effects of 17α-ethynylestradiol-induced cholestasis on the pharmacokinetics of doxorubicin in rats:...
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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: firstname.lastname@example.org
(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.,
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,