**1. Introduction**

Flavonoids are a large group of polyphenolic antioxidants present in various human foods, such as vegetables, fruits, and tea. Quercetin is a major plant flavonol, a subclass of flavonoids with a 3-hydroxyflavone structure; it is present in high levels in onions, kale, broccoli, and tea [1,2]. Quercetin is mostly present in foods in the form of glycosides, which are efficiently hydrolyzed in the small intestine to release quercetin aglycone when ingested [3]. Dietary consumption of quercetin is estimated to be between 25 and 50 mg per day, accounting for approximately 70% of the total dietary flavonol and flavonone intake [4–6]. Moreover, it is well recognized that quercetin has diverse biological effects, including antioxidative, antiviral, antiulcer, and anticancer activities [7–10]. These activities have led to its consumption in various dosages and forms (e.g., 200–1000 mg aglycone per capsule/tablet [11]) as dietary supplements.

However, a recent analysis reported that the increased demand and consumption of dietary supplements is likely associated with a risk of adverse events. Indeed, a high number of adverse events (i.e., 23,000 emergency department visits per year in the United States) are attributed to dietary supplements [12]. In particular, flavonoids can modulate the activity of major ATP-binding cassette (ABC) efflux transporters [13]. For example, several studies have consistently shown that quercetin interacts with both P-glycoprotein (P-gp) [14–16] and multidrug resistance-associated protein 1 (MRP1) [17], inhibiting the efflux of substrates in the specific transporter-overexpressing cells in vitro or increasing the bioavailability/brain accumulation of substrate drugs in vivo by affecting the transporters' activity. Moreover, our research group recently reported that repeated pretreatment with quercetin could upregulate the human multidrug resistance protein 1 (MDR1) gene via a vitamin D receptor-dependent pathway in Caco-2 cells [18]. Therefore, the increasing use of dietary supplements containing quercetin emphasizes the need to investigate the potential clinical interactions that can be induced by the flavonoid.

Among ABC transporters, breast cancer resistance protein (BCRP; encoded by the ABCG2 gene) is a major efflux transporter abundantly expressed at the apical membrane of intestinal/kidney epithelial cells and hepatocytes. The transporter functions as a physiological barrier against oral absorption as well as a determinant of the disposition of substrate drugs [19]. Recently, several studies have attempted to determine whether quercetin interacts with BCRP. Sesink et al. reported that the flavonoid can be transported by the mouse Bcrp1 transporter in MDCKII/mBcrp1 cells [20]; moreover, its presence was shown to inhibit the cellular accumulation of the BCRP substrates Hoechst 33342 and mitoxantrone in BCRP-overexpressing MCF-7 cells [21,22]. However, such observations in cell systems cannot be directly translated to substantial effects on efflux transporter activity in vivo. For example, a study reported that coadministration of topotecan (a BCRP substrate) with the flavonoids chrysin or 7,8-benzoflavone (potent inhibitors of the transporter in BCRP-overexpressing MCF-7 cells) resulted in no significant effects on the pharmacokinetics of the substrate in rats or P-gp-knockout mice [23]. Therefore, considering that no apparent in vitro to in vivo association regarding BCRP inhibition by flavonoids was found [23], a more pharmacokinetic-based understanding of the interaction of quercetin with BCRP is needed. To our knowledge, the in vivo pharmacokinetic inhibition of BCRP by quercetin has not been previously reported.

Therefore, the objective of this study was to conduct an integrated study including in vitro and in vivo pharmacokinetic assessments on the inhibition of BCRP by quercetin. Here, we showed that quercetin can increase the cellular accumulation and associated cytotoxicity of the BCRP substrate mitoxantrone in human cervical cancer HeLa cells. Importantly, the high inhibitory potency of quercetin in limiting transporter-mediated efflux was demonstrated using the kinetic parameters (e.g., IC<sup>50</sup> and Ki) associated with the efflux. Finally, the in vivo pharmacokinetics of the possible inhibition were studied in rats using sulfasalazine, a selective BCRP probe that was previously proven to show increased absorption by impaired BCRP function [24–26].

#### **2. Materials and Methods**

#### *2.1. Materials*

Quercetin (Figure 1), mitoxantrone (MX), Ko143, and sulfasalazine were purchased from Sigma-Aldrich (St Louis, MO, USA). Prazosin was purchased from Tokyo Chemical Industry (Tokyo, Japan). High-performance liquid chromatography-grade methanol and formic acid were purchased from Fisher Scientific (Pittsburgh, PA, USA) and Fluka (Cambridge, MA, USA), respectively.

**Figure 1.** Chemical structure of quercetin.
