaelis-Menten
equation with a Michaelis-Menten constant (Km) of 599 µM and maximum hydrolysis rate (Vmax)
of 8.38 µM/min. The effects of CPT-11, FT, 5-FU, CDHP and Oxo on p-NPA (500 µM) hydrolysis
were examined by incubating with the S9 fraction of HepG2 cells (Fig. 4B). CPT-11 was found to
be a potent inhibitor of p-NPA hydrolysis, whereas FT, 5-FU, CDHP and Oxo did not inhibit p-NPA
hydrolysis.
Effect of S-1 on the hydrolysis of CPT-11 by rat liver microsomes. Production of SN-38
from 10 µM CPT-11 over a 2-hour incubation period with rat liver microsomes in the absence of
S-1 is shown in Fig. 5A. A linear regression of the concentration versus time plot from 15 min
onwards yielded a steady-state velocity (r2 = 0.99). The kinetics of production of total forms of
SN-38 from CPT-11 at a range of concentrations (0.1 to 300 µM) was investigated at 1 hour (Fig.
5B). The Km and Vmax of hydrolysis of CPT-11 with rat liver microsomes were unchanged by
administration of S-1 for 7 consecutive days. The Km and Vmax values for rat liver microsomes in
the absence or presence of S-1 were 4.08 µM and 466 nM/h or 5.23 µM and 472 nM/h,
respectively.
Effect of S-1 on P-gp, MRP2 and BCRP protein expression in the liver. Potential
changes to the expression of P-gp, MRP2 and BCRP protein in the crude plasma membrane from
rat liver in the absence or presence of S-1 were explored by Western blot analyses (Fig. 6A). The
presence of S-1 significantly increased the level of BCRP protein in the crude plasma membrane
preparation of the rat liver. The relative amount of BCRP with S-1 was about 1.6-fold higher than
that without S-1. By contrast, administration of S-1 had no significant effect on the P-gp and MRP2
protein levels.
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Discussion
The efficacy of combination therapy with various anticancer agents has now been
generally recognized. However, there is little information on the pharmacokinetic interaction of
combination chemotherapy. Recently, we reported the pharmacokinetic changes of CPT-11 and its
metabolites in a colorectal cancer patient given oral S-1 (Yokoo et al., 2006). Here, we investigated
the effect of S-1 on the pharmacokinetics of CPT-11 and its metabolites in rats in order to explore
the possible mechanism of drug interaction.
Our results show that oral coadministration of S-1 causes a significant decrease in the
plasma concentration of an active metabolite, SN-38. In particular, Cmax and AUC of the SN-38
lactone form were decreased by coadministration of S-1 (Table 1). Furthermore, coadministration
of S-1 resulted in a significant increase in the biliary excretion of total form of SN-38 (Fig. 3). The
predominat biliary excretion of SN-38 was in SN-38 carboxylate form, rather than SN-38 lactone
form. The rate of biliary excretion of SN-38 carboxylate form increased on coadministration of S-1,
however, that of SN-38 lactone form was not increased. Accordingly, as an increase in biliary
excretion of total form of SN-38 could be resulted from the increased biliary excretion of
carboxylate form. By contrast, the total volume of bile secretion was unchanged between rats given
CPT-11 alone or in combination with S-1 (data not shown, P = 0.84). On the other hand, P-gp,
MRP2 and BCRP expressed at the bile canalicular membrane appear to be responsible for the
biliary excretion of CPT-11 and its metabolites (Sugiyama et al., 1998; Nakatomi et al., 2001).
Thus, S-1 mediated up-regulation of one or more of these membrane proteins might explain the
increased biliary excretion of SN-38. A previous pharmacokinetic study of colon cancer patients
demonstrated that cyclosporin, a modulator of P-gp and MRP2, reduces the clearance of CPT-11
and increases the AUC of CPT-11 after coadministration (Chester et al., 2003). Furthermore, it was
reported that the biliary excretion and the intestinal exsorption of CPT-11 or SN-38 were
significantly inhibited by coadministration of cyclosporin in rats (Arimori et al., 2003).
Figure 3 shows that the rate of biliary excretion of the SN-38 carboxylate form increased
upon coadministration of S-1. The total amount of SN-38 carboxylate form excreted in the absence
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or presence of S-1 was 9.7% and 14.9% of the dose 4 hour after administration, respectively (P <
0.01). Fig. 6 shows that the BCRP protein level in crude plasma membrane from the liver with S-1
was 1.6-fold higher than that without S-1 (P < 0.05). On the other hand, the mRNA level of BCRP
was measured in both the intestine and kidney with or without S-1, and we found that the mRNA
level was not affected by administration of S-1 in both tissues (data not shown). Therefore, an
increase in the level of BCRP protein might result in increased biliary excretion of the SN-38
carboxylate form. In previous studies, sulfasalazine, various aryl hydrocarbon receptor agonists
and progesterone were found to significantly increase the level of BCRP protein in vitro (van der
Heijden et al., 2004; Ebert et al., 2005; Wang et al., 2006). By contrast, 17β-estradiol significantly
decreases the level of BCRP protein in vitro (Wang et al., 2006). However, there are few previous
reports of drug-related regulation of BCRP protein levels in vivo (Han and Sugiyama, 2006). Here,
we report the S-1 mediated induction of BCRP protein in vivo. Our study shows that the rate of
biliary excretion of SN-38 carboxylate form is significantly increased by coadministration of S-1,
while that of SN-38 lactone form is unaltered. These results suggest BCRP may display a higher
affinity for SN-38 carboxylate form relative to SN-38 lactone form. Further studies are required to
confirm this hypothesis.
Although the rate of biliary excretion of SN-38 carboxylate form increased upon
coadministration of S-1, Cmax and AUC of SN-38 lactone form decreased. The predominant
CPT-11 metabolite in rat liver is SN-38 carboxylate form, rather than SN-38 lactone form. Four
hours after i.v. administration of CPT-11 in the absence of S-1, the concentration of the SN-38
carboxylate and lactone forms in the liver was 0.7 ± 0.2 and 0.2 ± 0.1 ng/g of tissue, respectively.
Thus, the ratio of the carboxylate or lactone forms of SN-38 to the total amount of SN-38 was
77.8% and 22.2%, respectively. Similar results were obtained after coadministration of CPT11 with
S-1 (data not shown). These results suggest that the induction of the BCRP protein level by
coadministration of S-1 increased the rate of biliary excretion of SN-38 carboxylate form, followed
by hydrolytic cleavage of the lactone, yielding the carboxylate form of SN-38 in the liver. The
lower rate of secretion of SN-38 lactone form from liver to blood compared to that of SN-38
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carboxylate form could explain the lower Cmax and AUC of SN-38 lactone form.
Previous studies of drug interaction between CPT-11 and 5-FU suggested that 5-FU
inhibits the enzymatic hydrolysis of CPT-11 to SN-38 in human and rats (Sasaki et al., 1994;
Umezawa et al., 2000). However, the hydrolysis of p-NPA was not inhibited by components of S-1
and 5-FU (Fig. 4). Furthermore, administration of S-1 did not affect the hydrolysis activity of rat
liver microsomes (Fig. 5). Therefore, inhibition of the hydrolysis activity by coadministration of
S-1 cannot account for the pharmacokinetic parameters of SN-38.
The piperidine ring of CPT-11 is oxidized to 7-ethyl-10-[4-N-(5-aminopentanoic
acid)-1-piperidino]carbonyloxycamptothecin and 7-ethyl-10-(4-amino-1-piperidino)
carbonyloxycamptothecin by cytochrome P450 (CYP) 3A4 and CYP3A5 in the liver (Santos et al.,
2000). A decrease in the Cmax and AUC of SN-38 lactone form could be caused by enhanced
activity of CYP3A upon coadministration of S-1. Indeed, a previous study showed that chronic oral
administration of FT (100 mg/kg daily for 20 days) resulted in the induction of CYP3A in rat liver
(Yamazaki et al., 2001). However, no increase in the concentration of CYP3A-derived metabolites
of CPT-11 could be detected (data not shown). We therefore conclude that the effect of
coadministration of S-1 on CYP3A activity can be ignored.
SN-38 is conjugated with glucuronic acid by hepatic UGT1A to form the inactive
metabolite SN-38G (Rivory and Robert, 1995). Induction or activation of UTG1A by treatment
with S-1 is a potential mechanism for the observed drug interaction. If this is the case, we might
anticipate an increase in the plasma concentration of SN-38G. However, we found that the plasma
concentration of SN-38G was lower upon coadministration of S-1, suggesting that S-1 has no effect
on UGT1A activity.
In summary, we have demonstrated significant drug interaction between CPT-11 and S-1
in rats. In particular, the Cmax and AUC of SN-38 lactone form were markedly reduced by
coadministration of S-1. Induction of BCRP protein was observed in the rat liver with S-1, resulting
in an increased rate of biliary excretion of SN-38 carboxylate form. We suggest our data reveals the
underlying mechanism of drug interaction between CPT-11 and S-1 which has been observed in
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previous clinical case reports. Further studies are needed to establish the most effective regimen for
combination therapy based on the pharmacokinetic and pharmacodynamic data.
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References
Arimori K, Kuroki N, Hidaka M, Iwakiri T, Yamsaki K, Okumura M, Ono H, Takamura N, Kikuchi
M and Nakano M (2003) Effect of P-glycoprotein modulator, cyclosporin A, on the
gastrointestinal excretion of irinotecan and its metabolite SN-38 in rats. Pharm Res
20:910-917.
Chester JD, Joel SP, Cheeseman SL, Hall GD, Braun MS, Perry J, Davis T, Button CJ and Seymour
MT (2003) Phase I and pharmacokinetic study of intravenous irinotecan plus oral
ciclosporin in patients with fuorouracil-refractory metastatic colon cancer. J Clin Oncol
21:1125-1132.
Ebert B, Seidel A and Lampen A (2005) Identification of BCRP as transporter of benzo[a]pyrene
conjugates metabolically formed in Caco-2 cells and its induction by Ah-receptor agonists.
Carcinogenesis 26:1754-1763.
Goto A, Yamada Y, Yasui H, Kato K, Hamaguchi T, Muro K, Shimada Y and Shirao K (2006)
Phase II study of combination therapy with S-1 and irinotecan in patients with advanced
colorectal cancer. Ann Oncol 17:968-973.
Hamada A, Aoki A, Terazaki H, Ito K, Yokoo K, Sasaki Y and Saito H (2005) Pharmacokinetic
changes of irinotecan by intestinal alkalinization in an advanced colorectal cancer patient.
Ther Drug Monit 27:536-538.
Han Y and Sugiyama Y (2006) Expression and regulation of breast cancer resistance protein and
multidrug resistance associated protein 2 in BALB/c mice. Biol Pharm Bull 29:1032-1035.
Humerickhouse R, Lohrbach K, Li L, Bosron WF and Dolan ME (2000) Characterization of
CPT-11 hydrolysis by human liver carboxylesterase isoforms hCE-1 and hCE-2. Cancer
Res 60:1189-1192.
Ikeda K, Yoshisue K, Matsushima E, Nagayama S, Kobayashi K, Tyson CA, Chiba K and
Kawaguchi Y (2000) Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytochrome
P-450 2A6 in human liver microsomes in vitro. Clin Cancer Res 6:4409-4415.
Kawato Y, Aonuma M, Hirota Y, Kuga H and Sato K (1991) Intracellular roles of SN-38, a
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 30, 2007 as DOI: 10.1124/dmd.107.015164
at ASPE
T Journals on M
arch 26, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD 15164
18
metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11.
Cancer Res 51:4187-4191.
Nakatomi K, Yoshikawa M, Oka M, Ikegami Y, Hayasaka S, Sano K, Shiozawa K, Kawabata S,
Soda H, Ishikawa T, Tanabe S and Kohno S (2001) Transport of
7-ethyl-10-hydroxycamptothecin (SN-38) by breast cancer resistance protein ABCG2 in
human lung cancer cells. Biochem Biophys Res Commun 288:827-832.
Ohtsu A, Baba H, Sakata Y, Mitachi Y, Horikoshi N, Sugimachi K and Taguchi T (2000) Phase II
study of S-1, a novel oral fluorophyrimidine derivative, in patients with metastatic
colorectal carcinoma. S-1 Cooperative Colorectal Carcinoma Study Group. Br J Cancer
83:141-145.
Rivory LP, Chatelut E, Canal P, Mathieu-Boue A and Robert J (1994) Kinetics of the in vivo
interconversion of the carboxylate and lactone forms of irinotecan (CPT-11) and of its
metabolite SN-38 in patients. Cancer Res 54:6330-6333.
Rivory LP and Robert J (1995) Identification and kinetics of a beta-glucuronide metabolite of
SN-38 in human plasma after administration of the camptothecin derivative irinotecan.
Cancer Chemother Pharmacol 36:176-179.
Santos A, Zanetta S, Cresteil T, Deroussent A, Pein F, Raymond E, Vernillet L, Risse ML, Boige V,
Gouyette A and Vassal G (2000) Metabolism of irinotecan (CPT-11) by CYP3A4 and
CYP3A5 in humans. Clin Cancer Res 6:2012-2020.
Sasaki Y, Ohtsu A, Shimada Y, Ono K and Saijo N (1994) Simultaneous administration of CPT-11
and fluorouracil: alteration of the pharmacokinetics of CPT-11 and SN-38 in patients with
advanced colorectal cancer. J Natl Cancer Inst 86:1096-1098.
Shirao K, Ohtsu A, Takada H, Mitachi Y, Hirakawa K, Horikoshi N, Okamura T, Hirata K, Saitoh S,
Isomoto H and Satoh A (2004) Phase II study of oral S-1 for treatment of metastatic
colorectal carcinoma. Cancer 100:2355-2361.
Shirasaka T, Nakano K, Takechi T, Satake H, Uchida J, Fujioka A, Saito H, Okabe H, Oyama K,
Takeda S, Unemi N and Fukushima M (1996) Antitumor activity of 1 M tegafur-0.4 M
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 30, 2007 as DOI: 10.1124/dmd.107.015164
at ASPE
T Journals on M
arch 26, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD 15164
19
5-chloro-2,4-dihydroxypyridine-1 M potassium oxonate (S-1) against human colon
carcinoma orthotopically implanted into nude rats. Cancer Res 56:2602-2606.
Shirasaka T, Shimamoto Y and Fukushima M (1993) Inhibition by oxonic acid of gastrointestinal
toxicity of 5-fluorouracil without loss of its antitumor activity in rats. Cancer Res
53:4004-4009.
Sugiyama Y, Kato Y and Chu X (1998) Multiplicity of biliary excretion mechanisms for the
camptothecin derivative irinotecan (CPT-11), its metabolite SN-38, and its glucuronide:
role of canalicular multispecific organic anion transporter and P-glycoprotein. Cancer
Chemother Pharmacol 42 Suppl:S44-49.
Takasuna K, Hagiwara T, Hirohashi M, Kato M, Nomura M, Nagai E, Yokoi T and Kamataki T
(1996) Involvement of beta-glucuronidase in intestinal microflora in the intestinal toxicity
of the antitumor camptothecin derivative irinotecan hydrochloride (CPT-11) in rats. Cancer
Res 56:3752-3757.
Takechi T, Fujioka A, Matsushima E and Fukushima M (2002) Enhancement of the antitumour
activity of 5-fluorouracil (5-FU) by inhibiting dihydropyrimidine dehydrogenase activity
(DPD) using 5-chloro-2,4-dihydroxypyridine (CDHP) in human tumour cells. Eur J
Cancer 38:1271-1277.
Umezawa T, Kiba T, Numata K, Saito T, Nakaoka M, Shintani S and Sekihara H (2000)
Comparisons of the pharmacokinetics and the leukopenia and thrombocytopenia grade after
administration of irinotecan and 5-fluorouracil in combination to rats. Anticancer Res
20:4235-4242.
Van den Brande J, Schoffski P, Schellens JH, Roth AD, Duffaud F, Weigang-Kohler K, Reinke F,
Wanders J, de Boer RF, Vermorken JB and Fumoleau P (2003) EORTC Early Clinical
Studies Group early phase II trial of S-1 in patients with advanced or metastatic colorectal
cancer. Br J Cancer 88:648-653.
van der Heijden J, de Jong MC, Dijkmans BA, Lems WF, Oerlemans R, Kathmann I, Schalkwijk
CG, Scheffer GL, Scheper RJ and Jansen G (2004) Development of sulfasalazine resistance
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 30, 2007 as DOI: 10.1124/dmd.107.015164
at ASPE
T Journals on M
arch 26, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD 15164
20
in human T cells induces expression of the multidrug resistance transporter ABCG2
(BCRP) and augmented production of TNFα. Ann Rheum Dis 63:138-143.
Wang H, Zhou L, Gupta A, Vethanayagam RR, Zhang Y, Unadkat JD and Mao Q (2006)
Regulation of BCRP/ABCG2 expression by progesterone and 17β-estradiol in human
placental BeWo cells. Am J Physiol Endocrinol Metab 290:E798-807.
Wiseman LR and Markham A (1996) Irinotecan. A review of its pharmacological properties and
clinical efficacy in the management of advanced colorectal cancer. Drugs 52:606-623.
Yamazaki H, Komatsu T, Takemoto K, Shimada N, Nakajima M and Yokoi T (2001) Rat
cytochrome p450 1A and 3A enzymes involved in bioactivation of tegafur to 5-fluorouracil
and autoinduced by tegafur in liver microsomes. Drug Metab Dispos 29:794-797.
Yokoo K, Watanabe H, Hamada A, Masa K, Saito H, Terazaki H and Sasaki Y (2006) Effect of S-1
on pharmacokinetics of irinotecan in a patient with colorectal cancer. Clin Pharmacol Ther
80:422-424.
Yoshigae Y, Imai T, Horita A, Matsukane H and Otagiri M (1998) Species differences in
stereoselective hydrolase activity in intestinal mucosa. Pharm Res 15:626-631.
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Footnotes
This work was supported, in part, by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports, and Culture of Japan
To whom correspondence should be addressed: Professor Hideyuki Saito, Ph.D. Department of
pharmacy, Kumamoto University Hospital, 1-1-1 Honjo, Kumamoto 860-8556, Japan, Phone &
Fax: 81-96-373-5820; E-mail: [email protected]
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Legends for figures
Figure 1. Metabolic pathway of the carboxylate and lactone forms of CPT-11.
Figure 2. Plasma concentration–time profiles of CPT-11 and its metabolites in the absence or
presence of S-1. Plasma concentration-time profiles of the carboxylate form (A, D, G), lactone
form (B, E, H) and total forms (i.e. carboxylate form plus lactone form) (C, F, I) of CPT-11 (○, ●),
SN-38 (�, ▲) and SN-38G (□, ■) in rats after receiving CPT-11 at 10 mg/kg i.v. administration in
the absence (○, �, □) or presence of S-1 (●, ▲, ■) at a dosage of 10 mg/kg/day. All data are
expressed as mean ± S.D., n = 5.
Figure 3. Biliary excretion of CPT-11 and its metabolites in the absence or presence of S-1.
Cumulative amounts versus time profiles of the carboxylate form (A, D, G), lactone form (B, E, H)
and total forms of (i.e. carboxylate form plus lactone form) (C, F, I) of CPT-11 (○, ●), SN-38 (�,
▲) and SN-38G (□, ■) in rats after receiving CPT-11 at 10 mg/kg i.v. administration in the absence
(○, �, □) or presence of S-1 (●, ▲, ■) at a dosage of 10 mg/kg/day. All data are expressed as means
± S.D., n = 5. The differences were analyzed statistically using Student’s unpaired t test. ∗ p < 0.05;
∗∗ p < 0.01.
Figure 4. Rate of p-NPA hydrolysis and the relative inhibition of p-NPA hydrolysis in the
HepG2 cell S9 fraction. (A) Rate of p-NPA hydrolysis as a function of p-NPA concentration (0.1
to 5 mM). (B) The relative inhibition (%) of p-NPA hydrolysis in HepG2 cell S9 fraction by
CPT-11, FT, 5-FU, CDHP and Oxo. All data are expressed as means ± S.D., n = 3.
Figure 5. Production of SN-38-incubation time profiling and enzyme kinetics in rat liver
microsomes. (A) Generation of SN-38 by incubating CPT-11 (10 µM) with rat liver microsomes in
the absence of S-1. (B) Production of SN-38 from CPT-11 in rat liver microsomes in the absence
(�) or presence of S-1 (▲) at a dosage of 10 mg/kg/day. All data are expressed as means ± S.D., n
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= 3.
Figure 6. Effect of S-1 on the protein concentration of P-gp, MRP2 and BCRP in the crude
membrane fraction of rat liver. (A) Western blot analyses for P-gp, MRP2 and BCRP in the crude
membrane fraction of rat liver in the absence or presence of S-1 at a dosage of 10 mg/kg/day. (B)
The quantitative data mean ± S.D. of densitometry measurements of the crude membrane fraction
of rat liver in the absence (open bars) or presence of S-1 (filled bars) at a dosage of 10 mg/kg/day.
The values observed in the absence of S-1 were arbitrarily defined as 100%. All data are expressed
as means ± S.D. for 4 rats in each group. The differences were analyzed statistically using Student’s
unpaired t test. ∗ p < 0.05.
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Table 1. Pharmacokinetic parameters of carboxylate form, lactone form and total forms (i.e.
carboxylate form plus lactone form) of CPT-11, SN-38 and SN-38G in rats administrated of
CPT-11 (10 mg/kg by i.v. bolus injection) without or with S-1 (10 mg/kg/day by oral administration
once a daily for 7 consecutive days). All data are expressed as means ± S.D., n = 5.
The differences were analyzed statistically using Student’s unpaired t test.
Parameters Without S-1 With S-1 % of Change P Value
CPT-11 Carboxylate form 1172.4 ± 403.5 773.9 ± 239.0 -34.0 0.0943347.9 ± 1148.3 1955.0 ± 920.9 -41.6 0.067
Lactone from 6167.5 ± 1351.9 4838.4 ± 698.2 -21.6 0.0876953.4 ± 1684.1 5428.9 ± 1282.8 -21.9 0.146
Total form 8363.3 ± 3178.1 5206.7 ± 821.7 -37.7 0.06411037.6 ± 1470.0 7383.9 ± 2160.1 -33.1 0.014
SN-38 Carboxylate form 118.8 ± 65.0 112.4 ± 23.9 -5.4 0.844219.3 ± 26.8 236.2 ± 59.9 7.7 0.581
Lactone from 144.1 ± 15.8 100.7 ± 8.9 -30.1 <0.001235.8 ± 43.6 156.2 ± 15.0 -33.8 0.005
Total form 259.6 ± 63.9 203.0 ± 28.6 -21.8 0.108468.2 ± 74.8 390.4 ± 72.7 -16.6 0.134
SN-38G Carboxylate form 75.0 ± 21.54 45.3 ± 19.5 -39.6 0.051244.0 ± 64.7 152.8 ± 68.5 -37. 4 0.062
Lactone from 216.3 ± 113.4 134.4 ± 27.1 -37.9 0.155410.8 ± 181.7 337.2 ± 82.3 -17.9 0.433
Total form 232.4 ± 72.6 174.0 ± 41.0 -25.1 0.156
Cmax (ng/mL)AUC (ngįh/mL)
Cmax
Cmax
Cmax
Cmax
Cmax
Cmax
Cmax
Cmax
644.1 ± 249.2 490.0 ± 146.7 -23.9 0.268
AUC
AUC
AUC
AUC
AUC
AUC
AUC
AUC
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CPT-11 Lactone Form
O
O
O
NN N O
NO
OH
OH
CPT-11 Carboxylate Form
Neutral / Base
Acid
SN-38 Lactone Form SN-38 Carboxylate Form
SN-38G Lactone Form SN-38G Carboxylate Form
O
O
OH
NOH
N
C2H5
OH C2H5
OH
C2H5
C2H5
O
O
OH
NO
N
C2H5
OH C2H5
OHO
OH
OH
COOH
OH
H
H
H
H
H
O
O
O
NOH
N
C2H5
OH C2H5
O
O
O
NN N O
NO
C2H5
OH C2H5
O
O
O
NO
N
C2H5
OH C2H5
O
OH
OH
COOH
OH
H
H
H
H
H
Neutral / Base
Acid
Neutral / Base
Acid
Fig. 1
CES CES
UGT1A UGT1A
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Carboxylate Form Lactone Form Total Form
A B C
D E F
G H I
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
Pla
sma
Con
cent
ratio
n of
CP
T-1
1(n
g/m
L)
Pla
sma
Con
cent
ratio
n of
CP
T-1
1(n
g/m
L)
Pla
sma
Con
cent
ratio
n of
CP
T-1
1(n
g/m
L)
Pla
sma
Con
cent
ratio
n of
SN
-38
(ng/
mL)
Pla
sma
Con
cent
ratio
n of
SN
-38
(ng/
mL)
Pla
sma
Con
cent
ratio
n of
SN
-38
(ng/
mL)
Pla
sma
Con
cent
ratio
n of
SN
-38G
(ng/
mL)
Pla
sma
Con
cent
ratio
n of
SN
-38G
(ng/
mL)
Pla
sma
Con
cent
ratio
n of
SN
-38G
(ng/
mL)
102
103
104
105
102
103
104
105
102
103
104
105
102
103
104
102
103
104
102
103
104
102
103
104
102
103
104
102
103
104
Fig. 2
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Cum
ulat
ive
Am
ount
of C
PT
-11
(% o
f tot
al d
ose)
Cum
ulat
ive
Am
ount
of C
PT
-11
(% o
f tot
al d
ose)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
Cum
ulat
ive
Am
ount
of C
PT
-11
(% o
f tot
al d
ose)
Cum
ulat
ive
Am
ount
of S
N-3
8G(%
of t
otal
dos
e)
Cum
ulat
ive
Am
ount
of S
N-3
8G(%
of t
otal
dos
e)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
Cum
ulat
ive
Am
ount
of S
N-3
8G(%
of t
otal
dos
e)
Cum
ulat
ive
Am
ount
of S
N-3
8(%
of t
otal
dos
e)
Cum
ulat
ive
Am
ount
of S
N-3
8(%
of t
otal
dos
e)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
0 1 2 3 4Time (h)
Cum
ulat
ive
Am
ount
of S
N-3
8(%
of t
otal
dos
e)
Lactone Form
0
20
40
60
80B
0
2
4
6
8
10H
0
5
10
15
20E
Carboxylate Form
0
20
40
60
80A
0
2
4
6
8
10G
0
5
10
15
20D
∗∗
∗∗
Total Form
0
20
40
60
80C
0
2
4
6
8
20I
0
5
10
15
20F
∗∗
∗∗∗∗
∗∗
∗∗∗
∗∗
Fig. 3
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1 10 100 10000
10
20
30
40
50
60
Concentration (µM)
% o
f Inh
ibiti
on o
f p-N
PA
Hyd
roly
sis
CPT-11FT
CDHPOxo
5-FU
Fig. 4
B
0 1 2 3 4 5 60
1
2
3
4
5
6
7
8
p-NPA Concentration (mM)
p-N
PA
Hyd
roly
sis
(µM
/min
)
0 0.2 0.4 0.6 0.8 1.0 1.2
0
1
2
3
4
5
6
p-NPA Concentration (mM)
p-N
PA
Hyd
roly
sis
(µM
/min
)
A
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Fig. 5
0 30 60 90 120 150
0 100 200 300
Time (h)
Concentration of CPT-11 (µM)
SN
-38
Pro
duct
ion
(nM
/h)
600
400
200
0
SN
-38
Pro
duct
ion
(nM
)
600
400
200
0
A
B
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BCRP
Without S-1 With S-10
50
100
150
200 ∗
Rel
ativ
e pr
otei
n le
velMRP2
Without S-1 With S-10
50
100
150
200
Rel
ativ
e pr
otei
n le
velP-gp
Without S-1 With S-10
50
100
150
200
Rel
ativ
e pr
otei
n le
vel
Fig. 6
Without S-1 With S-1
P-gp
MRP2
BCRP
A
B
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 30, 2007 as DOI: 10.1124/dmd.107.015164
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