An estimated 1.5 million pregnant women were living with HIV in 2011 . According to the WHO, all infected women, regardless of their clinical stage, should receive combination antiretroviral treatment throughout gestation to prevent HIV mother-to-child transmission (MTCT) . Together with other evidence-based interventions, antiretroviral pharmacotherapy in pregnancy reduces the percentage of HIV-positive infants from 20–45% to 1–2% .
Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, represents a backbone of combination anti-HIV therapy . To improve its pharmacokinetic properties, TFV is administered in the form of disoproxil fumarate. Despite being classified as pregnancy category B drug, WHO has incorporated tenofovir disoproxil fumarate (TDF) into recent guidelines for prophylaxis of HIV MTCT  and its use in pregnancy tends to increase [5,6]. It must be stressed out that medication of pregnant women requires special attention to guarantee adequate and well tolerated therapy throughout gestation; among others, changes in pharmacokinetics during pregnancy  and quantification of drug transport across the placenta  need to be taken into account.
Several members of ATP-binding cassette (ABC) drug efflux transporters have been localized in the apical, maternal-facing membrane of the placenta where they pump their substrates from the trophoblast cells back to the maternal circulation, thus limiting permeation of substrate drugs from mother to fetus . To date, the best-described ABC transporters in the placenta are: P-glycoprotein (ABCB1/MDR1) , Breast Cancer Resistance Protein (ABCG2/BCRP)  and Multidrug Resistance-Associated Protein 2 (ABCC2/MRP2) . It has been well documented that these transporters affect transplacental passage of many clinically used compounds, including antiretrovirals . Therefore, detailed knowledge on drug interactions with placental ABC transporters is required to complete their safety profile and to guarantee adequate and well tolerated medication of pregnant woman and her child . Interactions of TFV and TDF with ABCB1 and ABCC2 have been investigated in several studies, however, providing inconsistent results depending on the method used [14–20]. To our knowledge, data clearly describing ABCG2-mediated transport of TFV or TDF is still lacking. In addition, influence of ABC transporters on transplacental passage of these compounds has not been evaluated so far.
In the present study, we employed the in-vitro model of MDCKII cells transduced with human ABC transporters to investigate whether TFV and/or TDF are substrates of human ABCB1, ABCG2 or ABCC2. Furthermore, using the model of in-situ perfused rat placenta we aimed to elucidate potential effect of these transporters on TFV/TDF passage from mother to fetus.
Reagents and chemicals
Tenofovir [TFV, (1R)-9-(2-Phosphonylmethoxypropyl)-adenine)] and tenofovir disoproxil fumarate [TDF; bis(isopropyloxycarbonyloxymethyl)9-(2-Phosphonylmethoxypropyl)-adenine] were kindly provided by Gilead Sciences, Inc. (Foster City, California, USA). Radiolabeled [adenine-2,8–3H]tenofovir ([3H]TFV) and [adenine-8–3H] tenofovir disoproxil fumarate ([3H]TDF) were purchased from Moravek Biochemicals (California, USA). Dual ABCG2 and ABCB1 inhibitor, GF120918, was kindly provided by GlaxoSmithKline (Greenford, UK). Indomethacin, nonselective inhibitor of ABCC(s), was purchased from Sigma–Aldrich (St. Louis, Missouri, USA). Pentobarbital (Nembutal) was purchased from Abbott Laboratories (Abbott Park, Illinois, USA). All other chemicals were of analytical grade.
MDCKII (Madine-Darby Canine Kidney) parental cell line and MDCKII cells stably transduced with cDNA of human MDR1 (MDCKII-ABCB1), BCRP (MDCKII-ABCG2) or MRP2 (MDCKII-ABCC2) were obtained from Netherlands Cancer Institute (Dr A. Schinkel) and cultured in DMEM complete high-glucose medium with L-glutamine, supplemented with 10% fetal bovine serum.
Pregnant Wistar rats were purchased from Biotest Ltd. (Konarovice, Czech Republic) and were maintained in 12/12-h day/night standard conditions with water and pellets ad libitum. Experiments were performed on day 21 of gestation. Fasted rats were anesthetized with pentobarbital (Nembutal; Abbott Laboratories) in a dose of 40 mg/kg administered into the tail vein. All animal experiments were approved by the Ethical Committee of the Faculty of Pharmacy in Hradec Kralove (Charles University in Prague, Czech Republic) and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (1996) and the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes.
Transport experiments in vitro
Transport assays were performed on microporous polycarbonate membrane filters (3.0 μm pore size, 24 mm diameter; Transwell 3414; Costar, Corning, New York, New York, USA) as described previously . MDCKII-parental and MDCKII cells expressing ABCB1, ABCG2 or ABCC2 were seeded at a density of 1.0 × 106 cells per well and cultured for 72 h to confluence, including daily medium replacement. One hour before starting the experiment, cells were washed with phosphate-buffered saline (37°C) and incubated with 2 ml of Opti-MEM (Invitrogen, Carslbad, California, USA) either alone or containing the dual ABCB1 and ABCG2 inhibitor GF120918 (2 μmol/l). The experiment was started by replacing the medium in the donor compartment (either apical or basolateral) with fresh Opti-MEM (37°C) containing the tested compound, TFV or TDF, or combination of TDF with GF120918 (2 μmol/l). Both TFV and TDF were traced by [3H]TFV and [3H]TDF, respectively, achieving the final activity of 0.04 μCi/ml. The lowest point of the concentration range (3.1 nmol/l for TFV and 33.3 nmol/l for TDF) was determined by the specific activity of radioisotopes required for analysis. Aliquots of 50 μl were collected at 2, 4 and 6 h from the acceptor compartment and radioactivity was measured by liquid scintillation counting (Tri-Carb 2900 TR Perkin Elmer). At the end of the experiment leakage of FITC-dextran was analysed and was accepted up to 1% per hour. The percentage of radioactivity appearing in the acceptor compartment relative to stock solution initially added to the donor compartment was calculated. Ratios of basal-to-apical to apical-to-basal translocation after 6-h incubation (r) were calculated as described elsewhere [21,22].
Dual perfusion of the rat placenta in situ
The method of dually perfused rat term placenta was used as described previously . In brief, one uterine horn was excised and submerged in heated Ringer's saline. A catheter was inserted into the uterine artery proximal to the blood vessel supplying a selected placenta and connected with the peristaltic pump. Kreb's perfusion liquid containing 1% dextran was brought from the maternal reservoir at a rate 1 ml/min. The uterine vein, including anastomoses to other fetuses, was ligated behind the perfused placenta and cut so that maternal solution could leave the perfused placenta. The selected fetus was separated from the neighbouring ones by ligatures. The umbilical artery was catheterized by use of a 24-gauge catheter connected to the fetal reservoir and perfused at a rate of 0.5 ml/min. The umbilical vein was catheterized in a similar manner, and the selected fetus was removed. Before the start of each experiment, the fetal vein effluent was collected into preweighed glass vial to check for a possible leakage of perfusion solution from the placenta. In the case of leakage, the experiment was terminated. Maternal and fetal perfusion pressures were maintained at levels close to physiological values and monitored continuously throughout the perfusion experiments as described previously. At the end of experiment, placenta was perfused with radioactivity-free buffer for 10 min, excised from the uterine tissue, and dissolved in tissue solubilizer (Solvable; PerkinElmer Life and Analytical Sciences), and its radioactivity was measured to detect tissue-bound TFV or TDF.
Two types of perfusion systems were used in this study:
Open-circuit perfusion system was employed to study fetal-to-maternal (F > M) and maternal-to-fetal (M > F) clearances of TFV or TDF at various concentrations. TFV (50 nmol/l or 500 μmol/l) and TDF (50 nmol/l, 100 or 500 μmol/l) was added to either maternal (M>F studies) or fetal (F>M studies) reservoir immediately after successful surgery. After 5-min stabilization period the sample collection started (time 0). Fetal effluent was sampled into preweighed vials in 5 min interval, concentrations were measured radiometrically and transplacental clearance was calculated from all measured intervals as described below.
Closed-circuit (recirculation) perfusion system was employed to identify placental transporter(s) responsible for active transport of TDF from the fetal circulation. Both maternal and fetal sides of the placenta were infused with either nonsaturating (50 nmol/l) or saturating (500 μmol/l) concentrations of [3H]TDF and after short-time stabilization period, the fetal perfusate (10 ml) was recirculated for 60 min. Samples (250 μl) were collected every 10 min from the maternal and fetal reservoirs, and concentrations of [3H]TDF were measured. This experimental setup ensures steady concentration on the maternal side of the placenta and enables investigation of fetal/maternal ratio; any net transfer of the substrate implies transport against a concentration gradient and provides evidence of active transport. To determine the effect of efflux transporters on placental passage of TDF, GF120918 (2 μmol/l), a dual inhibitor of ABCB1 and ABCG2 , or indomethacin (0.28 mmol/l), a nonspecific inhibitor of ABCCs , were added to both maternal and fetal reservoirs and the fetal/maternal concentration ratio at equilibrium was calculated.
Pharmacokinetic analysis of efflux transport activity in the placenta
Organ clearance concept was applied to quantify M > F and F > M transport of TFV and TDF in open-circuit perfusion system . Averaged data from the intervals of 10–35 min were used for the following calculations. M > F transplacental clearance (Cl mf) normalized to placenta weight was calculated according to Eq. (1).
where C fv is drug concentration in the umbilical vein effluent, Q f is the umbilical flow rate, C ma is concentration in the maternal reservoir, and W p is the wet weight of the placenta. F > M transplacental clearance (Cl fm) was calculated according to Eq. (2).
where C fa is drug concentration in the fetal reservoir entering the perfused placenta via the umbilical artery.
In in-vitro cell-based studies, two-side unpaired Student's t-test was employed to compare apical-to-basal translocation and basal-to-apical-translocation at time point 6 h. In in-situ placenta perfusion studies, statistical significance was examined by two-side unpaired Student's t-test or one-way ANOVA followed by Bonferroni's test. All data were assessed using GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego, California, USA).
TFV transport across MDCKII parental and ABCB1, ABCG2, ABCC2-overexpressing cells
We first determined transepithelial transport of [3H]TFV at the concentration of 3.1 nmol/l through the monolayers of parental and ABCB1, ABCG2 or ABCC2-overexpressing cells. No asymmetry in basal-to-apical vs. apical-to-basal transport of TFV was observed in MDCKII parental cells (r = 0.90). Transports across MDCKII-ABCB1, ABCG2 and ABCC2 monolayers were equivalent to that in parental cell line (r = 0.89, 0.79 and 0.89, respectively) (Table 1). These findings show that TFV is not a substrate of any of the transporters investigated.
TDF transport across MDCKII parental and ABCB1, ABCG2, ABCC2-overexpressing cells
Transepithelial transport of [3H]TDF through MDCKII parental and ABCB1, ABCG2, ABCC2-overexpressing cells was measured at a concentration of 33.3 nmol/l. In the parental cells, basal-to-apical/apical-to-basal transport ratio (r) of 2.38 was observed likely resulting from activity of endogenous canine transporters as reported previously . Compared with MDCKII parental cells, significantly larger basal-to-apical/apical-to-basal ratios were observed in ABCB1 and ABCG2-overexpressing cells (r = 5.47 and 6.24, respectively) but not in ABCC2 cells (r = 3.06). Increase in TDF concentration (10 μmol/l) significantly reduced this ratio in both ABCB1 and ABCG2 cells (r = 4.56 and r = 4.78, respectively) indicating partial saturation of both transporters. Furthermore, addition of a dual ABCB1 and ABCG2 inhibitor, GF120918 (2 μmol/l), completely abolished the asymmetry in translocation of TDF in respective cell lines at both concentrations tested (33.3 and 10 μmol/l) reaching transport ratio values of approximately 1. These findings demonstrate that TDF is a substrate of human ABCB1 and ABCG2 but not of ABCC2 (Table 2).
Open circuit perfusion experiments: effect of inflow concentrations on transplacental clearance of TFV and TDF
The maternal or fetal side of the placenta was infused with various concentration of TFV (50 nmol/l or 500 μmol/l) or TDF (50 nmol/l, 100 or 500 μmol/l). No statistically significant differences between M>F and F>M clearances were observed at either low or high concentration of TFV, suggesting linear mechanism in transplacental transport of this compound. On the contrary, increase in TDF concentration resulted in significant changes in transplacental clearances in both M>F and F>M directions, confirming involvement of a capacity-limited transport mechanisms (Fig. 1). Less than 5% of TFV or TDF dose was detected in the placenta after perfusion experiments, suggesting limited tissue binding and negligible effect on clearance calculation.
Closed circuit perfusion experiments: effect of concentration and inhibitors on TDF transport across the placenta
To identify placental transporter(s) responsible for elimination of TDF from the fetal circulation, both sides of placenta were perfused with low nonsaturating concentration of TDF in closed circuit experimental setup in either absence or presence of inhibitors. In the absence of inhibitors, we observed significant decrease in TDF concentration in fetal perfusate, confirming active transport of this compound from fetal to maternal side of the placenta against concentration gradient. This decline was fully blocked by coinfusion with 500 μmol/l TDF confirming saturable transport. Furthermore, transport of TDF from fetus to mother was significantly blocked by GF120908 (2 μmol/l), whereas co-administration of indomethacin (0.28 mmol/l) did not show any effect (Fig. 2).
Although TFV is categorized as a pregnancy B drug by FDA, it is frequently used in the treatment of pregnant women with HIV infection. Nevertheless, transport of this compound, and its prodrug TDF, across the placenta from mother to fetus and the role of placental ABC drug efflux transporters in this event have not been systematically investigated to date. In this study we employ both in-vitro and in-situ experimental approaches to characterize interactions of TFV and TDF with the best-described ABC transporters localized in the placenta, that is ABCB1, ABCG2 and ABCC2, and to quantify their role in the transplacental pharmacokinetics of both compounds.
Using in-vitro transport experiments in MDCKII cells overexpressing human ABCB1, ABCG2 or ABCC2 we did not record any asymmetry in transepithelial translocation of TFV, indicating this compound is not a substrate of any of these transporters (Table 1). This correlates well with previously published data by Ray et al.  who used identical experimental model to exclude interactions between ABCB1 and TFV. We also support recent findings by Cihlar et al.  and Rodriguez-Novoa et al.  who suggested no role of ABCC2 in TFV pharmacokinetics. In addition, we provide the first evidence that ABCG2 does not mediate TFV transport.
TDF, on the contrary, showed strikingly different behaviour in comparison with TFV. When investigating its transport across ABCB1-transduced cells in basal-to-apical vs. apical-to-basal direction, TDF achieved transport ratio (r) 2–2.6 times higher than that observed in the parental cell line. Furthermore, application of GF120918 (2 μmol/l) abolished this asymmetry, resulting in translocation of TDF across the cell monolayer by mechanism of passive diffusion (r ≈ 1). These findings clearly confirm TDF as a substrate of ABCB1 transporter as suggested recently [17,18]. Based on our findings, TDF seems to be a substrate as strong as colchicine, model substrate often used for in-vitro assays , topotecan  or other antiretrovirals including zidovudine , abacavir  or lopinavir .
Similarly, in-vitro studies in ABCG2-transduced MDCKII cells revealed that TDF is transported by ABCG2. To our knowledge we provide the first evidence that pharmacokinetics of TDF can be affected by ABCG2 transporter. Conversely, Janneh et al. have recently reported  that application of dipyridamole, a nonspecific inhibitor of ABCB1 and ABCG2, resulted in insignificant increase in TDF accumulation within peripheral mononuclear cells suggesting that TDF is not a substrate of ABCG2. However, since their method failed to reveal transport of TDF by ABCB1 either, we speculate that low sensitivity of the experimental approach prevented disclosure of interaction between TDF and both ABC transporters in their study.
To confirm our in-vitro findings on the organ level, we employed the method of dually perfused rat term placenta, a well established method to study placental pharmacology [23,24,32,33] and physiology [34,35]. As both rat and human placenta express abundant amounts of ABCB1  and ABCG2 , we introduced this model as a suitable tool to investigate the role of placental ABCB1  and ABCG2  in placental disposition of drugs. In open-circuit perfusion setup, clearances of TFV in both fetal-to-maternal and maternal-to-fetal directions were comparable and independent of drug concentration. These findings indicate linear pharmacokinetics without involvement of transporter-mediated mechanism(s) (Fig. 1) and correspond well with our results obtained in vitro. Furthermore, low values of TFV transplacental clearances suggest its restricted passage across the placenta when compared with antipyrine, a marker of passive diffusion . This is in accordance with observations by Nirogi et al.  who found TFV in the placenta but not in amniotic fluid and fetal tissues when administered as a single fixed dose of efavirenz-emtricitabine-TDF in rats. Poor TFV transport was also observed in other biological membranes such as the intestine , blood–brain barrier  or blood–cerebrospinal barrier  most likely due to physical–chemical properties of the molecule, that is its anionic charge at physiological pH and low lipid-solubility of the nonionized fraction. Therefore, it is reasonable to assume that this molecule cannot readily cross cell membranes by passive diffusion; however, involvement of other transporters, such as equilibrative nucleoside transporters and/or concentrative nucleoside transporters, in TFV pharmacokinetics cannot be excluded.
Once absorbed, TDF is cleaved by nonspecific esterases, thus occurring in the systemic circulation predominantly as TFV . However, esterase-mediated degradation of TDF can be inhibited by concomitantly administered treatment or substances normally present in nutrition such as fruit esters [17,18]. It can be hypothesized that TDF, at least to some extent, can also circulate in the maternal blood and reach the placental barrier; therefore, in this study we investigated placental transfer of TDF as well. In contrast to TFV, we observed great asymmetry in TDF transplacental clearances between mother to fetus and fetus to mother. In detail, transport from fetus to mother was 11.7 times faster than that in the opposite direction. In addition, placental transport of this drug was concentration-dependent in both directions indicating involvement of a saturable transport mechanism. Furthermore, employing closed-circuit perfusion setup with low, nonsaturating TDF concentrations, we observed transport of the drug from fetal to maternal circulation against concentration gradient confirming active transport mechanism. This transport was significantly reduced by administration of GF120908, a common ABCG2 and ABCB1 inhibitor, confirming ability of these transporters to mediate passage of TDF from fetus to mother (Fig. 2). Taking these results together with findings from in-vitro transport experiments in MDCKII-transduced cells, we conclude that both ABCB1 and ABCG2 affect TDF transplacental kinetics. On the contrary, indomethacin, a nonspecific inhibitor of ABCC(s), did not affect TDF transplacental transport, indicating that ABCC2 does not modulate transplacental passage of TDF. As drug–drug interactions on ABC transporters may substantially affect the fate of drugs in organism, our findings should be taken into account when TDF is co-administered with compounds whose membrane transport is mediated by ABCB1 and/or ABCG2 such as antiretrovirals  or other drugs administered in pregnancy . Differences in ABCB1 expression between HIV-infected and noninfected women  as well as genetic polymorphisms leading to altered expression and function of the protein in the placenta  should also be considered when substrate drugs are prescribed.
In summary, our cell-based results suggest that TFV does not interact with human ABCB1, ABCG2 or ABCC2 transporters, whereas TDF was shown to be a dual substrate of both ABCB1 and ABCG2 but not of ABCC2. Our in-situ experiments in perfused rat placenta confirmed these findings, suggesting that ABCB1 and ABCG2, but not ABCC2, play an important role in efflux of TDF from fetus to mother. We propose limited mother-to-fetus transport of both TFV and TDF. While placental transport of TFV is restricted passively, by physical–chemical properties of the molecule, limited mother-to-fetus passage of TDF is actively mediated by placental ABCB1 and ABCG2 transporters, pumping this compound from trophoblast back to maternal circulation.
Z.N. performed in-situ placenta perfusion experiments, analysed the data and participated in writing the manuscript; L.C. designed and performed in-vitro cell-based experiments, analysed the data and participated in writing the manuscript; M.C. cultivated the cells, designed and performed in-vitro cell-based experiments and critically revised the article; F.S. designed and supervised the experiments, analysed the data and participated in writing the article.
This research was financially supported by Czech Science Foundation (GACR P303/120850) and Grant Agency of Charles University (GAUK 695912/C/2012 and SVV/2013/267-003). We thank Gilead Sciences, Inc. (333 Lakeside Drive Foster City, California 94404, USA) for providing TFV and TDF. We also wish to thank Dana Souckova and Renata Exnarova for skilful assistance with the perfusion experiments.
Conflicts of interest
There are no conflicts of interest.
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