The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.
- Klíčová slova
- ATP-binding cassette transporters, Concentrative nucleoside transporters, Entecavir, Equilibrative nucleoside transporters, Placental transfer,
- MeSH
- ABC transportér z rodiny G, člen 2 metabolismus MeSH
- dítě MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- membránové transportní proteiny metabolismus MeSH
- nádorové proteiny metabolismus MeSH
- nádory prsu * metabolismus MeSH
- nukleosidy metabolismus farmakologie MeSH
- P-glykoprotein metabolismus MeSH
- P-glykoproteiny metabolismus MeSH
- placenta * metabolismus MeSH
- potkani Wistar MeSH
- protein spojený s mnohočetnou rezistencí k lékům 2 MeSH
- proteiny přenášející nukleosidy metabolismus farmakologie MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům metabolismus MeSH
- těhotenství MeSH
- uridin MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 4-nitrobenzylthioinosine MeSH Prohlížeč
- ABC transportér z rodiny G, člen 2 MeSH
- ABCB1 protein, human MeSH Prohlížeč
- ABCC2 protein, human MeSH Prohlížeč
- ABCG2 protein, human MeSH Prohlížeč
- entecavir MeSH Prohlížeč
- membránové transportní proteiny MeSH
- nádorové proteiny MeSH
- nukleosidy MeSH
- P-glykoprotein MeSH
- P-glykoproteiny MeSH
- protein spojený s mnohočetnou rezistencí k lékům 2 MeSH
- proteiny přenášející nukleosidy MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům MeSH
- uridin MeSH
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.
- Klíčová slova
- NBMPR, breast cancer resistance protein, equilibrative nucleoside transporters, inhibition, selectivity,
- MeSH
- ABC transportér z rodiny G, člen 2 antagonisté a inhibitory metabolismus MeSH
- antivirové látky metabolismus farmakokinetika MeSH
- biologický transport účinky léků MeSH
- buněčné linie MeSH
- buňky MDCK MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- nádorové proteiny antagonisté a inhibitory metabolismus MeSH
- P-glykoproteiny antagonisté a inhibitory metabolismus MeSH
- placenta účinky léků metabolismus MeSH
- potkani Wistar MeSH
- psi MeSH
- těhotenství MeSH
- thioinosin analogy a deriváty farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- psi MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 4-nitrobenzylthioinosine MeSH Prohlížeč
- ABC transportér z rodiny G, člen 2 MeSH
- ABCB1 protein, human MeSH Prohlížeč
- ABCG2 protein, human MeSH Prohlížeč
- antivirové látky MeSH
- nádorové proteiny MeSH
- P-glykoproteiny MeSH
- thioinosin MeSH
Evidence on equilibrative nucleoside transporter 1 (ENT1) and microRNA-21 (miR‑21) is not yet sufficiently convincing to consider them as prognostic biomarkers for patients with pancreatic ductal adenocarcinoma (PDAC). Here, we investigated the prognostic value of ENT1/ENT1, miR-21, and neurogenic locus homolog protein 3 gene (NOTCH3) in a well-defined cohort of resected patients treated with adjuvant gemcitabine chemotherapy (n = 69). Using a combination of gene expression quantification in microdissected tissue, immunohistochemistry, and univariate/multivariate statistical analyses we did not confirm association of ENT1/ENT1 and NOTCH3 with improved disease-specific survival (DSS). Low miR-21 was associated with longer DSS in patients with negative regional lymph nodes or primary tumor at stage 1 and 2. In addition, downregulation of ENT1 was observed in PDAC of patients with high ENT1 expression in normal pancreas, whereas NOTCH3 was upregulated in PDAC of patients with low NOTCH3 levels in normal pancreas. Tumor miR‑21 was upregulated irrespective of its expression in normal pancreas. Our data confirmed that patient stratification based on expression of ENT1/ENT1 or miR‑21 is not ready to be implemented into clinical decision-making processes. We also conclude that occurrence of ENT1 and NOTCH3 deregulation in PDAC is dependent on their expression in normal pancreas.
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.
- MeSH
- adenosin metabolismus MeSH
- biologický transport fyziologie MeSH
- dideoxynukleosidy metabolismus MeSH
- ekvilibrační přenašeč nukleosidů 1 metabolismus MeSH
- ekvilibrační přenašeč nukleosidů 2 metabolismus MeSH
- krysa rodu Rattus MeSH
- látky proti HIV metabolismus MeSH
- lidé MeSH
- membránové transportní proteiny metabolismus MeSH
- nádorové buněčné linie MeSH
- nukleosidy metabolismus MeSH
- placenta metabolismus MeSH
- potkani Wistar MeSH
- proteiny přenášející nukleosidy metabolismus MeSH
- těhotenství MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- abacavir MeSH Prohlížeč
- adenosin MeSH
- dideoxynukleosidy MeSH
- ekvilibrační přenašeč nukleosidů 1 MeSH
- ekvilibrační přenašeč nukleosidů 2 MeSH
- látky proti HIV MeSH
- membránové transportní proteiny MeSH
- nukleosidy MeSH
- proteiny přenášející nukleosidy MeSH
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.
- Klíčová slova
- cAMP/protein kinase A signaling pathway, concentrative nucleoside transporters, differentiation, epigenetics, gene regulation, human placenta,
- MeSH
- buněčná diferenciace účinky léků fyziologie MeSH
- ekvilibrační proteiny přenášející nukleosidy genetika metabolismus MeSH
- gestační stáří * MeSH
- karbazoly farmakologie MeSH
- lidé MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- messenger RNA metabolismus MeSH
- nádorové buněčné linie MeSH
- placenta účinky léků metabolismus MeSH
- pyrroly farmakologie MeSH
- těhotenství MeSH
- upregulace MeSH
- vývojová regulace genové exprese účinky léků fyziologie MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cif nucleoside transporter MeSH Prohlížeč
- ekvilibrační proteiny přenášející nukleosidy MeSH
- karbazoly MeSH
- KT 5720 MeSH Prohlížeč
- membránové transportní proteiny MeSH
- messenger RNA MeSH
- pyrroly MeSH
A series of 143 salicylanilides substituted in positions 4 and 5 and in positions 3' and 4' was synthesized. The compounds were evaluated for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium avium. To describe the structure-antimycobacterial activity relationships (QSARs), an approach based on the combination of the Free-Wilson and Hansch methods was employed (the substituent constants were used in the case of the substituents on the phenyl ring; indicator parameters were used for the substituents on the acyl moiety). The relationships between the antimycobacterial activity and physico-chemical parameters of all substituents were also explored. The quadratic representation of lipophilicity parameters did not lead to significant correlations.
- MeSH
- antibakteriální látky chemická syntéza chemie farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- Mycobacterium účinky léků MeSH
- salicylanilidy chemická syntéza chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- salicylanilidy MeSH
