Transplacental Transfer Of Emtricitabine Mediated By Cell Membrane Transporters

ObjectiveAcquired immunodeficiency syndrome(AIDS)is a global epidemic.In 2017,there are about 36.9 million people infected with HIV worldwide.According to UNAIDS statistics,43%of HIV carriers in 2018 are women,and most women are in childbearing age.World health organization(WHO)recommends all HIV-positive pregnant women and lactating women should receive antiretroviral drugs to slow the progression of disease and avoid vertical transmission.Untreated HIV infection causes progressive CD4+T cell loss and a wide range of immunological abnormalities,leading to an increased risk of infectious and oncological complications.Emtricitabine(FTC)is an efficient and well-tolerated antiretroviral nucleoside drug FTC is a first-line antiviral drug that WHO recommends for HIV-infected pregnant women to control HIV viral load and reduce the risk of mother-to-child transmission.In order to reduce the occurrence of drug resistance,WHO recommends several antiretroviral drugs with different pharmacological effects,which is called high-efficiency combined antiretroviral therapy.The use of highly active antiretroviral therapy in HIV-infected pregnant women can effectively control the maternal viral load and reduce the risk of mother-to-child transmission.However,there may be drug-drug interactions on the placenta that cause changes of drug concentration on the fetal side.A decrease drug concentration may result in embryo toxicity and a decrease in drug concentration may cause antiviral failure.FTC is a highly polar compound,although many studies have reported that the placental permeability of FTC is well,but its transport mechanism is not very clear.The aim of this study was to clarify which transporters participate in the FTC across the blood-placental barrier;to explore whether the antiretroviral drug tenofovir(TFV)-efavirenz(EFV)affects FTC accumulation in Be Wo cells and human primary trophoblasts(PHTCs);and to investigate whether the protein expression of equilibrative nucleoside transporter(ENT)1 of different gestational ages to predict the transport of FTC during pregnancy.MethodWe used BeWo cells and PHTCs to screen for transporters involved in the placenta transport of FTC,transient or stable high expression of cell models as well as siRNA was applied to investigate the interactions between FTC and placenta-related transporters.The intracellular drug concentration of FTC was measured by liquid chromatography-mass spectrometry(LC-MS/MS).Real-time quantitative PCR and western blotting were used to detect the mRNA expression of nucleoside transporters and the protein expression of ENT/Entl in human and mouse placentas of different gestational ages.The accumulation of drugs in cells is not only related to the transport of transporters,but also to drug-drug interactions between drugs.Therefore,we investigated whether TFV or EFV affects FTC accumulation in BeWo cells and PHTCs.Results1.Placental transport mechanism of FTCWe used BeWo cells to study whether transporters participate in the transplacental transfer of FTC.The results on Be Wo cells suggested that uptake transporters ENTs,concentrative nucleoside transporters(CNTs),organic cation transporters(OCTs),and efflux transporters breast cancer resistance protein(BCRP),and multidrug resistance-assoiated proteins(MRPs)may be involved in transfer of FTC.Accumulation inhibition experiments on PHTCs also suggestted that ENTs and OCTs may be involved in FTC transport.2.Interaction of FTC with placenta-related transportersFirstly,siRNA was transfected into BeWo cells,and the accumulation of FTC in BeWo cells was reduced by knocking down the expression of ENT 1,which confirmed the involvement of ENT1 in FTC transport.Then we used different concentrations NBTI of ENTs inhibitor to prove that ENT2 did not participate in FTC transport.Finally,cell models using transient transfected or stable high transporter expression of transporter gene demonstrated that FTC was a substrate for CNT1,organic cation/carnitine transporter(OCTN1),BCRP,and MRP 1/2/3,but not a substrate for CNT3,OCTN2,and multidrug resistance protein(MDR)1.Besides,the uptake mediated by CNT1 followed Michaelis-Menten kinetics with the Km and Vmax values of 162 ± 54.1 ?M and 201 ± 15.2 pmol/mg protein/min,respectively.And the uptake kinetics of FTC by OCTN1 displayed atypical dynamics under Eadie-Hofstee analysis.At low concentration(10-400 ?M),the and Vmax values were 489±119 ?M and 0.364±0.0709 nmol/mg protein/min;whereas at high concentration(800-5000 ?M),the Km and Vmax values were 13.3±1.18 mM and 6.01±0.450 nmol/mg protein/min.3.Changes in the expression of nucleoside transporters in different stages of human and mouse placentas and the influence of combined antiretroviral drugs on transport of FTC.The expression of five kinds of nucleoside transporters(NTs)mRNA in placentas of human and mouse at different stages of pregnancy were detected.The CNT1 mRNA in human term placentas were significantly increased compared with first-trimester placentas,and other nucleoside transporters did not change significantly;the expression of Entl and Cnt3 mRNA in gestational day(GD)18 placentas of mice were significantly lower than that of GD15 and the expression of Ent2 mRNA in GD18 placentas were significantly higher than that in GD15,and there were no difference in Cntl/2 mRNA expression.Secondly,the experimental results showed,regardless of human or mouse,the expression of ENT/Entl protein in placentas did not change significantly during different pregnancy stages.Finally,whether the combination of antiviral drugs would affect the accumulation of FTC on BeWo cells and PHTCs,the experimental results showed that co-existence of TFV and/or EFV did not affect the accumulation of FTC on BeWo cells and PHTCs.ConclusionOur comprehensive study of the placental transfer of FTC demonstrated that ENT1,CNT1,OCTN1,BCRP and MRP1/2/3 are involved in FTC transport.Considering the location of transporters in syncytiotrophoblasts,we deduced that:(i)ENT1,CNT1 and OCTN1 contributed to FTC uptake from the maternal circulation to trophoblasts;(ii)ENT1,CNT1 and MRP1 helped FTC move from trophoblasts to the fetal circulation;(iii)BCRP and MRP1/2/3 might be involved in the efflux of FTC from trophoblasts to the maternal circulation.Co-existence of TFV or EFV did not affect FTC accumulation in BeWo cells and PHTCs.We provided,for the first time,information on how FTC transfers across the placenta.