Effects of 17α-ethynylestradiol-induced cholestasis on the pharmacokinetics of doxorubicin in rats: reduced biliary excretion and hepatic metabolism of doxorubicin

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  • 2013

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

    Xenobiotica, 2013; 43(10): 901907! 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 usedfor 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

    SpragueDawley rats (67 weeks old; weighing 200230 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) kg1 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) kg1 as free base

    was manually administered via the jugular vein over 1 min

    to control (n 6) and EEC (n 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, eachmetabolic 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 02, 26, 612 and 1224 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 concentrationtime curve from time

    0 to the last measured time, 24 h (AUC024 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 kg1 as free base) was administered as intravenous

    bolus followed by a constant-rate infusion of 834 and

    560 mg h1 kg1 as free base in distilled water for controland 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 mL1 in both groups of rats) wasattained 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): 901907

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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 MichaelisMenten 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 ofmethanol containing final doxorubicin concentrations of 0.5,

    1, 5, 10, 50 and 100 mM; 50 mL of 0.1 M Srensen phosphatebuffer (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 mL1 ofdaunorubicin.

    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 mL1 were measured in fresh plasmafrom control and EEC rats (n 5; each) (Choi et al., 2006).A Spectra/Por 4 membrane (mol. wt. cutoff 1214 KDa;

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

    dialysis cell into two compartments and 1 mL of plasma and

    isotonic Srensen 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 plasmacompartment. After 24 h, a 100 mL of sample was collectedfrom 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 mL1 ofdaunorubicin hydrochloride was added to 100mL of abiological 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 min1. 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 mL1 basedon a signal to noise ratio of >3.0. The coefficient variationof doxorubicin (from 0.01 to 100 mg mL1) was below 9.35%.

    Pharmacokinetic analysis

    The total area under the plasma concentrationtime curve

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

    trapezoidal ruleextrapolation 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...

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