Differential Preincubation Effects of Nicardipine on OATP1B1- and OATP1B3-Mediated Transport in the Presence and Absence of Protein: Implications in Assessing OATP1B1- and OATP1B3-Mediated Drug–Drug Interactions

Impaired transport activity of hepatic OATP1B1 and OATP1B3 due to drug–drug interactions (DDIs) often leads to increased systemic exposure to substrate drugs (e.g., lipid-lowering statins). Since dyslipidemia and hypertension frequently coexist, statins are often concurrently used with antihypertensives, including calcium channel blockers (CCBs). OATP1B1/1B3-related DDIs in humans have been reported for several CCBs. To date, the OATP1B1/1B3-mediated DDI potential of CCB nicardipine has not been assessed. The current study was designed to assess the OATP1B1- and OATP1B3-mediated DDI potential of nicardipine using the R-value model, following the US-FDA guidance. IC50 values of nicardipine against OATP1B1 and OATP1B3 were determined in transporter-overexpressing human embryonic kidney 293 cells using [3H]-estradiol 17β-D-glucuronide and [3H]-cholecystokinin-8 as substrates, respectively, with or without nicardipine-preincubation in protein-free Hanks’ Balanced Salt Solution (HBSS) or in fetal bovine serum (FBS)-containing culture medium. Preincubation with nicardipine for 30 min in protein-free HBSS buffer produced lower IC50 and higher R-values for both OATP1B1 and OATP1B3 compared to in FBS-containing medium, yielding IC50 values of 0.98 and 1.63 µM and R-values of 1.4 and 1.3 for OATP1B1 and OATP1B3, respectively. The R-values were higher than the US-FDA cut-off value of 1.1, supporting that nicardipine has the potential to cause OATP1B1/3-mediated DDIs. Current studies provide insight into the consideration of optimal preincubation conditions when assessing the OATP1B1/3-mediated DDIs in vitro.


Introduction
Organic anion transporter polypeptide (OATP) 1B1 and OATP1B3 are basolateral uptake transport proteins in human hepatocytes. Many clinically used drugs are substrates of OATP1B1 and OATP1B3, including widely prescribed lipid-lowering medicines [1]. Reduced transport activity of OATP1B1 and OATP1B3 due to drug-drug interactions (DDIs) and genetic variations have been reported to increase systemic exposure to statins and statin-induced myopathy [2,3].
Hypertension and dyslipidemia are two major risk factors for cardiovascular diseases that frequently coexist in patients [4]. Calcium channel blockers (CCBs) and statins are often prescribed for the treatment of hypertension [5] and dyslipidemia [6], respectively. Since simultaneous treatment of hypertension and dyslipidemia is beneficial to reduce the overall cardiovascular risk [4], concurrent administration of CCBs and statins is likely. For example, the single-pill therapy combining amlodipine, a CCB, and atorvastatin has 2.2. Cell Culture HEK293 stable cell lines expressing FLAG-tagged OATP1B1 (HEK293-FLAG-OATP1B1) [24] or FLAG-tagged OATP1B3 (HEK293-FLAG-OATP1B3) [25] have been published previously. Both HEK293 stable cell lines were cultured in DMEM culture media supplemented with 9% FBS (abbreviated as FBS-DMEM), antibiotic antimycotic, and 600 µg/mL G418 as published previously [24,25].

Transport Studies and IC 50 Determination
HEK293-FLAG-OATP1B1 and HEK293-FLAG-OATP1B3 cells (less than 40 passages) were seeded in poly-L-lysine coated 24-well plates at a density of 1.5 × 10 5 cells per well and allowed to grow for 48 h until confluent before the transport studies [26]. [ 3 H]-E 2 17βG (1 µM, 2 min) and [ 3 H]-CCK-8 (1 µM, 3 min) were used as substrates for OATP1B1 and OATP1B3 in HEK293-FLAG-OATP1B1 and HEK293-FLAG-OATP1B3 cells, respectively. Substrate accumulation time was at a linear uptake range and accumulation was determined in HBSS buffer containing 10 mM HEPES (pH 7.4), in the absence of protein, similar to that published previously [18,27]. E 2 17βG and CCK-8 concentrations were below the K m values for OATP1B1 and OATP1B3, respectively [28], hence, the determined IC 50 values approximate the K i values. In the preincubation scenario, cells were preincubated with the vehicle control, 0.1% DMSO (v/v) or nicardipine at indicated concentrations ranging from 0.05 to 4 µM for designated times, in either the DMEM culture medium, which contains 9% FBS or in HBSS buffer supplemented with 10 mM HEPES (pH 7.4). After preincubation, cells were washed three times with HBSS buffer and incubated with HBSS buffer containing substrate, in the absence of vehicle control or inhibitor. In the coincubation scenario, substrate accumulation was determined in the presence of vehicle control or nicardipine without preincubation. In the pre plus coincubation scenario, substrate accumulation was determined in the presence of the vehicle control or nicardipine, following preincubation at the same concentration of the vehicle control or nicardipine described above and subsequently washed. At the end of substrate incubation, cells were washed three times with ice-cold HBSS buffer and lysed in PBS containing 0.5% Triton X-100. Liquid scintillation counting was determined and normalized to the protein concentration, after correcting for any nonspecific binding in the poly-L-lysine-coated blank plates, as published previously [18,27].
where E is the remaining substrate transport at a given inhibitor concentration (C). IC 50 is the inhibitor concentration causing a response halfway between the maximally inhibited (bottom) response and the maximal (top) response.

Prediction of OATP-Mediated DDIs Using the R-Value Model
The R-value, which represents the predicted AUC ratio of the substrate drug in the presence versus absence of an inhibitor drug, was calculated based on Equation (2), following the US-FDA final guidance [15] with the parameters summarized in Table 1. A f u, plasma of 0.05 is the unbound fraction of nicardipine in human plasma [29]. I in, max is the estimated total maximum plasma concentration of nicardipine at the inlet to the liver and is estimated based on Equation (3), where I max is the maximum plasma concentration (C max ) of the inhibitor in the systemic circulation. Nicardipine doses were titrated, beginning with 20 mg, 3 times daily; doses in the range of 20 to 40 mg 3 times a day have been shown to be effective [29]. Steady-state C max values following nicardipine 20, 30, and 40 mg doses, 3 times a day, are shown in Table 1. The f a is the fraction absorbed. Nicardipine is completely absorbed following oral dosing [29], therefore, a f a of 1 is used for nicardipine. F g is the fraction that escaped gut metabolism (F g ). The k a is the absorption rate constant. Since values for F g and k a are unknown for nicardipine, values of F g = 1 and k a = 0.1/min were used as a worst-case estimate, according to the US-FDA guidance [15]. Q h is the hepatic blood flow rate (1500 mL/min) [32]. R B is the blood-toplasma concentration ratio. In human blood, 12-18% of total nicardipine was present in erythrocytes [30]. Using a hematocrit of 0.45 as a default value, the R B of nicardipine ranges from 0.63 to 0.67. R B of 0.63 was used as a worst-case scenario.
As K i approximates IC 50 , the R-value was determined as shown below:

Statistical Analysis
For the statistical analysis shown in Figure 1, fold changes vs. control and associated standard errors (SEs) were estimated using linear mixed models with a random effect (experimental date) and a fixed group effect, allowing for group-specific variances, similar to those published previously [14,18]. In multiple comparisons, p-values were adjusted using Bonferroni's method. A two-sided p-value of <0.05 defines statistical significance. SAS software (version 9.4, Cary, NC, USA) was used for statistical analyses. ter preincubation with 0.5, 2, and 4 µM nicardipine for 10 min, 30 min, and 1 h in HBSS buffer or FBS-containing medium, as indicated in the legend. Substrate accumulation was determined in the absence of nicardipine after washing. Linear mixed-effects models were fit to the data, as described in the "Materials and Methods" (n = 3 in triplicate). * and ** indicate statistically significant differences vs. CTL with Bonferroni-adjusted p-values < 0.05 and 0.01, respectively.
The preincubation effects of nicardipine (0.5-4 µM, 10 min −1 h) on OATP1B1-or OATP1B3-mediated transport were determined in two different preincubation media, HBSS buffer, and FBS-containing medium. As shown in Figure 1, the greatest inhibitory effects on OATP1B1 and OATP1B3 caused by nicardipine-preincubation were observed, in both, after nicardipine preincubation in the HBSS buffer. For OATP1B1, preincubation with 0.5 µM nicardipine significantly decreased OATP1B1-mediated transport after 30 min and 1 h preincubation in HBBS buffer by 0.78 ± 0.04 and 0.82 ± 0.04-fold of the control, respectively ( Figure 1A); however, 0.5 µM nicardipine had no effect after a 10 min prein- Substrate accumulation was determined in the absence of nicardipine after washing. Linear mixed-effects models were fit to the data, as described in the "Materials and Methods" (n = 3 in triplicate). * and ** indicate statistically significant differences vs. CTL with Bonferroni-adjusted p-values < 0.05 and 0.01, respectively.
The preincubation effects of nicardipine (0.5-4 µM, 10 min-1 h) on OATP1B1-or OATP1B3-mediated transport were determined in two different preincubation media, HBSS buffer, and FBS-containing medium. As shown in Figure 1, the greatest inhibitory effects on OATP1B1 and OATP1B3 caused by nicardipine-preincubation were observed, in both, after nicardipine preincubation in the HBSS buffer. For OATP1B1, preincubation with 0.5 µM nicardipine significantly decreased OATP1B1-mediated transport after 30 min and 1 h preincubation in HBBS buffer by 0.78 ± 0.04 and 0.82 ± 0.04-fold of the control, respectively ( Figure 1A); however, 0.5 µM nicardipine had no effect after a 10 min preincubation in HBSS buffer ( Figure 1A), or at any preincubation duration in the FBS-containing medium ( Figure 1B). Preincubation with nicardipine at 2 and 4 µM, in both the HBSS buffer ( Figure 1A) and in the FBS-containing medium ( Figure 1B), significantly reduced OATP1B1mediated transport of [ 3 H]-E 2 17G, at all preincubation time points, although a greater effect was seen in HBSS buffer than in the respective counterparts for FBS-containing medium ( Figure 1A,B).
For OATP1B3, preincubation with nicardipine at 0.5 µM did not significantly affect OATP1B3-mediated transport under any treatment condition ( Figure 1C,D). However, in the HBSS buffer, preincubation with 2 or 4 µM nicardipine significantly decreased OATP1B3mediated transport at all time points (10 min-1 h), with transport activity ranging from 0.41 ± 0.04 to 0.47 ± 0.03, and 0.24 ± 0.03 to 0.31 ± 0.02-fold of the control for 2 and 4 µM nicardipine, respectively ( Figure 1C). In the FBS-containing medium, small, yet significant, reductions in OATP1B3-mediated transport were observed after preincubation with nicardipine at 4 µM for 30 min and at 2 or 4 µM for 1 h ( Figure 1D).
In the HBSS buffer, for both 2 or 4 µM nicardipine preincubations, the inhibitory effects were greater on both OATP1B1-and OATP1B3-mediated transports following 30 min and 1 h preincubations than after 10 min preincubation, yet inhibition was similar following 30 min and 1 h for both transporters for each nicardipine concentration. Hence, a 30 min preincubation timepoint was chosen in the subsequent studies to determine the effects of nicardipine preincubation on the IC 50 values for OATP1B1 and OATP1B3.

Effects of Nicardipine Preincubation on IC 50 Values against OATP1B1 and OATP1B3 in HBSS and FBS-Containing Medium
IC 50 values for nicardipine against OATP1B1-mediated transport of [ 3 H]-E 2 17βG ( Figure 2) and OATP1B3-mediated transport of [ 3 H]-CCK-8 ( Figure 3) were determined by coincubation with either the vehicle control or nicardipine (up to 4 µM maximum aqueous soluble concentration) and either with (pre plus coincubation) or without (coincubation) 30 min nicardipine preincubation in HBSS buffer or FBS-containing medium. For comparison purposes, the preincubation data in Figure 1 and coincubation data in Figure 2 were replotted in Figure S2.
The IC 50 value of nicardipine against OATP1B1 was decreased by 3.6-fold (3.55 vs. 0.98 µM) and 1.8-fold (2.80 vs. 1.57 µM) after nicardipine preincubation in HBSS buffer and the FBS-containing medium, respectively ( Figure 2 and Table 2). For OATP1B3, a 30 min preincubation followed by coincubation with nicardipine in HBSS buffer yielded an IC 50 value of 1.63 µM. Notably, IC 50 values for nicardipine against OATP1B3 could not be confidently estimated in other conditions, including with coincubation-only and pre plus coincubation in the FBS-containing medium, due to the less efficient inhibitions ( Figure 3 and Table 2).

Prediction of OATP-Mediated DDIs Using Static R-Value Model
The R-values for nicardipine against OATP1B1 and OATP1B3 at doses ranging from 20 to 40 mg are summarized in Table 2. For both OATP1B1 and OATP1B3, the R-values were higher in the pre plus coincubation condition than in the coincubation condition for each dose. In the pre plus coincubation condition, the IC 50 values from inhibitor-preincubation in the HBSS buffer (pre+co-IC 50 , HBSS ) were lower than those for the FBS-DMEM (pre+co-IC 50 , FBS ), resulting in higher R-values at each dose for the HBSS buffer.
Under the pre plus coincubation condition, for OATP1B1, the R-values determined from pre+co-IC 50 , HBSS,OATP1B1 were higher than the US-FDA recommended DDI cut-off value of 1.1, at all doses, ranging from 1.2 to 1.4, which supports the OATP1B1-mediated DDI potential of nicardipine, even at a low dose of 20 mg. For OATP1B3, the R-values were only higher than the DDI cut-off value of 1.1 when using the pre+co-IC 50 , HBSS values at higher doses of 30 and 40 mg, suggesting a DDI potential against OATP1B3 at higher doses but not at the lowest dose of 20 mg.  The IC50 value of nicardipine against OATP1B1 was decreased by 3.6-fold (3.55 vs. 0.98 µM) and 1.8-fold (2.80 vs. 1.57 µM) after nicardipine preincubation in HBSS buffer and the FBS-containing medium, respectively ( Figure 2 and Table 2). For OATP1B3, a 30 min preincubation followed by coincubation with nicardipine in HBSS buffer yielded an IC50 value of 1.63 µM. Notably, IC50 values for nicardipine against OATP1B3 could not be confidently estimated in other conditions, including with coincubation-only and pre plus coincubation in the FBS-containing medium, due to the less efficient inhibitions (Figure 3 and Table 2

Discussion
The current study is the first to characterize the IC 50 values of nicardipine against OATP1B1 and OATP1B3 and report that nicardipine has the potential to cause OATP1B1and OATP1B3-mediated DDIs after following the current US-FDA recommended R-value model.
Interestingly, for the first time, the current study demonstrated differential inhibitorpreincubation effects in a protein-free HBSS buffer compared to an FBS-containing medium. Using the same preincubation time, the preincubation-induced inhibitory effects of nicardipine on OATP1B1-and OATP1B3-mediated transport appear to be concentration-dependent (Figure 1), with greater inhibitory effects observed at higher concentrations. A major component of FBS is bovine serum albumin (BSA) (~2.5 mg/mL in FBS [33]). The BSA (final concentration~0.25% (w/v)) in the FBS-DMEM preincubation medium provides a source for protein binding. In addition to the DMEM medium, in the HBSS buffer, the nicardipine preincubation effects were also greater when preincubation was conducted in the absence of FBS, compared to those in the presence of FBS, for both OATP1B1 (Figures S3A and 1A) and OATP1B3 (Figures S3B and 1C). Since nicardipine is highly protein-bound (f u = 0.05), the presence of BSA is anticipated to reduce the unbound concentrations of nicardipine and, therefore, may lead to a reduced inhibitory effect in the FBS-containing medium, compared to the protein-free HBSS buffer. More inhibitor drugs with a different f u would need to be tested in this way in order to draw definitive conclusions. The current data, reported herein, show the complexity involved in determining the inhibition potency of OATP1B1 and OATP1B3 in the preincubation condition and underline the value in considering the proteins present in the preincubation medium when testing preincubation effects.
Predicting transporter-mediated DDIs has continued to be challenging [34,35], where false negative predictions or large underestimations of DDIs have been reported [35,36]. Of note, all three reported DDIs of CCB against OATP1B1/3 drug substrates [8][9][10] were falsely predicted as negative [35,36]. Based on reported in vitro IC 50 values for nisoldipine [36], verapamil [37], and mibefradil [36] against OATP1B1 and/or OATP1B3, these CCBs are not anticipated to cause clinical DDIs against OATP1B1/3 substrates [35,36]. However, during in vivo investigations in humans, the coadministration of nisoldipine significantly increased the AUC of telmisartan, a substrate of OATP1B3 [38], by 2.32-fold [9]. Coadministration of verapamil increased the AUC of pravastatin by 1.32-fold [8], while coadministration of mibefradil increased the AUC of pravastatin and atorvastatin by 1.32-fold and 4.43-fold, respectively [10] (Table 3). Pravastatin is a substrate of OATP1B1 [13], and atorvastatin is a substrate of both OATP1B1 [13] and OATP1B3 [23]. Pravastatin is a metabolically stable statin [39], while atorvastatin it is a substrate of CYP3A4 [40]. The DDI of mibefradil against atorvastatin has been postulated to be due to CYP inhibition [10]. However, hepatic uptake processes via OATPs have been shown to contribute predominantly to the hepatic elimination of atorvastatin at a subtherapeutic dose [41]. Subjects with the OATP1B1 c. 521CC genotype, which has reduced transport activity, has an increased AUC for pravastatin and atorvastatin of 1.9-and 2.4-fold of the reference, respectively (reviewed in [42]). Thus, a potential role of hepatic OATP1B1/3 in the DDI of mibefradil against pravastatin and atorvastatin cannot be excluded. The reported IC 50 values of nisoldipine, verapamil, and mibefradil against OATP1B1 and OATP1B3 were all determined without inhibitor preincubation [36,37]. In the current study, adding a 30 min nicardipine preincubation step led to reduced IC 50 -and increased R-values, compared to no preincubation step, against OATP1B1 and OATP1B3 ( Table 2). The inhibitor preincubation step shifted the prediction of DDI from no DDI potential or DDI potential only at the highest dose of nicardipine for OATP1B1 to DDI potential even at the lower doses (Table 2). Substrate-dependent inhibition of OATP1B1 has been reported [26], where E 2 17βG has been recognized as a sensitive probe substrate of OATP1B1 in vitro. Recently, coproporphyrin I (CP-1), an endogenous substrate of OATP1B [43], has been reported as a promising endogenous biomarker with which to evaluate in vivo inhibition of OATP1B1 in humans [44]. The effects of nicardipine on OATP1B1/3-mediated transport in vitro are worth evaluating using CP-1 as the substrate for potential translational purposes. To the best of our knowledge, no in vivo studies on nicardipine against OATP1B1 and/or OATP1B3 drug substrates have been reported. The current prediction performance using the R-value model warrants further verification using a physiologically based pharmacokinetic model (PBPK) and a clinical DDI study of nicardipine against OATP1B1/3 substrate, such as with statins.
The CCB amlodipine has been predicted to have no DDI potential based on the IC 50 values against OATP1B1, determined without an inhibitor-preincubation step [45]. Isradipine and nifedipine, both at 20 µM, have been reported to inhibit OATP1B1-mediated transport by 47.5% [23] and >50% [23], respectively. Diltiazem does not inhibit OATP1B1, even at 100 µM [45]. Based on our report, herein, on the potential DDI of nicardipine against OATP1B1 and OATP1B3 and the previously reported apparent high chance of CCBs causing DDIs with OATP substrate drugs [35,36], the OATP1B1-and OATP1B3-mediated DDI potential of other CCBs warrants further evaluation, following the addition of an inhibitor preincubation step.
In conclusion, the present study reports that preincubation with nicardipine in a protein-free HBSS buffer elicits the lowest IC 50 values against OATP1B1 and OATP1B3, compared to coincubation-only or preincubation in FBS-containing medium. The current study predicts that nicardipine has the potential to cause hepatic OATP1B-mediated DDIs. Adding an inhibitor preincubation step may help to mitigate the false-negative predictions that are often associated with CCBs and warrants further studies with other CCBs alongside verification in clinical studies.
Author Contributions: Participated in research design: R.K. and W.Y.; Conducted experiments: R.K. and V.T.; Performed data analysis: R.K., C.X., V.T. and W.Y.; Wrote or contributed to writing the manuscript: R.K., C.X. and W.Y. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.