*2.9. Quantification Using LC-MS*/*MS*

Chromatographic quantification of sulfasalazine and prazosin was carried out using an LC-tandem mass spectrometry (LC-MS/MS) system equipped with a Waters e2695 high-performance liquid chromatography system (Milford, MA, USA) and an API 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA). Briefly, an aliquot (50 µL) of a sample was vortex-mixed with an acetonitrile solution containing glipizide (300 ng/mL, internal standard); this was followed by centrifugation (16,100 × g for 5 min at 4 ◦C). An aliquot (5 µL) of the supernatant was directly injected into the LC-MS/MS system. Separations were carried out using a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.7 mL/min using a reversed-phase high-performance LC column (Agilent Poroshell 120, EC-C18 2.7 µm, 4.6 × 50 mm). The following transitions were used for analyte detection: *m*/*z* 399.0 → *m*/*z* 380.8 for sulfasalazine and *m*/*z* 384.1 → *m*/*z* 95.0 for prazosin. For the internal standard glipizide, the transition *m*/*z* 445.8 → *m*/*z* 320.9 was used. The limits of quantification were 10 ng/mL for sulfasalazine and 50 nM for prazosin.

## *2.10. Data Analysis*

#### 2.10.1. In Vitro Kinetic Analysis

The apparent permeability coefficient (Papp) of prazosin was estimated using the following equation (Equation (1)):

$$P\_{app} = \frac{1}{A} \times \frac{1}{\mathcal{C}\_0} \times \frac{dQ}{dt} \tag{1}$$

where *dQ*/*dt*, A, and C<sup>0</sup> represent the transport rate, the surface area of the insert, and the initial concentration of the compound in the donor compartment, respectively. The efflux ratio (ER) was calculated by dividing the B-to-A apparent permeability coefficient (Papp, B-to-A) by the A-to-B apparent permeability coefficient (Papp, A-to-B). In the inhibition studies, the percentage of the control efflux ratio (%ER) was also calculated by dividing the value for ER in the presence of the inhibitor by that in the absence of the inhibitor (i.e., in the control). When necessary, the half maximal inhibitory concentration (IC50) was determined by nonlinear regression analysis using WinNonlin Professional 5.0.1 software (Pharsight Corporation, Mountain View, CA, USA) and the following equation (Equation (2)):

$$\mathbf{V} = \left. V\_{\text{max}} - (V\_{\text{max}} - V\_0) \times \left[ \frac{\left[I\right]^n}{\left[I\right]^n + \left(I \mathbf{C}\_{50}\right)^n} \right] \tag{2}$$

where V, Vmax, V0, [I], and n represent the rate of transport in the presence of the inhibitor, the maximal rate of transport, the basal rate of transport, the concentration of the inhibitor, and the Hill coefficient, respectively. When it was necessary to convert the IC<sup>50</sup> to the inhibitory constant (Ki), the following equation (Equation (3)) [32] was used under the assumption that competitive inhibition existed between the substrate and the inhibitor:

$$K\_i = \frac{IC\_{50}}{1 + \frac{[S]}{K\_m}} \tag{3}$$

where [S] is the concentration of the substrate and K<sup>m</sup> represents the Michaelis–Menten constant.

#### 2.10.2. Non-Compartmental Pharmacokinetic Analysis

Standard non-compartmental pharmacokinetic analysis was carried out using WinNonlin Professional 5.0.1 software (Pharsight, Cary, NC, USA) to calculate the pharmacokinetic parameters, including the peak concentration (Cmax), time of the peak concentration (tmax), elimination half-life (t1/2), area under the plasma concentration–time curve from time zero to the last sampling point, 8 h (AUC8h), and elimination clearance (CL/F).

### *2.11. Statistical Analysis*

For the comparison of means among the groups, one-way ANOVA (analysis of variance; for cytotoxicity and bi-directional transport studies) followed by Tukey's post hoc test were used. In these in vitro studies, a value of *p* < 0.05 was considered statistically significant. For the comparison of means between the groups for in vivo studies, the two-tailed/unpaired Student's t-test was used and a value of *p* < 0.05 with a statistical power more than 0.8 (Minitab 19.2, Minitab Inc., State College, PA, USA) was considered statistically significant.

#### **3. Results**

#### *3.1. FACS-Cellular Accumulation Study*

The expression of BCRP in Hela cells was confirmed by RT-PCR and compared with other cells, which were known to express high (Caco-2 and MCF-7) or low (SW620) levels of BCRP (Supplementary Figure S1) [33,34]. In the FACS-cellular accumulation study, the potential of quercetin to inhibit BCRP was first investigated by observing the cellular uptake of mitoxantrone (MX). The cellular uptake of MX with or without quercetin was analyzed by flow cytometry. The fluorescence intensity of a single cell measured by flow cytometry can be a good indication of the amount of MX internalized by each cell. As shown in Figure 2A, the peak fluorescence intensity of MX uptake was shifted to a higher level when MX was co-administered with quercetin, suggesting the promotion of MX internalization in HeLa cells. In the MX single treatment group, the percentage of cells with a significant uptake of MX was higher by 17.2% at 4 h of treatment and 27.1% at 6 h of treatment than at 2 h of treatment with MX alone as a control. In contrast, the cellular uptake of MX in the presence of quercetin was considerably higher by 30.2% at 4 h of treatment and 35.9% at 6 h of treatment (co-treatment with 1 µM quercetin) and by 45.3% at 4 h of treatment and 67.4% at 6 h of treatment (co-treatment with 100 µM quercetin) than at 2 h of treatment with MX alone. We also tested the internalization of MX when co-administered with 1 µM Ko143, a BCRP inhibitor. The results showed a considerably high number of cells that internalized MX when 1 µM Ko143 was co-administered with MX (Figure 2B). Thus, quercetin significantly promoted the cellular uptake of MX in HeLa cells likely via the inhibition of BCRP-mediated efflux. μ μ μ μ

μ μ **Figure 2.** Representative histogram of mitoxantrone (MX) uptake in HeLa cells. (**A**) Flow cytometry measurement of MX fluorescence in HeLa cells incubated with MX alone (green line) or MX with 1 (red line) or 100 µM quercetin (blue line) for 2, 4, and 6 h. (**B**) Flow cytometry measurement of MX fluorescence in HeLa cells incubated with MX alone (green) or MX with 1 µM Ko143, a specific breast cancer resistance protein (BCRP) inhibitor (purple line), for 2, 4, and 6 h.

μ
