effects of 17α-ethynylestradiol-induced cholestasis on the pharmacokinetics of doxorubicin in rats:...

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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


    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


    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.


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


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


    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


    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



    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,