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  • Here we report the first attempts

    2022-01-15

    Here we report the first attempts to assess roles of NTs and ABC transporters in transfer of ribavirin across the placental barrier using human placental villous fragments and microvillous plasma membrane (MVM) vesicles, in situ dually perfused rat term placenta, and cell-based models.
    Materials and methods
    Results
    Discussion As anti-HCV treatments may be potentially used to reduce rates of vertical HCV transmission [7], [8], it is important to identify safe and effective pharmacotherapy regimens for both pregnant woman and developing foetus. For more than a decade ribavirin has been the backbone of various HCV drug combinations [4], [13], [79]. Nowadays, it is frequently combined with directly acting antivirals against HCV [4] and is essential for treating life-threatening viral infections such as respiratory syncytial virus or influenza virus [10]. Clear signals of ribavirin teratogenicity in animals have been detected [25], but not in humans [15]. Therefore, ribavirin remains a candidate for prevention of HCV vertical infection [14], [21], [22]. Knowledge of ribavirin transplacental kinetics mechanisms is required to assure safe use of ribavirin in prevention of vertical transmission of HCV [23]. Thus, the role of NTs in placental ribavirin pharmacokinetics has been previously investigated using in vitro (BeWo cells) and in vivo (murine model) experimental approaches [26], [27]. However, this study addresses a gap through use of experimental systems derived from human placenta and in situ dually perfused rat term placenta (in addition to in vitro models) to assess roles of NTs and ABC in transfer of ribavirin across the placental barrier. In an initial experimental approach, we applied the well-established and routinely used in vitro model of accumulation into BeWo KT182 [27], [32], [35], [80], [81]. We observed elevated accumulation of [3H]-ribavirin in the presence of NBMPR (Fig. 1a) at 37 °C, suggesting that CNTs may concentrate [3H]-ribavirin in the cells when ENTs are inhibited. This phenomenon has been previously shown in Xenopus leavis oocytes suggesting that CNTs mediate high affinity uptake concentrating ribavirin in cells. It generates concentration gradient for ENTs that mediate opposite action, i.e. outward transfer decreasing cellular ribavirin concentration. Therefore, when ENTs are inhibited, elevated ribavirin uptake can be observed [78]. Moreover, Na+ depletion and uridine (a high affinity substrate of all CNTs) significantly decreased [3H]-ribavirin uptake relative to controls (Fig. 1a), confirming involvement of CNTs [27]. In a previous study we observed Na+-sensitive uptake of adenosine (a CNT2 and CNT3 substrate), but not thymidine (a CNT1 and CNT3 substrate) concluding that the only CNT sub-group functionally expressed in BeWo cells is CNT2 [32]. Moreover, based on mRNA quantification, our clone of BeWo expresses SLC28A2 more strongly than SLC28A3 (respectively encoding CNT2 and CNT3), while no SLC28A1 transcript (encoding CNT1) was detected [81]. Therefore, we suggest that ribavirin is a substrate of CNT2, which mediates its uptake into BeWo cells. To avoid masking effects of CNT2 on [3H]-ribavirin uptake in NBMPR-treated cells we performed experiments at 4 °C to inhibit ATP-dependent cellular processes (Fig. 1b). Low temperature (4 °C) is a condition commonly used to indirectly assess involvement of active membrane transport in drug kinetics as shown in Caco-2 cell line [82], [83]. Moreover, Archer et al. have described that temperature below 15 °C abolishes the activity of CNTs in rat microvascular endothelial cells while ENTs preserve the sensitivity to NBMPR and dipyridamole [84]. Jarvis et al. have shown in guinea pig erythrocytes that CNTs-controlled transport was 70-fold higher at 37 °C than at 10 °C while ENTs-mediated transport was less temperature-dependent [85]. Moreover, using vesicles prepared from human erythrocytes it has been recently evidenced that uridine Km is comparable at ice-cold temperature and 23 °C while Vmax was decreased by only 23% suggesting functional state of ENTs at very low temperatures [86]. In line with this finding, ENT1-mediated ribavirin uptake at ice-cold temperature was confirmed in this experimental system [86]. Under these conditions, we found that NBMPR had similar effects at both concentrations, confirming that ENTs, particularly ENT1 probably as it is most strongly expressed in the placenta [32], also contribute to [3H]-ribavirin uptake into BeWo cells. The suggested ENT1- and CNT2-mediated transport of [3H]-ribavirin is consistent with results of previous experiments with BeWo cells [27] and non-placental models, e.g. OR6 cells, HHL-5 cells, human hepatocytes, and mice [26], [32], [44], [48], [49], [50], [51], [87]. Analysis of the concentration-dependence of ribavirin accumulation in BeWo cells (Fig. 2), indicated that CNTs were largely responsible for its accumulation in the presence of 100 µM NBMPR at ribavirin concentrations <1 µM as previously found in experiments with Xenopus oocytes expressing human NTs [78]. However, higher concentrations resulted in saturation and thus abolishment of NTs’ effects (Fig. 2).