Cellular Uptake of Levocetirizine by Organic Anion Transporter 4

Accepted Manuscript Cellular Uptake of Levocetirizine by Organic Anion Transporter 4 Saki Noguchi, Tomohiro Nishimura, Saya Mukaida, Leslie Z. Benet, Emi Nakashima, Masatoshi Tomi PII:

S0022-3549(17)30212-5

DOI:

10.1016/j.xphs.2017.03.026

Reference:

XPHS 704

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 15 February 2017 Revised Date:

16 March 2017

Accepted Date: 27 March 2017

Please cite this article as: Noguchi S, Nishimura T, Mukaida S, Benet LZ, Nakashima E, Tomi M, Cellular Uptake of Levocetirizine by Organic Anion Transporter 4, Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.xphs.2017.03.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Cellular Uptake of Levocetirizine by Organic Anion Transporter 4

2 Saki Noguchi,1 Tomohiro Nishimura,1 Saya Mukaida1, Leslie Z. Benet,1,2 Emi

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Nakashima,1 Masatoshi Tomi1

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1Faculty

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and Medicine, University of California San Francisco, San Francisco, California

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94143-0912

of Pharmacy, Keio University, Minato-ku 105-8512, Tokyo, Japan

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Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy

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Correspondence to: Masatoshi Tomi (TEL&FAX: +81-3-5400-2660; E-mail:

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[email protected])

Keywords:

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pharmacokinetics; drug transport; placenta; renal transport

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Organic

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(OAT);

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transport;

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Abstract: The pharmacokinetics of cetirizine, a non-sedating antihistamine, is

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profoundly affected by transporter-mediated membrane transport in the kidney. In

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this study, we aimed to investigate the transport mechanism of levocetirizine, the

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pharmacologically active enantiomer of cetirizine, via human organic anion

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transporter 4 (OAT4) expressed in the apical membrane of renal proximal tubules

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and the basal plasma membrane of placental syncytiotrophoblasts. In cells

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expressing human OAT4 under the control of tetracycline, levocetirizine uptake

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was increased by tetracycline treatment. On the other hand, OAT4 expression did

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not facilitate efflux of preloaded levocetirizine from the cells, either in the presence

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or absence of extracellular Cl-. The OAT4-mediated levocetirizine uptake was

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concentration-dependent with a Km of 38 µM. The uptake rate of levocetirizine via

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OAT4 was approximately twice that of racemic cetirizine, indicating stereoselective

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uptake

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[3H]dehydroepiandrosterone sulfate uptake was inhibited by dextrocetirizine as

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well as levocetirizine. Overall, our findings indicate that OAT4 mediates

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levocetirizine uptake, but is unlikely to mediate the efflux.

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levocetirizine.

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OAT4-mediated

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Introduction

35 Cetirizine, a second-generation non-sedating antihistamine, has a zwitterionic

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structure and is a racemate composed of levocetirizine (eutomer) and

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dextrocetirizine (distomer); both levocetirizine and the racemate are widely used

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in the relief of allergic symptoms. Cetirizine is listed in class 3 of

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Biopharmaceutical Drug Disposition and Classification System, which implies low

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susceptibility to metabolism.1 Indeed, about 70% of orally administered cetirizine

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is excreted unchanged in the urine over 72 h.2 Benedetti et al.3 estimated that renal

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clearance of levocetirizine and dextrocetirizine in humans was 3.6 times higher

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than the clearance by glomerular filtration. Hence, membrane transport in renal

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epithelial cells is profoundly involved in the pharmacokinetics of cetirizine.

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Therefore, it is important to clarify the roles of individual transporters in

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membrane transport of cetirizine.

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Interactions of cetirizine with two renal membrane drug transporters have

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been reported so far. Cetirizine distribution to the brain was increased in multidrug

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resistance (mdr) 1a/1b knockout mice, and MDR1 mediated cetirizine efflux from

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monolayers of MDCK cells transfected with human MDR1.4, 5 Thus, MDR1-mediated

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cetirizine secretion is expected to occur in the apical membrane of renal proximal

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tubules. On the other hand, organic cation transporter 2 (OCT2) on the basal side

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of renal proximal tubules could also be involved, because cetirizine was reported to

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inhibit OCT2-mediated uptake activity.6 Nonetheless, it remains possible that other

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drug transporters in renal proximal tubules also interact with cetirizine.

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Organic anion transporter 4 (OAT4; SLC22A11) is one of the solute carrier

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family of drug transporters, and is expressed in human kidney and placenta.7 In the

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kidney, OAT4 is located at the apical membrane of proximal tubules.8 OAT4 is

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unique, since it works as an uptake transporter for some substrates, such as urate

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and steroid sulfates,9,

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aminohippurate and olmesartan.9,

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uptake of levocetirizine was enhanced in OAT4-expressing cells.12 However,

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detailed analysis of the OAT4-mediated efflux and uptake of levocetirizine is

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needed to understand the direction of physiological OAT4-mediated transport of

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levocetirizine. Since orthologues of human OAT4 have not been found in rodents,

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the interaction of OAT4 with drugs cannot be determined from rodent studies.

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Therefore, it is important to estimate the role of OAT4 on renal drug clearance by

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means of studies using human OAT4-overexpressing cells. In this study, we aimed

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to investigate OAT4-mediated transport mechanism of levocetirizine by using

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OAT4-overexpressing cells.

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A meeting abstract reported that cellular

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Cell Culture

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Materials and Methods

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but as an efflux transporter for others, such as p-

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A tetracycline-inducible human OAT4-expressing cell line (T-REx OAT4-293

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cells) was established from Flp-In T-REx 293 cells (Life Technologies, Carlsbad,

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California) as previously described,11 and maintained in Dulbecco’s modified

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Eagle’s medium (Nacalai Tesque, Kyoto, Japan) with 10% fetal bovine serum, 400

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µg/mL hygromycin B, and 15 µg/mL blasticidin at 37°C, under 5% CO2 in air. OAT4

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expression was induced by culturing the cells in the presence of 1 µg/mL

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tetracycline for 24 h.

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Transport Study The uptake and efflux studies using T-REx OAT4-293 cells were conducted as

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previously described.11 After the uptake and efflux procedures, cells were

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solubilized in NaOH overnight and the resulting suspension was neutralized with

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HCl. Levocetirizine, cetirizine, and fexofenadine were quantitated by tandem mass

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spectrometry (API-3200, AB Sciex, Flamingham, Massachusetts) coupled to

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high-performance liquid chromatography (Shimadzu, Kyoto, Japan) (LC-MS/MS)

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according to the previous report, with some modifications.13 Samples were

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deproteinized with the same volume of acetonitrile containing 100 nM

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fexofenadine (internal standard), and an aliquot (10 µL) was injected into the

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LC-MS/MS. Chromatographic separation was performed on a Capcell Pak C18

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UG120 column (2.0 mm I.D. × 35 mm, 5 µm, Shiseido, Tokyo, Japan) at 40°C with a

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mobile phase of 20% acetonitrile containing 0.1% formic acid; the flow rate was

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0.2 mL/min. Mass spectrometric detection was performed by multiple reaction

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monitoring in the electrospray ionization positive ion mode, using 389.20 to

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201.10 m/z for levocetirizine and cetirizine, and 503.128 to 171.200 m/z for

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fexofenadine. Cellular uptake of DHEAS, sodium salt, [1,2,6,7-3H(N)] ([3H]DHEAS;

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70.5 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, Massachusetts)

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was measured as previously described.11 Protein concentration was determined

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with a bicinchoninic acid assay kit (Pierce, Rockford, Illinois) with bovine serum

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albumin as a standard.

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Uptake of substrates was expressed as the concentration ratio (µL/mg protein)

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of nmol/mg protein in the cells to nmol/µL in the medium. OAT4-mediated uptake

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was calculated by subtracting the uptake of substrate by tetracycline-untreated

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(mock) cells from that by tetracycline-treated (OAT4-expressing) cells.

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Kinetic Analysis

The kinetic parameters were estimated by fitting the OAT4-mediated uptake

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rate to the following equations by means of nonlinear least-squares regression

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analysis using the MULTI program:14 V = (Vmax × [S]) / (Km + [S]), where V, [S], Km,

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and Vmax represent uptake velocity, concentration of substrate, Michaelis constant,

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and maximum uptake velocity, respectively, and V = V0 / [1 + ([I] / IC50)n], where V,

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V0, [I], IC50, and n represent uptake velocity in the presence of inhibitor, uptake

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velocity in the absence of inhibitor, concentration of inhibitor, median inhibitory

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concentration, and Hill slope.

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Statistical Analysis

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Statistical significance of differences was evaluated using the unpaired,

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two-tailed Student’s t-test or one-way ANOVA followed by Dunnett’s test. Unless

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otherwise indicated, data are presented as mean ± SEM.

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Results and Discussion

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First, we verified that levocetirizine is taken up via human OAT4. The uptake of

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levocetirizine by OAT4-expressing cells increased time-dependently and was

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significantly higher than that of mock cells, indicating that OAT4 recognizes

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levocetirizine as a substrate (Fig. 1a). Since OAT4 can work as an organic anion

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exchanger using extracellular Cl- as the counterpart ion,15 e.g., with olmesartan,11

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we examined the effect of extracellular Cl- on OAT4-mediated uptake and efflux of

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levocetirizine. Cellular uptake of levocetirizine via OAT4 was not affected by

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extracellular Cl- (Fig. 1a). Further, OAT4 did not facilitate the outflow of preloaded

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levocetirizine from the cells either in the presence and absence of extracellular Cl-

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(Fig. 1b). These results suggest that OAT4 is involved in levocetirizine uptake, but

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not in its efflux. Thus, OAT4-mediated transport of levocetirizine appears to be

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similar to that of estrone sulfate and urate, which are taken up into the cells by

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exchange with intracellular dicarboxylates or hydroxyl ions,15 but are not exported

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from the cells via OAT4.8, 9 Therefore, we propose that OAT4 mediates reabsorption

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of levocetirizine from urine into proximal tubule cells.

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We then examined the concentration dependence of levocetirizine uptake via

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OAT4. Levocetirizine uptake via OAT4 was saturable with Km of 38.3±13.0 µM and

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Vmax of 1.51±0.37 nmol/mg protein/min (mean±SD) (Fig. 2). The maximum plasma

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unbound concentration of levocetirizine is no more than 0.1 µM after oral

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administration of 10 mg.16, 17 These results indicate that OAT4-mediated uptake of

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levocetirizine would not be saturated under clinically relevant conditions.

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Significant elevation of the plasma concentrations of cetirizine and pilsicainide,

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a class Ic antiarrhythmic agent, was observed in a patient with renal insufficiency

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after coadministration of the two drugs.6 Renal transporters involved in their

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excretion process are likely to mediate this interaction. However, renal OAT4 is

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unlikely to mediate excretion of levocetirizine (Fig.1). And the uptake of

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levocetirizine via OAT4 was neither inhibited nor facilitated by extracellular

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pilsicainide up to 100 µM, although the uptake of levocetirizine was inhibited by

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olmesartan, a substrate of OAT4,11 with an IC50 of 7.23±3.49 µM (mean±SD) (Fig. 3).

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In the clinical context, the maximum plasma unbound concentration of pilsicainide

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is not expected to exceed 100 µM.18 Based on these results, OAT4 is unlikely to be

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implicated in the interaction between cetirizine and pilsicainide.

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In order to examine if the interaction with OAT4 is stereoselective, we

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compared OAT4-mediated uptakes of cetirizine (racemate of levocetirizine and

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dextrocetirizine) and levocetirizine. As shown in Fig. 4a, OAT4-mediated uptake

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rate of levocetirizine (0.667 µL/mg protein/sec) estimated by linear regression

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was approximately 1.8 times greater than that of cetirizine (0.372 µL/mg

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protein/sec), indicating that OAT4 exhibits stereoselectivity for levocetirizine. We

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then measured the inhibitory effects of levocetirizine and cetirizine on

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OAT4-mediated uptake of DHEAS, a typical OAT4 substrate. The uptake of

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[3H]DHEAS via OAT4 was inhibited by levocetirizine and cetirizine with IC50 values

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of 104±14 µM and 118±17 µM, respectively (mean±SD) (Fig. 4b), while 1 mM

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fexofenadine, another second-generation antihistamine, did not show an inhibitory

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effect (Fig. 4b), even though both cetirizine and fexofenadine mainly exist as

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zwitterions at the experimental pH of 7.4.19,

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inhibition curves by levocetirizine and cetirizine were 0.981±0.122 and

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0.626±0.059, respectively (mean±SD). These results indicate that dextrocetirizine

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is unlikely to be taken up via OAT4, but is capable of inhibiting OAT4-mediated

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uptake. Baltes et al.17 reported that the renal clearance of levocetirizine (32.0

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mL/min) was almost half that of dextrocetirizine (61.4 mL/min) in humans.

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Although the difference in plasma unbound fractions between the cetirizine

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enantiomers accounts for the difference in renal clearance,3, 21 our results raise the

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possibility that OAT4-mediated reuptake of levocetirizine also contributes to the

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lower renal clearance of levocetirizine, compared with dextrocetirizine. Thus,

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OAT4-mediated transport should be taken into account in considering the renal

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elimination process of levocetirizine in humans.

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OAT4 can mediate both influx and efflux transport of some substrates,15 but our

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results strongly suggest that it mediates only uptake of levocetirizine, not efflux.

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Since OAT4 localized in the apical membrane of proximal tubules,8 this may imply

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that OAT4 is involved in reabsorption of levocetirizine across the proximal tubules.

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The difference in renal clearance of cetirizine enantiomers in humans is consistent

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with stereoselective uptake via OAT4. The present results thus help to improve our

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understanding of cetirizine renal transport at the molecular level.

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Acknowledgements

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This work was supported in part by JSPS KAKENHI Grant Numbers 16J05272,

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15K15007, 15K08595, 26282028. It was also funded in part by MEXT-Supported

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Program for Strategic Research at Private Universities; Keio University Doctorate

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Student Grant-in-Aid Program.

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13. Flynn CA, Alnouti Y, Reed GA. Quantification of the transporter substrate

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15. Burckhardt G. Drug transport by organic anion transporters (OATs). Pharmacol Ther 2012;136(1):106-130.

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20. Yasui-Furukori N, Uno T, Sugawara K, Tateishi T. Different effects of three transporting inhibitors, verapamil, cimetidine, and probenecid, on fexofenadine pharmacokinetics. Clin Pharmacol Ther 2005;77(1):17-23.

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21. Gupta A, Hammarlund-Udenaes M, Chatelain P, Massingham R, Jonsson EN.

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Figure Legends

272 Figure 1. Time-courses of levocetirizine uptake (a) and efflux (b) by

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OAT4-expressing cells. Uptake of levocetirizine (4 µM) (b) and efflux of preloaded

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levocetirizine by tetracycline-treated (circles, OAT4-expressing) and untreated

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(triangles, mock) T-REx OAT4-293 cells were measured in the presence (closed

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symbols) or absence (open symbols) of extracellular Cl- at 37°C. *p<0.05,

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significantly different from levocetirizine transport by mock cells. Each point

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represents the mean ± SEM (n=4, a; n=8-9, b).

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Figure 2. Concentration-dependent uptake of levocetirizine via OAT4. Uptake of

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levocetirizine (1-79 µM) by T-REx OAT4-293 cells was measured at 37°C for 1 min.

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Data were subjected to Michaelis–Menten (main) and Eadie–Scatchard (inset)

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analyses. Each point represents the mean ± SEM (n=4).

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Figure 3. Effects of pilsicainide and olmesartan on levocetirizine uptake via OAT4.

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Uptake of levocetirizine (4 µM) by the cells was measured in the presence or

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absence (control) of pilsicainide (closed circles) or olmesartan (open circles)

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(0.1-100 µM) at 37°C for 30 sec, under Cl--free conditions. *p<0.05, significantly

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different from control. Each point represents the mean ± SEM (n=4).

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Figure 4. (a) Uptake of levocetirizine and cetirizine via OAT4. Uptake of

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levocetirizine (closed circles) and cetirizine (open circles) (5 µM) by T-REx

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OAT4-293 cells was measured at 37°C. *p<0.05, significantly different from the

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uptake of cetirizine. (b) Effects of levocetirizine, cetirizine, and fexofenadine on

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[3H]DHEAS uptake via OAT4. Uptake of [3H]DHEAS (3 nM) by the cells was

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measured in the presence or absence of levocetirizine (closed circles), cetirizine

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(open circles), or fexofenadine (closed triangles) (1-1000 µM) at 37°C for 10 sec.

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*p<0.05, significantly different from [3H]DHEAS uptake in the presence of cetirizine.

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Each point represents the mean ± SEM (n=4-8, a; n=4, b).

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