Equilibrative Nucleoside Transporter 1 (ENT1, SLC29A1) Facilitates Transfer of the Antiretroviral Drug Abacavir across the Placenta

Abstract

Abacavir is a preferred antiretroviral drug for preventing mother-to-child human immunodeficiency virus transmission; however, mechanisms of its placental transfer have not been satisfactorily described to date. Because abacavir is a nucleoside-derived drug, we hypothesized that the nucleoside transporters, equilibrative nucleoside transporters (ENTs, ) and/or Na-dependent concentrative nucleoside transporters (CNTs, ), may play a role in its passage across the placenta. To test this hypothesis, we performed uptake experiments using the choriocarcinoma-derived BeWo cell line, human fresh villous fragments, and microvillous plasma membrane (MVM) vesicles. Using endogenous substrates of nucleoside transporters, [H]-adenosine (ENTs, CNT2, and CNT3) and [H]-thymidine (ENTs, CNT1, and CNT3), we showed significant activity of ENT1 and CNT2 in BeWo cells, whereas experiments in the villous fragments and MVM vesicles, representing a model of the apical membrane of a syncytiotrophoblast, revealed only ENT1 activity. When testing [H]-abacavir uptakes, we showed that of the nucleoside transporters, ENT1 plays the dominant role in abacavir uptake into placental tissues, whereas contribution of Na-dependent transport, most likely mediated by CNTs, was observed only in BeWo cells. Subsequent experiments with dually perfused rat term placentas showed that Ent1 contributes significantly to overall [H]-abacavir placental transport. Finally, we quantified the expression of in first- and third-trimester placentas, revealing that is the dominant isoform. Neither nor expression changed over the course of placental development, but there was considerable interindividual variability in their expression. Therefore, drug-drug interactions and the effect of interindividual variability in placental ENT1 expression on abacavir disposition into fetal circulation should be further investigated to guarantee safe and effective abacavir-based combination therapies in pregnancy.

Multiple Drug Transporters Contribute to the Placental Transfer of Emtricitabine

“…ENTs are widely expressed in various tissues, and ENTs contribute to the transport of endogenous nucleosides and various anticancer and antiviral nucleoside drugs, such as adenosine, gemcitabine, ribavirin, and abacavir (27)(28)(29)(30). Our data revealed that NBTI and dipyridamole (two ENT-specific inhibitors) strongly inhibited FTC uptake in BeWo cells and PHTCs and that NBTI inhibited FTC accumulation in JEG-3 cells (see Fig.…”

Quantification of ENT1 and ENT2 Proteins at the Placental Barrier and Contribution of These Transporters to Ribavirin Uptake

“…13 ENT1 protein was found to be the predominant nucleoside transporter in placental MVMs (Table 1), as has already been confirmed at the functional and expressional levels. [8][9][10][11][12][13] The expression of ENT2 protein at human placental MVMs was under the limit of quantification according to our quantification analysis criteria, but this does not directly mean the absence of ENT2 protein. In fact, a positive peak was detected for 3 of 4 donors when only the most sensitive SRM transition for human ENT2 was considered, with a mean value of 0.74 fmol/mg protein (Supplemental Table S2).…”

“…11 In the rat placental barrier, the functional expression of ENT2 in placental MVM vesicles was suggested by the observation that adenosine uptake is more potently inhibited by NBMPR at 100 mM than at 0.1 mM, 10 which is inconsistent with the observation in human placental MVMs. 12 The involvement of CNTs in the uptake of the nucleoside was undetected neither in rat placental MVM vesicles. 10 Absolute quantification of nucleoside transporter proteins in placental MVMs using liquid chromatography-tandem mass spectrometry (LC-MS/ MS) should enable us to resolve these discrepancies and estimate the transport capacities of individual transporters at the placental MVMs based on the absolute protein expression levels.…”

“…6,7 Among these transporters, ENT1, encoded by SLC29A1, is expressed at the microvillous membrane (MVM) of the syncytiotrophoblast layer in humans, mice, and rats 8-including ribavirin and abacavir. [12][13][14] The fetal distribution of ribavirin was significantly reduced in Ent1-knockout mice at 15 min after administration but no significant difference was seen at 2 h. 13 This may indicate that ENT1 affects the rate, but not the extent, of ribavirin distribution into the fetus, and thus may imply that other transporter proteins contribute to the fetal transfer of ribavirin. Candidate ribavirin transporters include human ENT1, ENT2 (encoded by SLC29A2), CNT2 (encoded by SLC28A2), and CNT3 (encoded by SLC28A3), which were shown to transport ribavirin when expressed in Xenopus oocytes.…”

“…8,11 On the other hand, in fresh villous fragments and MVM vesicles of human placenta, nitrobenzylthioinosine (NBMPR) at 0.1 and 100 mM both inhibited the uptake of nucleosides to the same extent, suggesting negligible uptake via ENT2, 12 because nanomolar NBMPR almost completely inhibits ENT1 but has little effect on ENT2, whereas 100 mM NBMPR inhibits ENT2 as well as ENT1. 17 As for CNTs, negligible involvement was suggested based on the Na þ -insensitivity of uptake of nucleosides in human placental MVM vesicles 12,18 and the lack of immunohistochemical detection in CNT proteins at the syncytiotrophoblast layer, 8 but this is in conflict with immunohistochemical detection of CNT1 protein in another study. 11 In the rat placental barrier, the functional expression of ENT2 in placental MVM vesicles was suggested by the observation that adenosine uptake is more potently inhibited by NBMPR at 100 mM than at 0.1 mM, 10 which is inconsistent with the observation in human placental MVMs.…”