BIOPHARMACEUTICS & DRUG DISPOSITION Biopharm. Drug Dispos. 35: 253–263 (2014) Published online 3 March 2014 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/bdd.1890

Mechanism of an unusual, but clinically significant, digoxin–bupropion drug interaction Jiake Hea,b, Yang Yua, Bhagwat Prasada, Xijing Chenb, and Jashvant D. Unadkata,* a

Department of Pharmaceutics, University of Washington, Seattle, Washington, 98195, USA Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, Jiangsu, 210009, P.R. China

b

ABSTRACT: An unusual, but clinically significant, digoxin (DIG)–bupropion (BUP) drug interaction (DDI), in which BUP increased DIG renal clearance by 80% is reported. To investigate the mechanism(s) of this unusual DDI, first the effect of BUP, its circulating metabolites or their combination on [3H]-DIG transport by cells expressing human P-gp or human OATP4C1 was determined. Second, the study asked whether this DDI could be replicated in the rat so that it could be used to conduct mechanistic studies. Then, the effect of BUP and its rat metabolites on [3H]-DIG transport were tested by cells expressing rat Oatp4c1. Bupropion and its metabolites had no effect on human P-gp mediated transepithelial transport of [3H]-DIG. Bupropion and hydroxybupropion (HBUP) significantly stimulated H-OATP4C1 mediated transport of [3H]-DIG. In addition, BUP cocktail (BUP plus its metabolites) significantly increased the H-OATP4C1 mediated transport of [3H]-DIG, and partially reversed the inhibition by 100 μM DIG. However, erythro-hydrobupropion (EBUP) and threo-hydrobupropion (TBUP) did not affect the [3H]-DIG uptake by H-OATP4C1 cells. Bupropion administration significantly increased digoxin renal clearance in rats. Surprisingly, bupropion significantly inhibited r-Oatp4c1 mediated transport of [3H]-DIG at clinically relevant unbound plasma concentrations of BUP or those observed in the rat study, while HBUP or TBUP did not. These data support our hypothesis that at clinically relevant plasma concentrations, bupropion and its metabolites activate H-OATP4C1 mediated DIG tubular secretion, and could possibly explain the increase in digoxin renal clearance produced by bupropion. While bupropion increased digoxin renal clearance in the rat, it appeared to do so by inhibiting r-Oatp4c1-mediated digoxin renal reabsorption. Copyright © 2014 John Wiley & Sons, Ltd. Key words: digoxin; bupropion; drug–drug interaction; H-OATP4C1/r-Oatp4c1; P-gp

Introduction Digoxin (DIG), a cardiac glycoside, used in the treatment of congestive heart failure, remains the most prescribed cardiac glycoside [1–4]. It has a narrow therapeutic window [5,6]. Thus, drug–drug

*Correspondence to: Department of Pharmaceutics, University of Washington, P.O. Box 357610, Seattle, WA 98195, USA. E-mail: [email protected]

Copyright © 2014 John Wiley & Sons, Ltd.

interactions (DDI) can result in toxic or sub-therapeutic digoxin plasma concentrations. To date, digoxin is known to interact with a wide variety of drugs such as cyclosporine, macrolides, verapamil, nonsteroidal antiinflammatory drugs (NSAIDs), angiotensinconverting-enzyme (ACE) inhibitors and angiotensin II receptor antagonists [7]. The majority of these DDIs are due to inhibition of P-glycoprotein (P-gp) [7–11], resulting in increased digoxin plasma concentration either due to increased intestinal absorption of digoxin (inhibition of intestinal P-gp) or Received 1 November 2013 Revised 30 December 2013 Accepted 9 January 2014

254 decreased elimination of digoxin (inhibition of biliary or renal P-gp). The only DDI reported in which digoxin plasma concentrations are reduced is when intestinal P-gp is induced by the chronic administration of a P-gp inducer [12,13]. Therefore, we were surprised to observe an unusual, but clinically significant, digoxin–bupropion (BUP) DDI in which a single oral dose of bupropion (BUP, 150 mg, extended release tablet) was administered 24 h before a single oral dose of digoxin (0.5 mg) [12]. Bupropion increased digoxin renal clearance by 80% and decreased digoxin plasma AUC by 40%. Digoxin is eliminated from the body primarily by renal excretion (70–85%) and some by non-CYP hepatic metabolism. Digoxin is eliminated renally by both filtration (115 ml/min) and net secretion (37 ml/min) [14]. Although the mechanisms by which digoxin is secreted by the kidneys is not clear, P-gp (located on the apical membrane of the kidney tubular epithelial cells) and OATP4C1 (located on the basal membrane of the kidney tubular epithelial cells) are thought to be involved in tandem to allow vectorial transport of digoxin from the blood to the kidney lumen. Bupropion, a dopamine-norepinephrine reuptake inhibitor, is used to treat depression and to aid smoking cessation. Bupropion is eliminated from the body primarily by metabolism through CYP2B6 and carbonyl reductase enzymes to the active metabolites hydroxybupropion (HBUP), erythrohydrobupropion (EBUP) and threo-hydrobupropion (TBUP), respectively [15,16]. In humans, bupropion has a long elimination half-life (~20 h) and after [14C]-BUP oral administration, 87% and 10% of the radioactive dose were recovered in the urine and feces, respectively [15,17,18]. Less than 1% of bupropion is eliminated unchanged in the urine or feces [15]. Therefore, our observed BUP–DIG DDI, after single dose administration of BUP and digoxin was a surprise, especially because bupropion was administered 24 h prior to digoxin administration. Because of this staggered administration, the mechanism of this interaction is unlikely to involve intestinal P-gp, as it is highly unlikely that any bupropion would have remained in the intestine at 24 h when digoxin was administered. Therefore, it was hypothesized that at clinically relevant plasma concentrations, bupropion or its metabolites, increased digoxin renal clearance (CLr) by activating OATP4C1 or P-gp mediated tubular secretion of Copyright © 2014 John Wiley & Sons, Ltd.

J. HE ET AL.

digoxin. To test this hypothesis, the study first determined the effect of bupropion and its metabolites on the in vitro digoxin transport by cells expressing human P-gp or human OATP4C1 (H-OATP4C1). Second, the study investigated whether a preclinical animal model, such as the rat, could replicate this DDI and then be used to conduct mechanistic studies. It was found that this DDI could be replicated in the rat. Then, the mechanism of this DDI was explored using rat Oatp4c1 (r-Oatp4c1) overexpressing cells.

Methods Materials [3H]Digoxin (20 Ci/mmol) was purchased from American Radiolabeled Chemicals (St Louis, MO, USA). Unlabeled digoxin as a powder was purchased from TCI America (Portland, OR, USA). Digoxin formulation (Lanoxin, 0.25 mg/ml) was purchased from Baxter (Deerfield, IL, USA). The BUP, HBUP, TBUP and EBUP were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Diazepam (internal standard) and analytical reagent grade ammonium formate were purchased from Sigma-Aldrich (St Louis, MO, USA). Analytical reagent grade formic acid, optima® LC-MS grade methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other reagents were of the highest grade available from commercial sources.

Animals Female Sprague–Dawley rats (9–11 weeks, 250–300 g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA, USA) and housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle, with free access to food and water. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Washington. All experimental procedures were conducted according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington, DC, 1996). Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

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MECHANISM OF DIGOXIN–BUPROPION DRUG INTERACTION

Cell culture Madin-Darby canine kidney II (MDCKII) cells stably transfected with rat r-Oatp4c1 and human H-OATP4C1 (generously provided by Dr Leggas, University of Kentucky) were cultured in minimum essential medium with Earle’s salts supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in the presence of 5% CO2. 5 μg/ml blasticidin was used as the selection pressure. The MDR1-LLC-PK1 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in the presence of 5% CO2 [19].

[3H]-digoxin transport studies Digoxin transport studies were conducted according to a published method with minor modification [20]. The r-Oatp4c1 and H-OATP4C1 over-expressing MDCKII cells were plated at a density of 5 × 105 cells/well on 6-well plates (Corning Life Sciences, Acton, MA) and grown for 48 h. Twenty four hours prior to the transport study, the cell culture medium was replaced with that containing 5 mM sodium butyrate to induce transporter expression. Krebs-Henseleit buffer containing 12.5 mM HEPES was used as the uptake buffer. The cells were washed three times (1 ml/each) with pre-warmed uptake buffer (37 °C) and transport was initiated by replacing the uptake buffer with that containing 20 nM [3H]-DIG with/ without varying concentrations of bupropion or its metabolites or cold digoxin (100 μM) and then incubated for the designated time at 37 °C. After washing the cells three times with ice-cold DPBS the cells were lysed with 1 ml 2% SDS. The intracellular content of [3H]-DIG (800 μl of the lysate) was determined by a Tri-Carb 3110TR Liquid Scintillation Analyzer (PerkinElmer, Waltham, MA) liquid scintillation counter and normalized to the protein concentration (BCA assay).

[3H]-DIG transepithelial transport study [3H]-Digoxin transepithelial transport (in both absorptive and secretory directions) was carried out by seeding MDR1-LLC-PK1 cells on a transwell permeable support (6.5 mm Insert 24-well plates, Corning Life Sciences, Pittston, PA) at a density of Copyright © 2014 John Wiley & Sons, Ltd.

1 × 105 cells/well. Fresh medium was added every day after plating. Transepithelial resistance was measured in each well using a Millicell ohmmeter (Millicell ERS, Millipore Corp., Billerica, MA). On day 3 after plating, wells with a resistance of 400 Ω or greater were used in the transport experiments. DPBS containing 10 mM HEPES was used as the uptake buffer. The cells were washed three time (1 ml/each) with pre-warmed uptake buffer (37 °C) and transport was initiated by replacing the uptake buffer with that containing 50 nM [3H]DIG with/without varying concentrations of BUP, BUP metabolites, BUP cocktail (i.e. combination of BUP and its metabolites) or tariquidar (1 μM, a P-gp inhibitor and a positive control) and then incubated for the designated time at 37 °C. Then 75 μl or 500 μl aliquots were taken from the opposite compartment and the total radioactivity was determined by Tri-Carb 3110TR Liquid Scintillation Analyzer (PerkinElmer, Waltham, MA) liquid scintillation counter. The paracellular flux was monitored by the nonpermeable marker, [14C] mannitol. The digoxin efflux ratio was calculated as: Efflux ratio ¼ Papp ðB  AÞ=Papp ðA  BÞ WherePapp ¼

dQ=dt ; C0 A

dQ/dt is the rate of permeation of the drug across the cells, C0 is the donor compartment concentration at time zero and A is the area of the cell monolayer.

Animal study protocol Under isoflurane anesthesia (3–5% induction, 1–2% maintenance at 1.0 l/min), the femoral artery (for blood sampling), vein (for drug administration) and the bladder (for urine collection) were catheterized. Anesthesia was maintained throughout the experiment. The anesthesia plane and the condition of the animal were monitored throughout the experiment by routine tail/toe pinching test, respiration rate and the palpebral reflex test. Each animal received an i.v. bolus of 0.02 mg/kg digoxin with and without 10 mg/kg bupropion (i.p.). The digoxin blood samples (0.2 ml) were collected in heparinized tubes via the femoral artery at 0, 5, 10, 30, 60, 120, 180, 240, 360, 480 and 600 min. After each sampling, a volume of 0.2 ml physiologic saline solutions were administered to sustain isotonic fluid balance. Urine samples were collected at Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

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0–120 min, 120–240 min, 240–360 min, 360–480 min and 480–600 min. Plasma and urine samples were stored at 20 °C until analysis by LC-MS/MS.

Results

Plasma and urine analysis

Bupropion cocktail or bupropion did not significantly affect human P-gp mediated transport of [3H]-DIG in MDR1-LLC-PK1 cells (Figure 1A and B). The integrity of the cell monolayer was confirmed by minimal mannitol permeation across the cell monolayer (data not shown).

An ACQUITY UPLC® Class-I equipped with a sample manager and binary solvent manager and thermostat (Waters Corporation, MA, USA), coupled with API 4000 (Applied Biosystems/Sciex, MA, USA) triple quadrupole mass spectrometer with an electro-spray ionization source, was used for the LC-MS/MS analysis. Chromatographic separation was conducted at ambient temperature with a gradient mobile phase programme to separate BUP, HBUP, EBUP, TBUP and digoxin and diazepam (internal standard) on an Agilent Zorbax SB-C8 4.6 × 100 mm 3.5 μm analytical column with a guard column (C8 4 × 2.0 mm, Phenomenex, Torrance, CA, USA). The mobile phase consisted of water (A) and methanol (B), both containing 5 mM ammonium formate (approximately pH 3.6). The flow rate was 0.9 ml/min and the mobile phase was run using the following gradient programme (B concentration in parentheses): 0–10.8 min (30– 38%), 10.8–11.0 min (38–68%), 11.0–13.8 min (68%), 13.8–14.0 min (68–90%), 14.0–15.9 min (90%) and 16.0–17.5 min (30%). The following multiple reaction monitoring (MRM) transitions were used: BUP, m/z 240 → 184; HBUP, m/z 256 → 238; EBUP/TBUP, m/z 242 → 168; DIG, m/z 799 → m/z 651; diazepam (internal standard), m/z 285 → 154.

[3H]-DIG transepithelial transport study by MDR1-LLC-PK cells

Data analysis Phoenix® WinNonlin® (Pharsight Corporation, Mountain View, CA) was used to estimate digoxin pharmacokinetics in the rat by the noncompartmental method. Statistical analysis was performed using SPSS v. 12.0 (SPSS, Chicago, IL). Unless otherwise stated, the results are presented as mean ± SD. After testing for homogeneity of variance, the Mann–Whitney U test was used to analyse the in vivo data as the variance in the data was found not to be homogenous. In contrast, the Student’s paired t-test was used to analyse the in vitro data as the variance was found to be homogenous and the data normally distributed. A value of p less than 0.05 was considered to be statistically significant. Copyright © 2014 John Wiley & Sons, Ltd.

3

Figure 1. [ H]-Digoxin efflux ratio in MDR1-LLC-PK1 in the presence and absence of tariquidar (1 μM), BUP cocktail or BUP (A and B). Bupropion cocktail and BUP did not signif3 icantly affect the MDR1 mediated transport of [ H]-DIG (A and B). Bupropion cocktail consisted of 40 nM BUP, 400 nM HBUP, 40 nM EBUP and 120 nM TBUP. Data are mean ± SD, 3 n = 3. ***p < 0.001 when compared with efflux ratio of [ H] DIG alone Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

MECHANISM OF DIGOXIN–BUPROPION DRUG INTERACTION

[3H]-DIG transport by H-OATP4C1 overexpressing MDCKII cells The time course study showed that [3H]-DIG transport by H-OATP4C1 overexpressing MDCKII cells was linear up to 45 min (data not shown). The uptake of [3H]-DIG by H-OATP4C1 overexpressing MDCKII cells was significantly inhibited by 100 μM digoxin indicating robust transport activity of H-OATP4C1 in these cells (Figure 2). Bupropion and its major metabolite, HBUP, significantly stimulated the H-OATP4C1 mediated transport of [3H]-DIG. This effect was largest at BUP and HBUP concentrations of 100 nM and 1 nM, respectively (Figure 2A and B). Moreover, at clinically relevant unbound plasma concentration, the BUP cocktail significantly stimulated the H-OATP4C1 mediated transport of [3H]-DIG by 55%, and partially but modestly reversed the inhibition by 100 μM digoxin (Figure 2C). However, at the concentrations tested, the other two metabolites of BUP, EBUP and TBUP, did not affect the [3H]-DIG uptake by H-OATP4C1 cells.

[3H]-DIG transport by r-Oatp4c1 overexpressing MDCKII cells The time course study showed that [3H]-DIG transport by r-Oatp4c1 overexpressing MDCKII cells was linear up to 30 min (data not shown). The uptake of [3H]-DIG by r-Oatp4c1 overexpressing MDCKII cells was significantly inhibited by 100 μM digoxin indicating robust transport activity of r-Oatp4c1 expressed in these cells (Figure 3). Bupropion significantly inhibited the r-Oatp4c1 mediated transport of [3H]-DIG at clinically relevant unbound plasma concentrations of the drug or those observed in the rat study. In contrast, HBUP or TBUP did not inhibit [3H]-DIG transport by r-Oatp4c1 in the concentration range 1 nM to 1 μM (Figure 3A and B).

Pharmacokinetics of digoxin in the presence or absence of bupropion in female rats The error (bias, %) or precision (CV, %) in the assay of digoxin, bupropion and its metabolites, based on quality control samples that spanned the calibration range, was < 15%. Co-administration of BUP and digoxin did not significantly affect the systemic plasma concentration, terminal plasma t1/2, plasma Copyright © 2014 John Wiley & Sons, Ltd.

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AUC, volume of distribution at steady-state (Vss), or total clearance (CL) of digoxin (Table 1, Figure 4). However, co-administration of BUP and digoxin significantly increased (51%, p = 0.032) the renal clearance (CLr) of digoxin (Table 1). HBUP was the major BUP metabolite in female rats, followed by TBUP, but EBUP was not detected.

Discussion Digoxin has been identified as both an r-Oatp4c1and H-OATP4C1 transporter substrate in kidney with a similar Km value, 8.0 μM and 7.8 μM, respectively, but it is not a substrate of the hepatic organic anion-transporting polypeptide transporters (OATP) OATP1A2, OATP1B1, OATP1B3 or OATP2B1 [21,22]. At the amino acid level, r-Oatp4c1 and H-OATP4C1 share 80.4% homology [22]. Digoxin is also a substrate of both rodent and human P-gp. Therefore, it was hypothesized that BUP increases the renal clearance of digoxin by acutely stimulating OATP4C1 (located on the basal membrane of the human kidney epithelial cells) or P-gp (located on the apical membrane of the kidney epithelial cells) mediated renal transport of digoxin. Preliminary studies with LLC-PK1 cells expressing human P-gp indicated no effect of bupropion or BUP cocktail on digoxin transport by these cells (Figure 1A and B). Similarly, bupropion metabolites had no effects on the efflux ratio of [3H]-DIG (n = 2, data not shown). Therefore, the study investigated the effect of bupropion or its metabolites (at clinically relevant plasma concentrations) on H-OATP4C1-mediated digoxin transport. In humans, the concentration range of BUP, HBUP, EBUP plus TBUP are around 0.02–0.6 μM, 0.1–1.68 μM, 0.07–0.56 μM, respectively [18,23]. The fraction of BUP, HBUP and TBUP unbound in human plasma is ~0.16, 0.23, 0.58, respectively [24,25]. Assuming the same is true for EBUP, the clinically relevant Cmin–Cmax unbound plasma concentrations of BUP, HBUP, EPUB plus TBUP are estimated to be 3 nM– 0.1 μM, 0.02–0.39 μM, 0.04–0.32 μM, respectively. As was hypothesized, bupropion and its major circulating metabolite, HBUP, stimulated the H-OATP4C1-mediated [3H]-DIG transport in the clinically relevant plasma concentration range (100 nM–1 μM for Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

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3

Figure 2. [ H]-Digoxin transport in H-OATP4C1 overexpression MDCKII cells in the presence and absence of DIG, BUP, BUP metabolites or BUP cocktail (A, B and C). Bupropion at 100 nM and 1 μM, and HBUP at 1 nM and 10 nM significantly increased 3 the H-OATP4C1 mediated transport of [ H]-DIG (A). Moreover, BUP cocktail significantly increased H-OATP4C1 mediated 3 transport of [ H]-DIG, and partially, but modestly, reversed H-OATP4C1 inhibition by 100 μM DIG (C). Bupropion cocktail consisted of 15 nM BUP, 125 nM HBUP, 50 nM EBUP and 50 nM TBUP. Data are mean ± SD, n = 3. *p < 0.05, **p < 0.01 when com3 pared with uptake of [ H] DIG alone. #p < 0.05 when compared with uptake of 100 μM DIG

Copyright © 2014 John Wiley & Sons, Ltd.

Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

MECHANISM OF DIGOXIN–BUPROPION DRUG INTERACTION

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3

Figure 3. [ H]-Digoxin transport by r-Oatp4c1 overexpressing MDCKII cells in the presence and absence of DIG, BUP or its metab3 olites (A and B). Bupropion (but not its metabolites) significantly inhibited r-Oatp4c1 mediated transport of [ H]-DIG at clinically relevant unbound plasma concentrations or those observed in the rat study (A). Data are mean ± SD, n = 3. *p < 0.05, **p < 0.01 3 when compared with uptake of [ H] DIG alone

Table 1. Digoxin pharmacokinetic parameters in rats when administered alone (i.v. 0.02 mg/kg, n = 4) or with bupropion (i.p. 10 mg/kg, n = 5)

t1/2 (h) AUC0-10h (ng × h/ml) AUC0-∞ (ng × h/ml) Vss (ml/kg) CL (ml/h/kg) CLr (ml/h/kg) CLnr (ml/h/kg)

DIG alone

DIG + BUP

4.59 ± 1.12 45.84 ± 7.38 56.66 ± 8.48 2359.29 ± 541.48 360.04 ± 62.83 77.01 ± 28.12 283.03 ± 57.26

4.77 ± 2.09 39.37 ± 15.35 50.38 ± 13.65 2929.91 ± 1553.82 418.28 ± 100.35 116.57 ± 11.10* 301.71 ± 105.32

Data are mean ± SD. *p < 0.05 when compared with digoxin alone.

Copyright © 2014 John Wiley & Sons, Ltd.

BUP and 1 nM–10 nM for HBUP). Moreover, at clinically relevant concentrations, the cocktail of BUP and its metabolites stimulated H-OATP4C1mediated [3H]-DIG transport. This stimulation modestly reversed the inhibition caused by a high concentration of cold digoxin (100 μM). The mechanism by which bupropion and its metabolites stimulate H-OATP4C1-mediated [3H]DIG transport needs to be investigated in detail. However, it is most likely due to allosterism. H-OATP4C1 has at least two binding sites, a digoxin and an estrone 3-sulfate binding site [26]. Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

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DIG plasma concentration (ng/ml)

50.00

0.02 mg/kg digoxin (i.v.) + 10 mg/kg bupropion (i.p.) 0.02 mg/kg digoxin (i.v.)

5.00

0.50 0

2

4

6

8

10

BUP plasma concentration (ng/ml)

1800.00

30.00

BUP HBUP

1500.00

25.00

TBUP

1200.00

20.00

900.00

15.00

600.00

10.00

300.00

5.00

0.00

0.00 0

2

4

6

8

10

HBUP and TBUP plasma concentration (ng/ml)

Time (h)

Time (h) Figure 4. (A) Digoxin plasma concentration versus time profiles when DIG was administered alone (i.v. 0.02 mg/kg, n = 4) or with bupropion (i.p. 10 mg/kg, n = 5). Bupropion did not significantly affect digoxin plasma concentrations. (B) The plasma concentration versus time profiles of BUP, HBUP and TBUP after co-administration of 10 mg/kg BUP (i.p.) and 0.02 mg/kg DIG (i.v.) (n = 5). EBUP was not detected. Data are mean ± SD

In addition, OATPs have been shown previously to be allosteric where the binding of one drug can increase the transport of another [27]. Thus, the binding of bupropion and its metabolites to OATP4C1 could possibly increase the transport of digoxin and therefore the renal secretion of digoxin. Typical of such allosteric interactions, higher concentrations of BUP or HBUP abolished the stimulation of [3H]DIG transport, presumably because at these higher concentrations OATP4C1 was inhibited. As to whether the stimulation of H-OATP4C1-mediated [3H]-DIG transport quantitatively explains the observed increase in digoxin renal clearance in humans caused by bupropion cannot be determined from our data. This is because the fidelity Copyright © 2014 John Wiley & Sons, Ltd.

of in vitro to in vivo extrapolation of such DDI has never been tested. If the in vivo magnitude of H-OATP4C1 stimulation by bupropion resembles that observed in vitro, such a DDI almost certainly cannot completely explain the DDI observed in vivo. In that event, we cannot discount other possibilities such as acute posttranslational modification of OATP4C1 by BUP or the involvement of other transporters, such as the organic solute transporters-α and -β (OST-α and OST-β) [28,29]. To elucidate the mechanisms of this BUP–DIG interaction in detail, we asked whether a preclinical animal model, such as the rat, could replicate this DDI. Digoxin is not metabolized by Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

MECHANISM OF DIGOXIN–BUPROPION DRUG INTERACTION

hepatic cytochrome P450 enzymes in human [30], and it is predominately cleared by the kidneys with 70–85% excreted unchanged in the urine [31]. However, digoxin is highly metabolized in rats to form digoxigenin bis-digitoxoside, digoxigenin mono-digitoxoside and digoxigenin, which account for > 60% of a dose [32,33]. Since Cyp3a2, the major enzyme involved in digoxin metabolism in rats, is not expressed in adult female rat, the Cyp3a2 metabolism of digoxin is expected to be minimal. For this reason, and because urinary catheterization can be more easily performed, we chose to study the DIG–BUP interaction in female rats. In our female rat study, the pharmacokinetic parameters of digoxin were consistent with previously reported values in the rat [34]. Although BUP significantly increased digoxin CLr, it did not have an effect on the systemic disposition of digoxin (AUC, CL or half-life) most likely because digoxin CLr is only a small fraction (0.2) of the total digoxin CL. The plasma protein binding of digoxin in humans and rats is ~25% and ~17%, respectively [14,35]. Considering the low plasma protein binding of digoxin, the mechanism of this BUP–digoxin interaction is unlikely to involve the competitive displacement of plasma protein and result in a change in the digoxin filtration

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clearance. Therefore, as outlined above, it was hypothesized that the net digoxin renal secretion clearance by r-Oatp4c1was increased by bupropion or its metabolites. Surprisingly, bupropion significantly but modestly inhibited [3H]-DIG transport into r-Oatp4c1 expressing cells in a concentration dependent manner (10 nM to 1 μM), while HBUP and TBUP had no effect. If BUP inhibits [3H]-DIG transport by r-Oatp4c1, it should reduce (not increase) digoxin renal clearance if r-Oatp4c1 is localized (as reported) at the basolateral membrane of the proximal tubular cells in the kidney [22]. However, after this study was initiated, the localization of r-Oatp4c1 was revised. Kuo et al. have shown that r-Oatp4c1 is primarily localized in the proximal straight tubules (S3) and is co-localized with P-gp. Therefore, Oatp4c1 is presumably an apical uptake transporter in the rat kidney [20]. If this localization is correct, our in vitro data are consistent with our in vivo rat data. That is bupropion increases the renal clearance of digoxin in the rat by inhibiting its reabsorption by r-Oatp4c1. In our rat study, the concentration range of BUP, HBUP and TBUP post-dose were 0.57–5.70 μM, 0.02–0.10 μM and 5–8 nM, respectively. The plasma protein binding of BUP is 75–80% in preclinical species [15]. Assuming the same is true for its metabolites, the

Figure 5. Mechanism of digoxin–bupropion DDI Copyright © 2014 John Wiley & Sons, Ltd.

Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd

262 estimated unbound concentrations range of BUP, HBUP and TBUP in our in vivo rat study were 0.11–1.14 μM, 4.7–20.9 nM and 1.0–1.6 nM, respectively. Therefore, the observed 51% increase in CLr in female rats could be due to an inhibition of r-Oatp4c1 expressed on the apical membrane of the proximal tubular cell in the rat kidney. Thus, given the different localization of r-Oatp4c1 and H-OATP4C1, the rat cannot serve as a representative animal model to further study the mechanisms of BUP–DIG DDI. As to why bupropion inhibits r-Oatp4c1 but stimulates H-OATP4C1 is not clear, but is likely due to species difference in the binding site(s). In conclusion, at clinically relevant plasma concentrations, BUP, HBUP and BUP cocktail increased the H-OATP4C1-mediated transport of [3H]-DIG, while EBUP and TBUP had no effect. In addition, bupropion inhibited r-Oatp4c1mediated transport of [3H]-DIG, while its metabolites did not. While we cannot discount the involvement of other transporters or mechanisms, our data suggest that stimulation of H-OATP4C1 mediated digoxin tubular secretion from the kidney or inhibition of r-Oatp4c1-mediated digoxin reabsorption could be contributing factors to the BUP–DIG DDI observed in humans and rats, respectively (Figure 5). To our knowledge, this is the first time that stimulation of a transporter has been observed to increase the clearance of a drug in vivo in humans. To prevent future clinically significant digoxin DDI, studies to identify other drugs that can stimulate H-OATP4C1 mediated digoxin transport are needed.

Acknowledgements The rat Oatp4c1 and human OATP4C1 cell line was generously provided by Dr Leggas, University of Kentucky. This work was supported by China Scholarship Council (CSC).

Conflict of Interest The authors declare no competing financial interest. Copyright © 2014 John Wiley & Sons, Ltd.

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Biopharm. Drug Dispos. 35: 253–263 (2014) DOI: 10.1002/bdd