Involvement of up-regulation of hepatic breast cancer resistance …dmd.aspetjournals.org/content/dmd/early/2007/05/30/dmd... · 2007-05-30 · breast cancer resistance protein (BCRP/ABCG2),

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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.)

DMD Fast Forward. Published on May 30, 2007 as doi:10.1124/dmd.107.015164

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

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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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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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


50

100

150

200

Rel

ativ

e pr

otei

n le

velP-gp


50

100

150

200

Rel

ativ

e pr

otei

n le

vel

Fig. 6


P-gp

MRP2

BCRP

A

B


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