2013

http://informahealthcare.com/xenISSN: 0049-8254 (print), 1366-5928 (electronic)

Xenobiotica, 2013; 43(10): 901–907! 2013 Informa UK Ltd. DOI: 10.3109/00498254.2013.783250

RESEARCH ARTICLE

Effects of 17a-ethynylestradiol-induced cholestasis on thepharmacokinetics of doxorubicin in rats: reduced biliary excretionand hepatic metabolism of doxorubicin

Young Hee Choi1, Yu Kyung Lee1, and Myung Gull Lee2

1College of Pharmacy and Research Institute of Pharmaceutical Sciences, Dongguk University-Seoul, Goyang, South Korea and2College of Pharmacy, The Catholic University of Korea, Bucheon, South Korea

Abstract

1. Since the prevalent hormonal combination therapy with estrogen analogues in cancerpatients has frequency and possibility to induce the cholestasis, the frequent combinationtherapy with 17a-ethynylestradiol (EE, an oral contraceptive) and doxorubicin (an anticancerdrug) might be monitored in aspect of efficacy and safety. Doxorubicin is mainly excreted intothe bile via P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) inhepatobiliary route and metabolized via cytochrome P450 (CYP) 3A subfamily. Also the hepaticMrp2 (not P-gp) and CYP3A subfamily levels were reduced in EE-induced cholestatic (EEC) rats.Thus, we herein report the pharmacokinetic changes of doxorubicin with respect to thechanges in its biliary excretion and hepatic metabolism in EEC rats.2. The pharmacokinetic study of doxorubicin after intravenous administration of itshydrochloride was conducted along with the investigation of bile flow rate and hepatobiliaryexcretion of doxorubicin in control and EEC rats.3. The significantly greater AUC (58.7% increase) of doxorubicin in EEC rats was due to theslower CL (32.9% decrease). The slower CL was due to the reduction of hepatic biliary excretion(67.0% decrease) and hepatic CYP3A subfamily-mediated metabolism (21.9% decrease) ofdoxorubicin. These results might have broader implications to understand the alteredpharmacokinetics and/or pharmacologic effects of doxorubicin via biliary excretion and hepaticmetabolism in experimental and clinical estrogen-induced cholestasis.

Keywords

17a-Ethynylestradiol, cholestasis, cytochromeP450, doxorubicin, hepatic metabolism,hepatobiliary excretion, multidrugresistance-associated protein 2,pharmacokinetics

History

Received 7 January 2013Revised 27 February 2013Accepted 4 March 2013Published online 10 April 2013

Introduction

The hormonal combination therapy with estrogen analogues

such as 17a-ethynylestradiol (EE), a synthetic estrogen used

for oral contraceptives, is prevalently being used for the

contraception in cancer patients and improves the efficacy of

cancer chemotherapy (Schwarz et al., 2009). Anticancer

actions of estradiol or EE on tumors in mice, rats and humans

have been documented (Key, 1995; Rajkumar et al., 2004;

Schwarz et al., 2009; Sivaraman et al., 1998; Yao et al., 2000).

However, it is true that there are controversial evidences of

EE therapy. In addition, EE therapy has been known to cause

intrahepatic cholestasis (Eloranta et al., 2001; Savander et al.,

2003) and decreases bile flow rate in experimental animal

models (Crocenzi et al., 2001; Rodriguez-Garay, 2003). Thus,

by these EE-induced changes, therapeutic effect of anticancer

drug might be influenced and it seems valuable to investigate

(Early Breast Cancer Trialists’ Collaborative Group, 2005).

Doxorubicin, an anthracycline anticancer drug, impairs

DNA synthesis during tumor cell division and is commonly

used for the treatment of ovarian cancer, mammary cancer,

lymphoma and osteosarcoma (Rocha et al., 2001; Smylie

et al., 2007). The extent of absolute oral bioavailability (F)

of doxorubicin is very low (less than 10%) likely due to the

extensive hepatic metabolism and biliary excretion in

rats (Choi et al., 2011). Doxorubicin is metabolized to

its metabolites, doxorubicinol and the forms of alycones,

mainly via cytochrome P450 (CYP) 3A subfamily and

their glucuronide conjugates (Lee & Lee, 1999; Speeg &

Maldonado, 1994). Recently, it was reported that down-

regulation of hepatic P-glycoprotein (P-gp) and multidrug

resistance-associated protein 2 (Mrp2) directly leads to a

reduction in hepatobiliary excretion of doxorubicin in rats

and/or humans (Cui et al., 1999; Hidemura et al., 2003;

Pauli Magnus & Meier, 2005).

The combination use of EE and doxorubicin was known to

enhance anticancer effect (Czeczuga-Semeniuk et al., 2004).

On the other hand, the induction of intrahepatic cholestasis

and reduction of bile flow by EE therapy might affect

anticancer effect of doxorubicin in the aspect of the elimin-

ation of doxorubicin via biliary excretion and hepatic

metabolism. In EE-induced cholestasis (EEC) in rats, an

animal model representing the cholestasis state, the impair-

ment of Mrp2 and reduction of CYP3A were caused by EE

Address for correspondence: Y. H. Choi, College of Pharmacy, DonggukUniversity-Seoul, Dongguk-lo 32, Ilsandong-gu, Goyang, Gyeonggi-do410-820, South Korea. Tel/Fax: 1-82-31-961-5212. E-mail: choiyh@dongguk.edu

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(Crocenzi et al., 2001; Lin et al., 2002; Micheline et al, 2002;

Trauner et al., 1997) and these changes might affect the

elimination of doxorubicin. Therefore, we herein focus on

the pharmacokinetic changes of doxorubicin in EEC rats.

Materials and methods

Chemicals

Doxorubicin hydrochloride was purchased from Bo-Ryung

Pharmaceutical Company (Seoul, South Korea). Daunorubi-

cin hydrochloride (internal standard for high-performance

liquid chromatographic (HPLC) analysis of doxorubicin), EE,

1,2-propanediol, dextran (mol. wt. 65 000), the reduced form

of b-nicotinamide adenine dinucleotide phosphate (NADPH;

as a tetrasodium salt) and tris(hydroxymethyl)aminomethane

(tris)-buffer were purchased from Sigma-Aldrich Corp.

(St. Louis, MO). Other chemicals were of reagent or HPLC

grade.

Animals

The protocols for all animal studies were approved by

Dongguk University Medical Center in Institutional Animal

Care and Use Committee (Seoul, South Korea). Female

Sprague–Dawley rats (6–7 weeks old; weighing 200–230 g)

were purchased from Charles River Company Korea (Orient,

Seoul, South Korea) and maintained in the same conditions as

a reported method (Choi et al., 2010).

The rats were randomly divided into two groups; control

and EEC groups. In EEC rats, intrahepatic cholestasis was

induced by the daily subcutaneous injection of EE (dissolved

in 1,2-propanediol) at a dose of 10 mg (in 4 mL) kg�1 for five

consecutive days. Control rats were injected with the same

volume of 1,2-propanediol alone (Jin et al., 2009).

Measurement of parameters in cholestasis

The degree of EEC was measured by the determination of bile

acids, alkaline phosphate and testosterone levels in serum

(analyzed by Green Cross Reference Laboratory, Seoul, South

Korea). For the estimation of bile flow rate, the common bile

duct of the rats was cannulated using polyethylene tubing and

the bile flow rate was estimated gravimetrically using the

volume of bile juice and collection periods (Choi et al., 2006).

The rats were euthanized and the total liver weight was

measured (Choi et al., 2006). All steps were conducted in

control and EEC rats.

Intravenous administration of doxorubicinhydrochloride to control and EEC rats

On day 6 after the start of the treatment with EE or 1,2-

propanediol, the surgical procedures including the cannula-

tion of the carotid artery (for blood sampling) and the

jugular vein (for drug administration in the intravenous study)

were conducted as similar as reported methods (Choi et al.,

2006, 2010).

After rats were recovered from the anesthesia and freely

moving, doxorubicin hydrochloride (dissolved in distilled

water) at a dose of 20 mg (in 2 mL) kg�1 as free base

was manually administered via the jugular vein over 1 min

to control (n¼ 6) and EEC (n¼ 7) rats. Blood samples

(approximately 0.22 mL, each) were collected via the carotid

artery at 0 (control), 1, 5, 15, 30, 60, 120, 180, 240, 300, 360,

400 and 480 min after the administration of doxorubicin

hydrochloride. After centrifugation of a blood sample, a

100-mL of supernatant was collected. At the end of 24 h, each

metabolic cage was rinsed with 10 mL of distilled water

and the rinsings were combined with the 24-h urine in urine

collector. All plasma and urine samples were stored at �70 �C(Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC)

until used for the analysis of doxorubicin.

Biliary clearance of doxorubicin after intravenousadministration of its hydrochloride to control andEEC rats

On day 6, the biliary excretion of doxorubicin was measured.

The procedures used for the cannulation of the carotid artery,

the jugular vein and the bile duct (for bile juice sampling)

were similar to reported methods (Choi et al., 2006, 2010).

Blood samples were collected as the same as the intravenous

study mentioned above. The bile samples were collected

between 0–2, 2–6, 6–12 and 12–24 h, respectively. The

volume of bile and the amount of doxorubicin excreted into

the bile were measured (Choi et al., 2006). The biliary

clearance (CLbile) was calculated by dividing the cumulative

amount of doxorubicin excreted into the bile up to 24 h by

the area under the plasma concentration–time curve from time

0 to the last measured time, 24 h (AUC0–24 h).

Net biliary clearance of doxorubicin after intravenousinfusion of its hydrochloride to control and EEC rats

The procedures used for the cannulation of the carotid artery,

the jugular vein and the bile duct were similar to reported

methods (Choi et al., 2006, 2010).

On day 6, a loading dose of doxorubicin hydrochloride

(3.76 mg kg�1 as free base) was administered as intravenous

bolus followed by a constant-rate infusion of 834 and

560 mg h�1 kg�1 as free base in distilled water for control

and EEC rats, respectively, using Harvard infusion pump

(PHD2000, South Natick, MA). After 60 min infusion, the

steady state plasma concentration (Css) of doxorubicin

(at approximately 0.1mg mL�1 in both groups of rats) was

attained and bile was collected at 20-min interval throughout

the experiment. Blood samples were taken at the bile

collection time periods (60, 80, 100 and 120 min after the

infusion was started; Choi et al., 2006). After 120 min

infusion, plasma samples were collected and the rats were

sacrificed. The liver was homogenized with four volumes of

the distilled water and the concentration of doxorubicin in

the liver was measured (Choi et al., 2006). The volume

of bile samples was measured gravimetrically with specific

gravity.

The apparent biliary clearance (CLbile/plasma) based on the

plasma concentration was calculated by dividing the biliary

excretion rate by Css in each collection time period. The net

biliary clearance (CLbile/liver) based on the liver concentration

was calculated by dividing the biliary excretion rate by the

liver concentration (doxorubicin concentration in the liver

at 120 min after starting infusion; Suzuki et al., 2006).

The tissue-to-plasma ratio (T/P) is represented as the ratio of

902 Y. H. Choi et al. Xenobiotica, 2013; 43(10): 901–907

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the liver concentration/plasma concentration of doxorubicin

(Choi et al., 2006).

Measurement of Vmax, Km and CLint for thedisappearance of doxorubicin in hepatic microsomalfractions from control and EEC rats

The hepatic and intestinal microsomes in control and

EEC rats were prepared based on a reported method (Choi

et al., 2010). The Vmax (the maximum velocity) and Km

(the apparent Michaelis–Menten constant; the concentration

at which the rate is one-half of the Vmax) for the disappearance

of doxorubicin were determined after incubating the above

microsomal fractions (equivalent to 1.0 mg protein); 5 mL of

methanol containing final doxorubicin concentrations of 0.5,

1, 5, 10, 50 and 100 mM; 50 mL of 0.1 M Sørensen phosphate

buffer (pH 7.4) containing 1 mM NADPH; and 0.1 M

phosphate buffer (pH 7.4) which adjusted the final volume

as 0.5 mL. The samples were incubated in a thermomixer

[37 �C, 50 oscillations per min (opm); Eppendorf, Hamburg,

Germany] for 15 min, and then the reaction was terminated by

the addition of 1 mL of acetonitrile containing 5 mg mL�1 of

daunorubicin.

Based on a non-linear regression method (Duggleby,

1995), the kinetic constants (Km and Vmax) for the disappear-

ance of doxorubicin were calculated and the intrinsic

clearance (CLint) for the disappearance of doxorubicin was

calculated by dividing the Vmax by the Km.

Rat plasma protein binding of doxorubicin usingequilibrium dialysis

Using equilibrium dialysis, the protein-binding values of

doxorubicin at 0.1 mg mL�1 were measured in fresh plasma

from control and EEC rats (n¼ 5; each) (Choi et al., 2006).

A Spectra/Por 4 membrane (mol. wt. cutoff 12–14 KDa;

Spectrum Medical Industries, Houston, TX) divided a 1 mL

dialysis cell into two compartments and 1 mL of plasma and

isotonic Sørensen phosphate buffer (pH 7.4) containing 3%

(w/v) dextran were spiked to each compartment, respectively.

A 10 mL of doxorubicin solution was spiked into the plasma

compartment. After 24 h, a 100 mL of sample was collected

from each compartment.

HPLC analysis of doxorubicin

Concentrations of doxorubicin in the samples were determined

by a slight modification of a reported HPLC analysis (Lee &

Lee, 1999). A 200 mL of acetonitrile containing 50 ng mL�1 of

daunorubicin hydrochloride was added to 100mL of a

biological sample. After vortex-mixing and centrifugation,

30 mL of the supernatant was directly injected onto a reversed-

phase column (XBridgeTM RP18; 150 mm. ‘.� 4.6 mm. i.d.;

particle size, 5 mm; Waters, Milford, MA). The mobile phase,

0.02 M phosphate buffer:acetonitrile (70:30, v/v), was run at

a flow-rate of 1.0 mL min�1. The excitation and emission

wavelengths of fluorescence detector were 460 and 580 nm,

respectively. The retention times of doxorubicin and dauno-

rubicin were 4.2 and 7.5 min, respectively, and the detection

limit of doxorubicin in rat plasma was 0.01 mg mL�1 based

on a signal to noise ratio of >3.0. The coefficient variation

of doxorubicin (from 0.01 to 100 mg mL�1) was below 9.35%.

Pharmacokinetic analysis

The total area under the plasma concentration–time curve

from time zero to infinity (AUC) was calculated using the

trapezoidal rule–extrapolation method (Chiou, 1978).

The following pharmacokinetic parameters using a non-

compartmental analysis (WinNonlin�; professional edition

version 2.1; Pharsight, Mountain View, CA) were calculated

based on the standard methods (Gibaldi & Perrier, 1982);

the terminal half-life, time-averaged total body, renal and

non-renal clearances (CL, CLR and CLNR, respectively),

mean residence time (MRT) and apparent volume of distri-

bution at a steady state (Vss).

Statistical analysis

To compare the means for the unpaired data, an unpaired

t-test was used as a typical manner. A p-value 50.05 was

considered to be statistically significant and all data are

expressed as mean� standard deviation (SD).

Results

Representative parameters of cholestasis

The parameters in control and ECC rats are listed in Table 1.

EEC rats had significantly smaller body weight gain (2 versus

38 g) on day 6. Relative liver weight was significantly heavier

(31.5% increase), but bile flow rate was significantly slower

(57.9% decrease) by cholestasis (EEC rats). In addition,

serum levels of alkaline phosphate and bile acids were

significantly higher (86.8% and 160% increase, respectively),

but the testosterone level was significantly lower (94.5%

decrease) in EEC rats. These observations suggest that EE

administration led to experimental cholestasis in rats.

Intravenous administration of doxorubicinhydrochloride to control and EEC rats

The mean arterial plasma concentration–time profiles

and pharmacokinetic parameters of doxorubicin following

intravenous administration of its hydrochloride at a dose

of 20 mg kg�1 as free base to control and EEC rats are

shown and listed in Figure 1 and Table 2, respectively.

After intravenous administration to EEC rats, the AUC was

significantly greater (58.7% increase), CL and CLNR were

significantly slower (32.9% and 33.6% decrease, respect-

ively), CLR was significantly faster (133% increase) and Ae0–

24h was significantly greater (280% increase) than that in

control rats. Note that the Ae0–24 h and CLR of doxorubicin

were almost negligible. The Vss values were considerably

large for both control and EEC rats, suggesting that the

affinity of doxorubicin to rat tissues were considerable.

Biliary clearance of doxorubicin after intravenousadministration of its hydrochloride to control andEEC rats

The ratios of doxorubicin dose excreted in 24-h bile were

29.8� 5.35% and 13.9� 6.98% for control and EEC rats,

respectively (Figure 2); they were significantly different. The

CLbile values are listed in Table 3. In ECC rats, the CLbile was

significantly slower (67.0% decrease) than that in control rats.

DOI: 10.3109/00498254.2013.783250 Pharmacokinetics of doxorubicin in cholestatic rats 903

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Net biliary excretion of doxorubicin afterintravenous infusion of its hydrochloride tocontrol and EEC rats

To investigate the changes in net hepatobiliary excretion

of doxorubicin at steady state, the net biliary clearances

of doxorubicin in control and EEC rats were measured and

are listed in Table 4. No significant differences were

observed between the control and EEC rats in the plasma

concentrations of doxorubicin at steady state (Css; 0.106

and 0.106 mg mL�1, respectively). In EEC rats, the bile flow

rate, biliary excretion rate, CLbile/plasma (representing the

apparent biliary clearance based on the plasma concentra-

tions) and CLbile/liver (representing the actual hepatobiliary

transport function of doxorubicin) were significantly slower

(70.2%, 64.4%, 63.4% and 76.4% decrease, respectively)

than those in control rats. In EEC rats, the concentration of

doxorubicin in the liver and the T/P ratio were significantly

higher and greater (204% and 210% increase, respectively)

than those in control rats.

Table 1. Characteristics of control and EEC rats.

Parameter Control rats (n¼ 4) EEC rats (n¼ 4)

Initial body weight (g) 220� 31.3 222� 46.3Final body weight (g) 258� 6.61 224� 10.8a

Relative liver weight (g final body weight�1) 0.0441� 0.00265 0.0580� 0.00568a

Bile flow rate (mL min�1 g liver�1) 2.90� 0.204 1.22� 0.105b

Serum level � �Alkaline phosphate (U L�1) 190� 39.2 355� 70.5b

Bile acids (mmol L�1) 13.4� 4.56 34.8� 7.32b

Testosterone (ng L�1) 1.78� 0.759 0.0975� 0.0624a

Data are given as the mean� standard deviation (SD). EEC, 17a-ethynylestradiol-induced cholestasis; U, unit.aEEC rats were significantly different (p50.01) from control rats.bEEC rats were significantly different (p50.001) from control rats.

Time (min)

0 6 12 18 24

Cum

ulat

ive

bilia

ry e

xcre

tion

of in

trav

enou

s do

se (

%)

0

5

10

15

20

25

30

35

*

**

*

Figure 2. Mean Cumulative biliary excretion of doxorubicin after intra-venous administration of its hydrochloride at a dose of 20 mg kg–1 tocontrol (f; n¼ 4) and EEC (*; n¼ 4) rats. Bars represent standarddeviations. *EEC rats were significantly different (p50.01) fromcontrol rats.

Time (min)

0 120 240 360 480

Pla

sma

conc

entr

atio

n of

dox

urub

icin

(µg

/ml)

0.01

0.02

0.05

0.1

0.2

0.5

1

2

5

10

20

50

100

Figure 1. Mean arterial plasma concentration–time profiles of doxo-rubicin after intravenous administration of its hydrochloride at a dose of20 mg kg–1 to control (f; n¼ 6) and EEC (*; n¼ 7) rats. Bars representstandard deviations.

Table 2. Pharmacokinetic parameters of doxorubicin after intravenousadministration of its hydrochloride at a dose of 20 mg kg�1 to control andEEC rats.

Parameter Control rats (n¼ 6) EEC rats (n¼ 7)

Initial body weight (g) 230� 35.6 225� 56.3Final body weight (g) 262� 7.53 220� 11.6a

AUC (mg min mL�1) 138� 18.5 219� 26.3b

Terminal half-life (min) 384� 229 657� 358MRT (min) 256� 162 368� 246CL (mL min�1 kg�1) 139� 18.5 93.3� 10.9b

CLR (mL min�1 kg�1) 0.675� 0.333 1.57� 0.451 c

CLNR (mL min�1 kg�1) 138� 17.7 91.7� 12.0b

Vss (mL kg�1) 37 600� 8700 34 200� 18 800Ae0–24 h (% of dose) 0.482� 0.239 1.83� 0.473 c

Data are given as the mean� standard deviation (SD). EEC, 17a-ethynylestradiol-induced cholestasis; AUC, total area under the plasmaconcentration–time curve from time zero to infinity; MRT, meanresidence time; CL, time-averaged total body clearance; CLR, time-averaged renal clearance; CLNR, time-averaged non-renal clearance;Vss, apparent volume of distribution at steady state; Ae0–24h, percentageof the dose excreted in the urine up to 24 h.

aEEC rats were significantly different (p50.01) from control rats.bEEC rats were significantly different (p50.05) from control rats.cEEC rats were significantly different (p50.001) from control rats.

904 Y. H. Choi et al. Xenobiotica, 2013; 43(10): 901–907

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Vmax, Km and CLint for the disappearance ofdoxorubicin in hepatic microsomes from controland EEC rats

Based on the relationships between concentrations of doxo-

rubicin and velocity for the disappearance of doxorubicin in

hepatic microsomes, the Vmax, Km and CLint for the disappear-

ance of doxorubicin from control and EEC rats are listed

in Table 5. The Vmax in EEC rats was significantly slower

(28.4% decrease) than that in control rats, suggesting that

the maximum velocity for the disappearance of doxorubicin

was significantly slower in EEC rats. However, the Km values

were comparable between the two groups of rats, suggesting

that the affinity of the enzyme(s) for doxorubicin was not

affected by EE treatment. As a result, the CLint in EEC rats

was significantly slower (21.9% decrease) than that in control

rats. These data suggest that the hepatic metabolism of

doxorubicin in EEC rats was significantly decreased by EE

treatment.

Measurement of rat plasma protein binding ofdoxorubicin using equilibrium dialysis

Protein-binding values of doxorubicin in fresh rat plasma

from control and EEC rats were 71.3� 21.5% and 68.9�31.7%, respectively; they were not significantly different.

The adsorption of doxorubicin to the equilibrium dialysis

apparatus, which included the semi-permeable membrane,

was almost negligible, and 91.3–99.3% of the spiked amounts

of doxorubicin were recovered from the both plasma and the

buffer compartments.

Discussion

Since EE showed an antitumor activity (Rajkumar et al.,

2004; Sivaraman et al., 1998), EE treatment has possibility

in cancer patients as one of the frequent medications with

co-administration of anti-cancer drugs. However, as a

clinical counterpart of oral contraceptive-induced cholestasis

(Rodriguez-Garay, 2003), an induction of cholestasis might

be a point to be monitored. Experimental intrahepatic

cholestasis induced by EE treatment is a widely used animal

model. Doxorubicin is mainly eliminated by biliary excretion

and metabolism in the liver (Choi et al., 2011). Thus, the

correlation of metabolic enzymes and transporters between

rats and humans are the important points. The sequential

homologies of CYPs (including CYP3A subfamily) between

rat and human are more than 70%; there are generally

conserved regions (for P-450 reductase, heme and signal

peptide) which cause this similarity (Soucek & Gut, 1992;

Zawaira et al., 2008). P-gp and Mrp2 in rats have orthologues

in humans, facilitating experimental approaches (Scheffer

et al., 2000; Stephens et al., 2001).

Considering that doxorubicin is a substrate of both P-gp

and Mrp2 (Hidemuraet al., 2003; Speeg & Maldonado, 1994),

it is reasonable to speculate the effects of P-gp and Mrp2 on

hepatobiliary excretion of doxorubicin in EEC rats. However,

the protein expression of P-gp was already reported to be

comparable between the control and EEC rats (Muhlfeld

et al., 2003). Thus, the effect of Mrp2 for the hepatobiliary

excretion of doxorubicin (Muhlfeld et al., 2003) was

considered in this study. In addition, even though Mrp3 was

reported to increase in ECC rats (Ruiz et al., 2007), Mrp3 was

not involved for the elimination of doxorubicin (Zelcer et al.,

2001). Down-regulation of Mrp2 in the liver is known to

cause cholestatic liver injury via inhibition of biliary excre-

tion of bile acids (Borst & Elferink, 2002; Crocenzi et al.,

2001). Based on the decreased mRNA levels of Mrp2

(Jin et al., 2009), the elevated hepatic level of bile acids as

well as diminished bile flow rate appear to be responsible for

the reduction of Mrp2 in EEC rats (Table 1). Also CYP

3A4 was inactivated by EE as mechanism-based manner

(Lin et al., 2002). Thus, the present in vivo and in vitro studies

using rats might provide a meaningful forecast of the effect of

Table 4. Parameters on net hepatobiliary clearance of doxorubicin incontrol and EEC rats at steady state.

ParameterControl rats

(n¼ 4)EEC rats(n¼ 4)

Initial body weight (g) 235� 35.1 230� 54.1Final body weight (g) 269� 6.29 224� 4.79a

Css (mg mL�1) 0.106� 0.00499 0.103� 0.00603Bile flow rate (mL min�1 g liver�1) 3.62� 0.210 1.08� 0.0731a

Biliary excretion rate(mg min�1 kg�1)

3.59� 0.469 1.28� 0.145a

CLbile/plasma (mL min�1 kg�1) 33.9� 4.26 12.4� 1.14a

CLbile/liver (g min�1 kg�1) 3.37� 1.28 0.795� 0.312b

Concentration in liver (mg g�1) 0.596� 0.153 1.81� 0.487b

T/P 5.61� 1.38 17.4� 4.28b

Data are given as the mean� standard deviation (SD). EEC, 17a-ethynylestradiol-induced cholestasis; CSS, plasma concentration atsteady state; CLbile/plasma, apparent biliary clearance; CLbile/liver, netbiliary clearance; T/P, ratio of liver concentration/plasmaconcentration.

aEEC rats were significantly different (p50.001) from control rats.bEEC rats were significantly different (p50.01) from control rats.

Table 3. Parameters on biliary clearance of doxorubicin in control andEEC rats.

Parameter Control rats (n¼ 4) EEC rats (n¼ 4)

Initial body weight (g) 223� 25.9 220� 16.5Final body weight (g) 252� 9.30 228� 21.4a

Relative liver weight(g final body weight�1)

0.0405� 0.627 12.4� 0.479a

CLbile (mL min�1 kg�1) 37.9� 2.73 12.5� 0.509b

Data are given as the mean� standard deviation (SD). EEC, 17a-ethynylestradiol-induced cholestasis; CLbile, time-averaged biliaryclearance.

aEEC rats were significantly different (p50.05) from control rats.bEEC rats were significantly different (p50.01) from control rats.

Table 5. Km, Vmax and CLint for the disappearance of doxorubicin inhepatic microsomes from control and EEC rats.

ParameterControl rats

(n¼ 4)EEC rats(n¼ 4)

Km (mM) 20.6� 1.20 19.0� 4.41Vmax (nmol min�1 mg protein�1) 0.461� 0.0206 0.330� 0.0711a

CLint (mL min�1 mg protein�1) 0.0224� 0.00204 0.0175� 0.00269a

Data are given as the mean� standard deviation (SD). EEC, 17a-ethynylestradiol-induced cholestasis; Km, apparent Michaelis–Mentenconstant (the concentration at which the rate is one-half of the Vmax);Vmax, maximum velocity; CLint, intrinsic clearance.

aEEC rats were significantly different (p50.05) from control rats.

DOI: 10.3109/00498254.2013.783250 Pharmacokinetics of doxorubicin in cholestatic rats 905

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EE-induced cholestasis on the pharmacokinetics of doxo-

rubicin with respect to the changes in Mrp2 and CYP3A

subfamily.

We first measured the in vivo pharmacokinetics of

doxorubicin after intravenous administration of its hydro-

chloride to control and EEC rats. The Ae0–24 hs of doxorubicin

were less than 1.83% of the dose in both groups of rats

(Table 2), suggesting that intravenous doxorubicin was almost

completely eliminated via the liver in rats. After intravenous

administration of doxorubicin hydrochloride, the significantly

greater AUC of doxorubicin in EEC rats was due to the

slower CL and CLNR than those in control rats (Table 2).

The significantly slower CLNR could be due to the decreased

biliary excretion (Figure 2 and Table 3) and hepatic metab-

olism of doxorubicin (Table 5) in EEC rats compared with

control rats.

To confirm the difference in the biliary excretion of

doxorubicin between control and EEC rats (Figure 2), we

measured the hepatobiliary clearance with continuous intra-

venous infusion. Hepatobiliary excretion consists of the

uptake from the sinusoidal side into the hepatocytes and

the efflux from the inside of the hepatocytes out into the

bile across the canalicular membrane. Since P-gp already

has been reported to be comparable between control and

cholestasis state and Mrp3 was not involved for the elimin-

ation of doxorubicin as mentioned above, Mrp2 activity in the

second step of hepatic biliary excretion was investigated in

this study. Both significantly slower CLbile/plasma and CLbile/

liver in EEC rats (Table 4) suggested that there is a difference

in doxorubicin transport ability via Mrp2 in hepatobiliary

excretion compared to control rats. As Mrp2 is located on the

bile canalicular membrane of hepatocytes (Hidemura et al.,

2003), Mrp2 might be included for the hepatobiliary excretion

of doxorubicin and the decreased mRNA levels of Mrp2

affected the reduced CLbile/liver of doxorubicin in EEC rats as

reported because the protein expression level of the trans-

porter determinates its function to a great degree (Jin et al.,

2009). Similarly, impairment in bile flow rate under

cholestasis was reported to be accompanied by down-

regulation of Mrp2 in the liver for the secretion of bile salts

and/or other bile constituents (Pauli-Magnus & Meier, 2005),

which plays an important role in elimination of drugs most

likely by biliary excretion (Borst & Elferink, 2002; Chan

et al., 2004; Vlaming et al., 2006). Furthermore, the organic

cation transporters as well as other unidentified phenomena

related to the uptake of doxorubicin seemed to be responsible

for the uptake of doxorubicin into the hepatocytes and affect

the hepatobiliary excretion (Okabe et al., 2005).

In aspect of the metabolic changes in doxorubicin in EEC

rats, the significantly slower CLNR of doxorubicin (Table 2)

could be due to the decreased CYP3A subfamily in EEC rats

(Lin et al., 2002). In in vitro hepatic microsomal studies,

the significant slower CLint was due to the decreased Vmax in

EEC rats (Table 5), indicating the maximum velocity for

the disappearance (primarily metabolism) of doxorubicin was

decreased in EEC rats because of the decreased CYP3A.

Since doxorubicin is a drug with a low hepatic extraction

ratio (50.24; Ballet et al., 1987), its hepatic clearance

(metabolism) depends on the hepatic intrinsic free drug

clearance (CL0int) for the disappearance of doxorubicin and

the free fraction of doxorubicin in the plasma, fu (Wilkinson

& Shand, 1975). The CL0int � fu is the intrinsic total drug

clearance. The significantly slower CLNR of doxorubicin in

EEC rats (Table 2) could have been supported by the slower

in vitro hepatic CLint for the disappearance of doxorubicin

(Table 4). The contribution of fu to CLint seemed to be

negligible because fus plasma were comparable between two

groups of rats. Thus, the decreased protein expression of

hepatic CYP3Asubfamily in EEC rats seemed to contribute

the changes in pharmacokinetics (including hepatic metabol-

ism) of doxorubicin in rats. Mottino et al. (2002) and Lin et al.

(2002) reported that EE reduced the Mrp2 and CYP3A

activities, respectively. These changes directly affected the

pharmacokinetic changes of doxorubicin: the decreased

biliary excretion of doxorubicin and bile flow rate by the

reduced Mrp2 activity and decreased CLint and increased

AUC (decreased CLNR) by the reduction of hepatic metab-

olism of doxorubicin via hepatic CYP3A.

The estimated CLR as fu of doxorubicin (CLR,fus) were

2.35 and 5.05 mL min�1 kg�1 in control and EEC rats,

respectively. These values, 2.35 and 5.05 mL min�1 kg�1,

were considerably slower than and close to the reported

glomerular filtration rate (as measured by creatinine clear-

ance), 5.24 mL min�1 kg�1, in rats (Davies & Morris, 1993).

The above data indicate that doxorubicin is reabsorbed in

rat renal tubules in control rats and excreted mainly via

glomerular filtration in ECC rats. In ECC rats, reabsorption

of doxorubicin was inhibited. The faster CLR,fu (by 115%) of

doxorubicin in EEC rats could be due to the up-regulated

Mrp2 in the kidney as a compensatory mechanism in

cholestatic state (Denk et al., 2004). Although the Ae0–24 h

values were almost negligible, Ae0-24 h and CLR in EEC rats

were significantly greater and faster than those in control rats,

respectively (Table 2), and these results support the compen-

satory mechanism of Mrp2 in the kidney.

In conclusion, the reduction of hepatic Mrp2 and CYP3A

subfamily causes the greater AUC (slower CLNR) of doxo-

rubicin in EEC rats due to the decreased biliary excretion

and hepatic metabolism of doxorubicin. Since a variety of

substrates are subjected to being transported via Mrp2 and/or

CYP3A subfamily, the regulation may have broader implica-

tions for the understanding of the altered pharmacokinetics

and/or pharmacologic effects of relevant substrate drugs

in experimental, as well as in clinical estrogen-induced

cholestasis.

Declaration of interest

This study was supported by the Dongguk University

Research Foundation Fund of 2010.

The authors declare that they have no conflicts of interest

to declare.

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