S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is Not a Selective Inhibitor of Equilibrative Nucleoside Transporters but Also Blocks Efflux Activity of Breast Cancer Resistance Protein.

PURPOSE: S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) is routinely used at concentrations of 0.10 µM and 0.10 mM to specifically inhibit transport of nucleosides mediated by equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2), respectively. We recently showed that NBMPR (0.10 mM) might also inhibit placental active efflux of [3H]zidovudine and [3H]tenofovir disoproxil fumarate. Here we test the hypothesis that NBMPR abolishes the activity of P-glycoprotein (ABCB1) and/or breast cancer resistance protein (ABCG2). METHODS: We performed accumulation assays with Hoechst 33342 (a model dual substrate of ABCB1 and ABCG2) and bi-directional transport studies with the ABCG2 substrate [3H]glyburide in transduced MDCKII cells, accumulation studies in choriocarcinoma-derived BeWo cells, and in situ dual perfusions of rat term placenta with glyburide. RESULTS: NBMPR inhibited Hoechst 33342 accumulation in MDCKII-ABCG2 cells (IC50 = 53 µM) but not in MDCKII-ABCB1 and MDCKII-parental cells. NBMPR (0.10 mM) also inhibited bi-directional [3H]glyburide transport across monolayers of MDCKII-ABCG2 cells and blocked ABCG2-mediated [3H]glyburide efflux by rat term placenta in situ. CONCLUSION: NBMPR at a concentration of 0.10 mM abolishes ABCG2 activity. Researchers using NBMPR to evaluate the effect of ENTs on pharmacokinetics must therefore interpret their results carefully if studying compounds that are substrates of both ENTs and ABCG2.

Interactions between Maraviroc and the ABCB1, ABCG2, and ABCC2 Transporters: An Important Role in Transplacental Pharmacokinetics.

Maraviroc is a chemokine receptor 5 (CCR5) inhibitor used in the treatment of human immunodeficiency virus (HIV) that also shows therapeutic potential for several autoimmune, cancer, and inflammatory diseases that can afflict pregnant women. However, only limited information exists on the mechanisms underlying the transplacental transfer of the drug. We aimed to expand the current knowledge base on how maraviroc interacts with several placental ATP-binding cassette (ABC) efflux transporters that have a recognized role in the protection of a developing fetus: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance protein 2 (ABCC2). We found that maraviroc does not inhibit any of the three studied ABC transporters and that its permeability is not affected by ABCG2 or ABCC2. However, our in vitro results revealed that maraviroc shows affinity for human ABCB1 and the endogenous canine P-glycoprotein (Abcb1) expressed in Madin-Darby canine kidney II (MDCKII) cells. Perfusion of rat term placenta showed accelerated transport of maraviroc in the fetal-to-maternal direction, which suggests that ABCB1/Abcb1 facilitates in situ maraviroc transport. This transplacental transport was saturable and significantly diminished after the addition of the ABCB1/Abcb1 inhibitors elacridar, zosuquidar, and ritonavir. Our results indicate that neither ABCG2 nor ABCC2 influence maraviroc pharmacokinetic but that ABCB1/Abcb1 may be partly responsible for the decreased transplacental permeability of maraviroc to the fetus. The strong affinity of maraviroc to Abcb1 found in our animal models necessitates studies in human tissue so that maraviroc pharmacokinetics in pregnant women can be fully understood. SIGNIFICANCE STATEMENT: Antiretroviral drug maraviroc shows low toxicity and is thus a good candidate for prevention of mother-to-child transmission of human immunodeficiency virus when failure of recommended therapy occurs. Using in vitro cell-based experiments and in situ dually perfused rat term placenta, we examined maraviroc interaction with the placental ABC drug transporters ABCB1, ABCG2, and ABCC2. We demonstrate for the first time that placental ABCB1 significantly reduces mother-to-fetus transport of maraviroc, which suggests that ABCB1 may be responsible for the low cord-blood/maternal-blood ratio observed in humans.

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

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, SLC29A) and/or Na+-dependent concentrative nucleoside transporters (CNTs, SLC28A), 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, [3H]-adenosine (ENTs, CNT2, and CNT3) and [3H]-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 [3H]-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 [3H]-abacavir placental transport. Finally, we quantified the expression of SLC29A in first- and third-trimester placentas, revealing that SLC29A1 is the dominant isoform. Neither SLC29A1 nor SLC29A2 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.

Interactions of protease inhibitors atazanavir and ritonavir with ABCB1, ABCG2, and ABCC2 transporters: Effect on transplacental disposition in rats.

Atazanavir and ritonavir are preferred protease inhibitors frequently used in combination antiretroviral therapy for prevention of HIV mother-to-child transmission. Although their use is associated with higher risk of congenital anomalies, factors affecting atazanavir and ritonavir placental transfer are not known. This study is the first attempt to evaluate whether the placental drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and/or multidrug resistance-associated proteins 2 (ABCC2), affect placental pharmacokinetics of atazanavir or ritonavir. Transport experiments across MDCKII cells expressing respective human ABC carrier showed that atazanavir is a substrate of ABCB1 and dual perfusion studies in a rat placenta confirmed this finding. In conclusion, we suggest that placental ABCB1 might reduce ATV maternal-to-fetal transfer and therefore represent a site for pharmacokinetic drug-drug interactions of ATV. Further studies in human placenta models are necessary to provide additional data closer to clinical environment.

Expression of Concentrative Nucleoside Transporters ( SLC28A) in the Human Placenta: Effects of Gestation Age and Prototype Differentiation-Affecting Agents.

Equilibrative ( SLC29A) and concentrative ( SLC28A) nucleoside transporters contribute to proper placental development and mediate uptake of nucleosides/nucleoside-derived drugs. We analyzed placental expression of SLC28A mRNA during gestation. Moreover, we studied in choriocarcinoma-derived BeWo cells whether SLC29A and SLC28A mRNA levels can be modulated by activity of adenylyl cyclase, retinoic acid receptor activation, CpG islands methylation, or histone acetylation, using forskolin, all- trans-retinoic acid, 5-azacytidine, and sodium butyrate/sodium valproate, respectively. We found that expression of SLC28A1, SLC28A2, and SLC28A3 increases during gestation and reveals considerable interindividual variability. SLC28A2 was shown to be a dominant subtype in the first-trimester and term human placenta, while SLC28A1 exhibited negligible expression in the term placenta only. In BeWo cells, we detected mRNA of SLC28A2 and SLC28A3. Levels of the latter were affected by 5-azacytidine and all- trans-retinoic acid, while the former was modulated by sodium valproate (but not sodium butyrate), all- trans-retinoic acid, 5-azacytidine, and forskolin that caused 25-fold increase in SLC28A2 mRNA; we documented by analysis of syncytin-1 that the observed changes in SLC28A expression do not correlate with the morphological differentiation state of BeWo cells. Upregulated SLC28A2 mRNA was reflected in elevated uptake of [3H]-adenosine, high-affinity substrate of concentrative nucleoside transporter 2. Using KT-5720 and inhibitors of phosphodiesterases, we subsequently confirmed importance of cAMP/protein kinase A pathway in SLC28A2 regulation. On the other hand, SLC29A genes exhibited constitutive expression and none of the tested compounds increased SLC28A1 expression to detectable levels. In conclusion, we provide the first evidence that methylation status and activation of retinoic acid receptor affect placental SLC28A2 and SLC28A3 transcription and substrates of concentrative nucleoside transporter 2 might be taken up in higher extent in placentas with overactivated cAMP/protein kinase A pathway and likely in the term placenta.

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption.

Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir.

Role of ABC and Solute Carrier Transporters in the Placental Transport of Lamivudine.

Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport.