Short Communication Inhibitory Effects of Terpenoids on Multidrug Resistance-Associated Protein 2- and Breast Cancer Resistance Protein-Mediated Transport

The possibility of interactions between natural products/supplements and conventional prescription medicines is one of the most important issues in pharmacotherapeutic safety. Recently, we reported that some terpenoids such as (R)-( )-citronellal and glycyrrhetic acid, which are present in herbal medicines, can act as inhibitors of P-glycoprotein (MDR1/ABCB1). In the present study, the effects of seven terpenoids on multidrug resistance-associated protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ ABCG2)-mediated transport were investigated in vitro. Membrane vesicles were prepared from MRP2 cDNA transfected Sf9 cells derived from pupal ovarian tissue of Spodoptera frugiperda, a fall armyworm, and BCRP cDNA transfected LLC-PK1 cells derived from porcine kidney. MRP2or BCRP-mediated efflux transport was measured as ATP-dependent accumulation of [H]estradiol 17-D-glucuronide (E217 G) into membrane vesicles collected by a rapid filtration technique. The effects of (R)-( )-citronellal, (S)( )-citronellol, -terpinene, terpinolene, ( )-pinene, abietic acid, and glycyrrhetic acid on the intravesicular accumulation of [H]E217 G were examined. Large decreases in the [ H]E217 G accumulation into vesicles from MRP2-overexpressing Sf9 cells were observed in the presence of glycyrrhetic acid and abietic acid, and their IC50 values were about 20 and 51 M, respectively. [H]E217 G accumulation into vesicles from BCRP-overexpressing LLC-PK1 cells was suppressed by only glycyrrhetic acid, with an IC50 value of about 39 M. Other terpenoids used in this study did not alter the ATP-dependent accumulation of [H]E217 G. These findings suggest that glycyrrhetic acid and abietic acid can potently inhibit MRP2or BCRP-mediated membrane transport and may interact with their substrates in pharmacokinetic processes. The ATP-binding cassette (ABC) transporter superfamily plays important roles in drug absorption and disposition. ABC transporters were originally implicated in multidrug resistance in tumor cells (Sarkadi et al., 2006). Further research has shown that these transporters are distributed throughout many normal tissues of the body. For example, P-glycoprotein (P-gp/ABCB1), multidrug resistanceassociated protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) exist on the apical membrane of intestinal epithelia and function as a defense system against xenobiotics (Benet et al., 1999; Borst et al., 2000; Maliepaard et al., 2001; Leslie et al., 2005). P-gp, MRP2, and BCRP have been reported to have well defined roles in the transport of clinically relevant drugs and to mediate cellular resistance to these drugs (Litman et al., 2001; Leslie et al., 2005). These ABC transporters can transport diverse substrates to the outside of cellular membranes using the hydrolytic energy of ATP as a driving force (Litman et al., 2001). Recently, the possibility of drug interactions has been increasing because of multiple and complex medications, and significant revisions of safety profiles in the product information have been frequently undertaken in clinical practice (Yoshida et al., 2006c). Moreover, herbal medicine and diet supplements made from natural products are widely used in patients treated with conventional prescription medicines. Recent advancements in biopharmaceutical research have revealed physiological and pharmacological aspects of transporters; however, drug interactions with other drugs, endogenous substrates, and food ingredients have not been fully clarified. Previously, we investigated possible interactions between Japanese traditional herbal medicines and conventional medicines (Kawakami et al., 2002) and reported that an extract of Zanthoxyli Fructus and some terpenoids can inhibit P-gp–mediated efflux transport in vitro and in vivo (Yoshida et al., 2005, 2006b). In the present study, the inhibitory effects of seven terpenoids (Fig. 1) that can be P-gp inhibitors (Yoshida et al., 2006a) on MRP2and BCRP-mediated transport were investigated using membrane vesicles. Materials and Methods Materials. [H]estradiol 17-D-glucuronide (E217 G, 55 Ci/mmol) was purchased from PerkinElmer, Inc. (Waltham, MA). ATP, AMP, creatine phosphate, and creatine phosphokinase were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Sf9 cells were maintained as a suspension culture at 37C with serum-free Excel 420 (Nichirei Corp., Tokyo, Japan) supplemented with an antibiotic-antimycotic mixture (Life Technologies Inc., Paisley, UK). (R)-( )-citronellal, (S)-( )-citronellol, and -terpinene were purchased from Sigma-Aldrich Corp. Terpinolene, ( )-pinene, and abietic acid were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Glycyrrhetic acid was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All the other reagents are commercially available and of analytical grade. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.019513. ABBREVIATIONS: ABC, ATP-binding cassette; P-gp, P-glycoprotein; MRP2, multidrug resistance-associated protein 2; BCRP, breast cancer resistance protein; E217 G, estradiol 17-D-glucuronide. 0090-9556/08/3607-1206–1211$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 7 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 19513/3356567 DMD 36:1206–1211, 2008 Printed in U.S.A. 1206 at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from