The inhibitory effect of antiretroviral drugs on the L-carnitine uptake in human placenta

Toxicology and Applied Pharmacology 368 (2019) 18–25

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The inhibitory effect of antiretroviral drugs on the L-carnitine uptake in human placenta

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Rona Karahoda, Martina Ceckova , Frantisek Staud Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University, Czech Republic

A R T I C LE I N FO

A B S T R A C T

Keywords: Carnitine Placenta Antiretrovirals Membrane transport Carnitine deficiency

In spite of remarkable reduction in the number of children born with HIV due to antiretroviral therapy, concerns remain on the short- and long-term effects of antiretroviral drugs at the feto-placental unit. Cardio- and skeletal myopathies have been reported in children exposed to antiretroviral drugs prenatally. These conditions have also been described in perturbed placental transfer of L-carnitine, an essential co-factor in fatty acid oxidation. Due to limited fetal and placental synthesis, carnitine supply is maintained through the placental carnitine uptake from maternal blood by the organic cation/carnitine transporters OCTN1 and OCTN2 (SLC22A4 and SLC22A5, respectively). The aim of our study was to investigate potential inhibition of placental carnitine uptake by a broad range of antiretroviral drugs comprising nucleoside/nucleotide reverse transcriptase inhibitors (lamivudine, zidovudine, abacavir, tenofovir disoproxil fumarate), non-nucleoside reverse transcriptase inhibitors (rilpivirine, efavirenz, etravirine), protease inhibitors (ritonavir, lopinavir, atazanavir, saquinavir, tipranavir), integrase inhibitors (raltegravir, dolutegravir, elvitegravir) and viral entry inhibitor, maraviroc. Studies in choriocarcinoma BeWo cells and human placenta-derived models confirmed predominant expression and function of OCTN2 above OCTN1 in L-carnitine transport. Subsequent screenings in BeWo cells and isolated MVM vesicles revealed seven antiretroviral drugs as inhibitors of the Na+-dependent L-carnitine uptake, corresponding to OCTN2. Ritonavir, saquinavir and elvitegravir showed the highest inhibitory potential which was further confirmed for ritonavir and saquinavir in placental fresh villous fragments. Our data indicate possible impairment in placental and fetal supply of L-carnitine with ritonavir and saquinavir, while suggesting retained placental carnitine transport with the other antiretroviral drugs.

1. Introduction

of Perinatal Transmission). In particular, mitochondrial toxicity from in utero exposure to several antiretrovirals, remains a major concern (Gingelmaier et al., 2009; Hernandez et al., 2017; 2012; Ross et al., 2012). L-carnitine is an important co-factor in β-oxidation, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane (Grube et al., 2005; McGarry and Brown, 1997). It plays a critical role in energy metabolism of tissues that are highly dependent on fatty acid oxidation as means of energy supply, such as heart, skeletal muscle, liver, gastrointestinal tract and feto-placental unit (Kim and Roe, 1992). Both the syncytiotrophoblast and embryo express fatty acid

Use of antiretroviral (ARV) drugs in pregnancy has greatly increased over the last two decades, resulting in a dramatic decline of new HIV infections among children aged 0–14 years. According to the latest data, 75% of HIV-infected pregnant women receive antiretroviral therapy, with expected increase up to 90% by 2020 (UN Joint Programme on HIV/AIDS (UNAIDS), 2017). In spite of evident and inevitable benefit in preventing mother-to-child HIV transmission, concerns remain about the maternal and fetal safety during antiretroviral therapy and the short- and long-term effects of these drugs on the developing fetus (Panel on Treatment of HIV-Infected Pregnant Women and Prevention

Abbreviations: ANOVA, one-way analysis of variance; ARV, antiretroviral; DMEM, Dulbecco's modified eagle medium; DMSO, dimethyl sulfoxide; MTCT, mother-tochild transmission; MVM, microvillous plasma membrane; NIH, National Institute of Health; HIV, human immunodeficiency virus; FAO, fatty acid oxidation; OCTN1, organic cation/carnitine transporter 1; OCTN2, organic cation/carnitine transporter 2; TEA, tetraethyl ammonium; FBS, fetal bovine serum; BCA, bicinchonic acid; SDS, sodium dodecyl sulphate; SLC, solute carrier ⁎ Corresponding author at: Department of Pharmacology and Toxicology, Charles University, Faculty of Pharmacy in Hradec Kralove, Akademika Heyrovskeho 1203, Hradec Kralove 500 05, Czech Republic. E-mail address: [email protected] (M. Ceckova). https://doi.org/10.1016/j.taap.2019.02.002 Received 9 November 2018; Received in revised form 31 January 2019; Accepted 5 February 2019 Available online 05 February 2019 0041-008X/ © 2019 Elsevier Inc. All rights reserved.

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the NIH, as a part of the NIH AIDS Reagent Program and comprise: ritonavir (RIT), lopinavir (LOP), atazanavir (ATA), saquinavir (SAQ), tipranavir (TIP), raltegravir (RAL), maraviroc (MVC), lamivudine (3TC), zidovudine (AZT), abacavir (ABC), tenofovir disoproxil fumarate (TDF), rilpivirine (RIL), efavirenz (EFA) and etravirine (ETR). Dolutegravir (DOL) and elvitegravir (ELV) were obtained from MedChemExpress Europe. All other chemicals were of analytical grade. Bicinchoninic acid assay (BCA assay) reagents were purchased from Thermo Scientific (Rockford, USA). Tri Reagent solution was obtained from Molecular Research Centre (Cincinnati, OH, USA).

oxidation (FAO) enzymes and besides carbohydrate metabolism, fatty acids represent an important energy source (Oey et al., 2003; Oey et al., 2006; Shekhawat et al., 2003; Rakheja et al., 2002). Due to limited carnitine synthesizing capacity, sufficient placental and fetal carnitine levels are ensured mainly through uptake from maternal circulation, achieved by the sodium-independent and sodium-dependent organic cation/carnitine transporter 1 (OCTN1, SLC22A4) and 2 (OCTN2, SLC22A5), respectively (Roque et al., 1996; Wu et al., 1999; Novak et al., 1981; Schmidt-Sommerfeld et al., 1985). Due to its high mRNA and protein expression in the apical membrane in human placenta and transport characteristics, OCTN2 is believed to be the major mechanism responsible for carnitine transport from maternal to fetal circulation (Grube et al., 2005; Lahjouji et al., 2004). In addition, impaired OCTN2 function has been linked to several pathologies. Specifically, defective OCTN2 function as a result of mutation is associated with primary carnitine deficiency in humans, manifested chiefly as cardio- and skeletal myopathies (Steuerwald et al., 2017; Gallant et al., 2017). The outcomes of impaired OCTN2-mediated transport were clearly demonstrated in OCTN2 null mice (known as juvenile visceral steatosis mouse), who show < 20% placental and fetal carnitine content compared to the OCTN2 wild-type mice, resulting to be lethal for the developing embryos (Shekhawat et al., 2004; Shekhawat et al., 2018). Significantly lower levels of free and total carnitine and acylcarnitines were also observed in heterozygous OCTN2+/− mice. Additionally, many xenobiotics such as anticonvulsants and fluoroquinolone antibiotics have been shown to inhibit OCTN2 function in vitro and ex vivo, potentially leading to secondary carnitine deficiency (Ohashi et al., 1999; Hirano et al., 2008; Wu et al., 2004; Verrotti et al., 1999). In the recent years, several cohort studies have reported neonate disorders in fetuses born to mothers receiving antiretrovirals during pregnancy. In particular, use of nucleoside analog reverse transcriptase and protease inhibitors has been linked to increased incidence of heart defects, myocardial remodeling as well as musculoskeletal defects (Van Dyke et al., 2016; Sibiude et al., 2015; Sibiude et al., 2014; GarciaOtero et al., 2016), suggesting mitochondrial dysfunction in exposed fetuses (Blanche et al., 2006; Lopaschuk et al., 2010). So far, no comprehensive study has been conducted to link these therapeutic outcomes with impaired FAO and hindered carnitine transport across placenta. Therefore, we hypothesize that the observed defects may be at least partly caused by affected transport of L-carnitine from mother to fetus, leading to suboptimal levels of the cofactor in fetal circulation and hence faulty FAO. Employing in vitro (uptake studies in choriocarcinoma cell line BeWo) and ex vivo (accumulation studies in fresh villous fragments and microvillous plasma membrane (MVM) vesicles isolated from human term placentas) approaches we aimed to screen a broad range of antiretroviral drugs for interaction with OCTN transporters. Our study comprises drugs involved in common therapeutic regimens of pregnant women, as well as ARV drugs with yet limited knowledge on safety profile in pregnant women. In detail, we have included: nucleoside/nucleotide reverse transcriptase inhibitors (lamivudine, zidovudine, abacavir, tenofovir disoproxil fumarate), non-nucleoside reverse transcriptase inhibitors (rilpivirine, efavirenz, etravirine), protease inhibitors (ritonavir, lopinavir, atazanavir, saquinavir, tipranavir), integrase inhibitors (raltegravir, dolutegravir, elvitegravir) and viral entry inhibitor, maraviroc.

2.2. Cells The human choriocarcinoma-derived BeWo cell line (b30 clone) was kindly provided by Prof. Ch. Albrecht (University of Bern, Switzerland) with permission from Dr. A. Schwartz (Washington University, St. Louis, USA). Cells were cultured in Dulbecco's Modified Eagle Medium (high glucose) supplemented with 10% FBS, at 37 °C/5% CO2. 2.3. RNA isolation, reverse transcription and quantitative PCR analysis Total RNA was isolated from weighed tissue samples or directly from BeWo b30 cells using Tri Reagent solution, according to the manufacturer's instructions. The purity of the isolated RNA was checked by the A260/A280 ratio, whereas the A260/230 ratio was used to evaluate contamination by organic solvents. RNA integrity was confirmed by electrophoresis on a 1.5% agarose gel and total RNA concentration was calculated by A260 measurement. Conversion of RNA into cDNA was done using the gb Reverse Transcription Kit from Generi Biotech s.r.o. (Hradec Kralove, Czech Republic) on a Bio-Rad T100TM Thermal Cycler (Hercules, CA, USA). Quantitative PCR (qPCR) analysis of SLC22A4 and SLC22A5 expression in BeWo b30 cells and human term placentas was performed using QuantStudioTM 6 (Thermo Fisher Scientific, Waltham, MA, USA). 0.5 μl of the obtained cDNA (25 ng/ul) was amplified in a 384-well plate, with total reaction volumes of 5 μl per well. PCR was performed using the TaqMan® Universal Master Mix II without UNG (Thermo Fisher Scientific, Waltham, MA, USA), and predesigned TaqMan® Real Time Expression PCR assays for human SLC22A4 (Hs00268200_m1) and SLC22A5 (Hs00929869_m1). Stable expression of the reference gene was verified before beginning the quantitative analysis. Each sample was amplified in triplicate, using the following PCR cycling profile: 95 °C for 10 mins, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. 2.4. Droplet digital PCR assay The cDNA of one comparator sample used in qRT-PCR was further absolutely quantified for the SLC22A4 and SLC22A5 genes, using ddPCR. The reaction mixture consisted of 10 μl of ddPCR™ Supermix for Probes (Bio-Rad, Hercules, CA, USA), 1 μl of the above-mentioned predesigned TaqMan assays (Thermo Fisher Scientific, Waltham, MA, USA) and 2 μl of cDNA, in a total volume of 20 μl. For each gene, three technical replicates were used in two different cDNA concentrations (12.5 ng/μl and 0.5 ng/μl). The mixtures were then loaded into sample wells in the DG8 Cartridges, whereas 70 μl of the Droplet Generation Oil was added to the oil wells. After sealing with DG8 Gasket, the cartridge was placed into the QX200 Droplet Generator. 40 μl of obtained droplets were transferred to 96 well-plate, heat-sealed using PX1 PCR Plate Sealer and amplified to end-point using T100™ Thermal Cycler. The following cycling conditions were used: single cycle of 95 °C for 10 mins, followed by 40 cycles of 94 °C for 30 s and 60 °C for 1 min; and finally a single cycle of 98 °C for 10 mins. The plate was then placed on QX200™ Droplet Reader, which reads the droplets from each individual well. Depending on fluorescence amplitude, the droplets are

2. Materials and methods 2.1. Chemicals and reagents The radiolabeled compound [3H]-L-carnitine (60 Ci/mmol) was purchased from M.G.P. spol. s.r.o. (Zlin, Czech Republic). Unlabeled carnitine and the non-specific inhibitor verapamil (VER), were purchased from MedChemExpress Europe through Scintilla s.r.o. (Jihlava, Czech Republic) and from Sigma-Aldrich (St. Louis, MO, USA), respectively. Most of antiretrovirals used in the study were obtained from 19

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temperature (21–25 °C) prior to uptake. 10 μl samples of MVM were pre-incubated for 10 min with or without 1 mM carnitine or 10/1 μM concentration of studied antiretrovirals in extravesicular buffer. Uptake of 34.3 nM [3H]-L-carnitine was initiated by adding the substrate diluted in extravesicular buffer (EVB: 145 mM NaCl, 5 mM HEPES and 5 mM Tris, pH 7.4; for Na+-free buffer KCl was used instead) to the preincubated MVM vesicles. Uptake was halted after 1 min or defined time points, by adding 2 ml ice-cold stopping buffer (130 mM NaCl, 10 mM Na2HPO4, 4.2 mM KCl, 1.2 mM MgSO4, 0.75 mM CaCl2, pH 7.4) and filtering through a 0.45 μM mixed cellulose ester filter (MF-Millipore membrane filter HAWP02500) under vacuum. Filters were washed with 10 ml stopping buffer, and the filter-associated radioactivity was determined. Protein-free controls (in which the MVM vesicle protein was replaced with IVB) were analyzed in parallel to determine tracer binding to the filter, which was subtracted from the total vesicle count. Unspecific binding of [3H]-L-carnitine to the plasma membrane was excluded by measuring time zero uptakes, which revealed comparable values to the protein-free controls.

scored as positive and negative. According to the number of positive droplets compared to negative, the concentration of the target gene is calculated in the QuantaSoft™ Software. For final evaluation of data, only wells in which the number of droplets obtained was higher than 13,000 were used. Concentration of target genes transcripts/μl RNA in our comparator sample was then used to deduct the transcript concentrations of the other samples analyzed by qRT-PCR. Expression levels are reported in number of SLC22A4 and SLC22A5 transcripts per μg of transcribed RNA. The QX200™ Droplet Digital™ PCR System, T100™ Thermal Cycler and all consumables and reagents were obtained from BioRad, Hercules, CA, USA (unless otherwise stated). 2.5. In vitro accumulation studies in BeWo b30 cells 2 × 105 of BeWo b30 cells were seeded on 24-well culture plates (TPP, Trasadingen, Switzerland) and cultured for 2 days until confluent. Uptake studies were performed as previously described (Ceckova et al., 2006). Briefly, before the start of the experiment, the cells were preincubated for 15 min in Na+ buffer (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2 2H2O, 0.8 mM MgSO4 7H2O, 5 mM glucose, 25 mM Tris; pH 7.4) alone or with the antiretrovirals of interest (10 μM concentration) and carnitine (1 mM). The cells were then incubated with [3H]-L-carnitine in Na+ buffer with or without the appropriate inhibitor, at a final activity of 0.25 μCi/ml. Accumulation was stopped after 15 min or at designated time points (for time-dependent studies) by quick aspiration of the radioactivity-containing buffer and washing twice with 0.5 ml of phosphate-buffered saline, after which the cells were lysed in 0.02% SDS. The intracellular concentrations of radioisotopes were subsequently determined and normalized against the protein content (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). Uptake is reported in pmol/mg protein. Uptakes in the absence of Na+ were performed using Na+-free buffer containing 140 mM M-methyl-D-glucamine instead of NaCl.

2.7. Ex vivo accumulation studies in fresh villous fragments from human placenta Ex vivo analysis of [3H]-L-carnitine uptake by the human placenta was performed using the method of accumulation in fresh villous fragments derived from human term placentas (Atkinson et al., 2006; Greenwood and Sibley, 2006; Neumanova et al., 2015). Briefly, placentas were collected after uncomplicated pregnancies from women at the University Hospital in Hradec Kralove, following written informed consent approved by the University Hospital Research Ethics Committees (201006S15P). Small fragments of villous tissue were dissected within 30 min of delivery. These were washed in Tyrode's buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5.6 mM glucose, pH 7.4) and then tied to hooks. Before starting the experiment, the fragments were allowed to stabilize for 30 min in a mixture of Tyrode's buffer and DMEM 1:1. Subsequently, uptake was performed in Tyrode's buffer in the presence/absence of antiretrovirals/ control inhibitors for 15 min. Vigorous washing in excess Tyrode's buffer (with or without antiretrovirals/control inhibitors) was applied to terminate accumulation and remove extracellularly bound drugs. Villous fragments were then immersed in distilled water overnight to allow release of the radioisotope, which was then counted. The fragments were further dissolved in 0.3 M NaOH solution for 6–8 h at 37 °C, following protein measurement using BCA assay. The accumulated radioactivity was then adjusted to the obtained protein content. The experiment was performed in two setups. We first investigated the time course of [3H]-L- carnitine uptake in the presence and absence of Na+ by measuring the uptake at 1, 15, 30, 60 and 120 min. Further, the effect of 1 mM carnitine and selected antiretrovirals (ritonavir 1 and 10 μM, saquinavir 1 and 10 μM, elvitegravir 1 μM, tipranavir 10 μM and lopinavir 10 μM) on [3H]-L-carnitine uptake after 15 min of incubation was investigated; the incubation length was chosen based on the previous step's results.

2.6. Preparation of microvillous plasma membrane (MVM) vesicles and uptake assays MVM vesicles were employed for further screening of the potential inhibitory effect of selected antiretroviral drugs on [3H]-L-carnitine uptake in the apical membrane of the human syncytiotrophoblast layer. Human placentas were obtained from uncomplicated pregnancies at term (after 38–40 weeks of gestation) delivered at the University Hospital in Hradec Kralove, after obtaining the women's written informed consent and with the approval of the University Hospital Research Ethics Committee (201,006 S15P). MVM vesicles were isolated by tissue homogenization followed by Mg2+ precipitation and differential centrifugation as described previously (Glazier et al., 1988; Ceckova et al., 2016). The final MVM pellet was resuspended in intravesicular buffer (IVB; 290 mM sucrose, 5 mM HEPES and 5 mM Tris, pH 7.4), vesiculated by passing 15 times through a 25-gauge needle, stored at 4 °C, and used in uptake experiments within 3 days of isolation or frozen at −80 °C and equilibrated to room temperature on the day of the experiment. Before the initiation of uptake experiments the comparable rate of uptake in fresh and thawed vesicles was verified. The protein concentrations in MVM vesicles and placental homogenate were determined using the BCA assay, and optimal purity was confirmed by measuring the enrichment of MVM by alkaline phosphatase activity compared to the placental homogenate. The alkaline phosphatase enrichment factor in MVM vesicles used in this study was 23.39 ± 6.56 (mean ± SD, n = 11 donors). Right side-out orientation of vesicles (> 80%) was evaluated by comparing specific alkaline phosphatase activity upon vesicular disruption by detergent, as described previously (Glazier et al., 1988). Uptake of [3H]-L-carnitine into MVM vesicles was measured at room temperature using rapid vacuum filtration approach (Glazier and Sibley, 2006). MVM vesicles (15–30 mg/ml) were equilibrated to room

2.8. Radioisotope analysis The concentrations of [3H]-L-carnitine in experimental samples were measured by liquid scintillation counting (Tri-Carb 2910 TR; Perkin Elmer, Waltham, MA, USA). 2.9. Statistical analysis Uptake studies on fresh villous fragments and vesicles prepared from human placentas were evaluated using paired t-test analysis. On the other hand, One-way ANOVA, followed by Dunnett's multiple comparisons test was used for evaluation of accumulation studies on the BeWo cells. All statistical analysis was performed using the Graph Pad 20

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across the apical MVM (Fig. 3C). 3.4. Ex vivo inhibition analysis of [3H]-L-carnitine uptake by human fresh villous placental fragments Fresh villous placental fragments were employed to further evaluate the results obtained from our in vitro studies on BeWo cells and ex vivo studies on MVM vesicles in the more complex placental tissue. Following confirmation of Na+-dependent [3H]-L-carnitine uptake in this model (Fig. 4A), we exposed the villous fragments to the ARV drugs which, at concentrations correlating to those achievable in clinical practice, provided the most significant inhibitory effect in previous experiments. Among them, the highest inhibitory potency was revealed in the case of protease inhibitors ritonavir and saquinavir (Fig. 4B).

Fig. 1. Expression of SLC22A4 and SLC22A5 genes encoding for OCTN1 and OCTN2, respectively in BeWo b30 cells (cell culture passages 1–10) and term placentas (n = 11 donors).

4. Discussion Prism 7.0 software; * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001). L-carnitine plays a pivotal role in energy supply through shuttling fatty acids across the mitochondrial membrane, allowing their β-oxidation (Grube et al., 2005; McGarry and Brown, 1997). Two membrane transporters, OCTN1 and OCTN2, of the solute carrier transporter (SLC) family have been described to play a role in the uptake of carnitine from maternal blood, thereby ensuring sufficient levels in the placenta and fetus. While the high capacity OCTN2 is believed to play a critical role in steady supply of carnitine to the trophoblast, OCTN1 is thought to contribute to a lower extent (Lahjouji et al., 2004). Mutations in SLC22A5 gene encoding for OCTN2 lead to primary carnitine deficiency which, because of its urgency, is currently evaluated in newborns through neonate screening programs in most of developed countries, allowing in time supplementation of L-carnitine (Gallant et al., 2017; Rasmussen et al., 2014). Likewise, drug-mediated placental inhibition of OCTN2 has been described and associated with secondary carnitine deficiency (Hirano et al., 2008; Wu et al., 2004). So far, genetic variations of OCTN1 have not been directly linked to carnitine deficiency and there is no evidence that OCTN1 would compensate for the impaired OCTN2 function, further confirming the dominant role of OCTN2 on total placental and fetal L-carnitine concentrations (Tahara et al., 2009; Lahjouji et al., 2001; Tamai, 2013). Whilst reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 can represent a recently suggested effective strategy to protect the heart against ischaemia-reperfusion-induced damage, insufficient levels of carnitine (below 10–20% of normal ranges) in the placenta and fetus have been associated with conditions in newborns such as cardiomyopathy, skeletal myopathy, hypoketotic hypoglycemia etc. (Steuerwald et al., 2017; Gallant et al., 2017; Shekhawat et al., 2004; 2018; Liepinsh et al., 2015). In addition, defective OCTN2 in a mouse model has been associated with reduced expression of growth factors and embryonic lethality (Shekhawat et al., 2018). Antiretroviral therapy in pregnant HIV-positive women is becoming an inevitable standard to help ensure protection of mother-to-child transmission (MTCT) of the virus (UN Joint Programme on HIV/AIDS (UNAIDS), 2017; Panel on Treatment of HIV-Infected Pregnant Women and Prevention of Perinatal Transmission; Townsend et al., 2014). During the recent years, several studies have been performed to address associations between use of antiretroviral therapy in pregnancy and development of neonate disorders. Reports on fetal mortality and stillbirths in HIV-pregnant women receiving ARV therapy are inconclusive and highly controversial. While some studies report increased risk of stillbirths in HIV-infected women on ARV therapy at the time of conception (Stringer et al., 2018; Chen et al., 2012), others dispute these findings (Cotter et al., 2006; Uthman et al., 2017). Moreover, the infection itself is suggested to be correlated with impaired birth outcomes (Lawn et al., 2016; Wedi et al., 2016). However, with particular interest to this study, four independent cohort studies have shown a link between prenatal use of zidovudine and incidence of heart defects and

3. Results 3.1. qRT-PCR and ddPCR analysis of hSLC22A4 and hSLC22A5 expression in BeWo b30 cells and human term placentas qRT- and ddPCR analysis was performed to verify the mRNA expression of SLC22A4 and SLC22A5 in BeWo cells (within passages used for in vitro uptake studies) and 11 samples of human term placentas (used in ex vivo experiments). SLC22A4 and SLC22A5 transcripts were detected in all the samples without significant variation among cellular passages and individual placentas. Evaluation of SLC22A4 and SLC22A5 expression revealed 46- and 27- times higher levels of SLC22A5 compared to SLC22A4 in BeWo and human placenta, respectively (Fig. 1). These results confirm OCTN2 as the predominantly expressed carnitine transporter in human term placenta as well as in the choriocarcinoma cell line BeWo. 3.2. In vitro inhibition studies of [3H]-L-carnitine uptake using BeWo cells BeWo cells were used to screen for potential inhibitory effect of a broad range of antiretrovirals on L-carnitine uptake. To characterize the uptake of [3H]-L-carnitine in this cellular model, time-dependent studies in presence and absence of Na+ were performed (Fig. 2B). They confirm significant contribution of the Na+-dependent mechanism in Lcarnitine uptake, which increased with time up to 60 min. A 15-minute incubation time was then used for subsequent inhibitory screening with antiretrovirals (Fig. 2A). As control inhibitor, 1 mM verapamil was used, whilst the saturation of the Na+-dependent L-carnitine uptake mechanism was confirmed by 1 mM cold carnitine. Several ARV drugs decreased the uptake of [3H]-L-carnitine in the presence of Na+ (OCTN2-mediated) (Fig. 2A), while not affecting the uptake in Na+-free (OCTN1-mediated) conditions (Fig. 2C). 3.3. Inhibitory effects of antiretroviral drugs on [3H]-L-carnitine uptake into human placental MVM vesicles To confirm the results obtained in our in vitro model, uptake experiments using MVM vesicles isolated from human term placentas were performed. Similar to BeWo cells, time-dependent accumulation studies revealed predominant contribution of Na+-dependent mechanism in L-carnitine uptake into the MVM vesicles (Fig. 3B). Based on this curve, one-minute uptake was chosen for evaluation of studied ARV drugs and their effect on the L-carnitine uptake into the vesicles (Fig. 3A). In drugs showing significant inhibitory effect, a follow up study was performed also at lower (1 μM) concentration. Among the studied ARV drugs, ritonavir, saquinavir and elvitegravir showed the highest inhibitory potential on the Na+-dependent L-carnitine uptake 21

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Fig. 2. [3H]-L-carnitine uptake studies in the BeWo (b30) choriocarcinoma cell line. (A) Effect of antiretroviral drugs, high concentration of carnitine (CAR, 1 mM) and unspecific inhibitor verapamil (VER, 1 mM) on 15 min [3H]-L-carnitine uptake in Na+-containing buffer. (B) Time-dependent uptake of L-carnitine in the presence/absence of Na+ (C) Effect of ARV drugs that were revealed as inhibitors of Na+-dependent carnitine uptake shown in Fig. 2A, on the cellular accumulation of carnitine in the Na+ free conditions. The data are presented as mean ± SD (n ≥ 3). Statistical significance was evaluated by One-way ANOVA, followed by Dunnett's multiple comparisons test;* (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001).

digital PCR enabling absolute quantification, we revealed 46- and 27times higher number of SLC22A5 transcripts than SLC22A4, in BeWo cells and placentas, respectively (Fig. 1). In addition, our in vitro and ex vivo time-dependent experiments of carnitine uptake in the presence or absence of Na+, demonstrate clear Na+-dependent mechanism in uptake of L-carnitine by BeWo cell line, MVM vesicles and fresh villous fragments isolated from human term placentas (Figs. 2B, 3B, 4A). Furthermore, the uptake was saturable in the presence of high (1 mM) concentration of carnitine (Figs. 2A, 4A). While these transport characteristics have been previously described in BeWo cells and MVM vesicles (Lahjouji et al., 2004; Rytting and Audus, 2005), this is the first study showing that fresh villous fragments also provide a valuable method to evaluate carnitine transport and OCTN function in the placenta. As the main part of the study, we focused on identifying potential antiretroviral drugs able to interact with OCTN transporters. The initial screenings in BeWo cells revealed seven compounds as inhibitors of Na+-dependent L-carnitine uptake, corresponding to OCTN2 (Fig. 2A). The same pattern of decreased L-carnitine uptake was observed ex vivo using MVM vesicles isolated from human term placenta. Specifically, OCTN2 inhibition was observed upon exposure to most of the protease inhibitors, two integrase inhibitors, maraviroc and the non-nucleoside reverse transcriptase inhibitor rilpivirine. No inhibitory effect on carnitine uptake was noted with other reverse transcriptase inhibitors. The ARV drugs that revealed ability to inhibit the carnitine uptake when applied consistently at 10 μM concentration, were further

myocardial remodeling in newborns (Van Dyke et al., 2016; Sibiude et al., 2015; 2014; Garcia-Otero et al., 2016). On the other hand, findings by the Antiretroviral Pregnancy Registry show no association between zidovudine use at any stage of pregnancy and risk of neither coronary heart disease nor ventricular septal defect (Vannappagari et al., 2016) was found. Nevertheless, cardiovascular defects in newborns have also been associated with the prenatal use of tenofovir, ritonavir-boosted lopinavir, lamivudine and atazanavir; with atazanavir and ritonavir further linked to musculoskeletal defect outcomes (Van Dyke et al., 2016). These are particularly important findings since the heart and skeletal muscle are highly dependent on mitochondrial metabolism and FAO as energy supply (Lopaschuk et al., 2010). In this study, we hypothesized that the observed conditions may be a result of defective transport of L-carnitine from mother to fetus, leading to suboptimal levels of the cofactor in fetal circulation and hence impaired fatty acid oxidation. Therefore, we aimed to perform screening of several antiretroviral drugs for the inhibition of OCTN2 function in order to provide a better understanding of their interaction on the level of carnitine supply and homeostasis. Functional expression of OCTN2 has been already found in BeWo cells and in human placenta, showing localization of this transporter predominantly on the maternal blood-facing, microvillous membrane of trophoblast (Grube et al., 2005; Lahjouji et al., 2004; Rytting and Audus, 2005). Gene expression of SLC22A5 as well as SLC22A4 encoding for OCTN2 and OCTN1, respectively, was verified also in the cellular passages and placentas used in this study. Following droplet

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Fig. 3. [3H]-L-carnitine uptake studies in MVM vesicles isolated from human term placentas. (A) Effect of studied antiretroviral drugs (10 μM) and control inhibitor verapamil (VER, 1 mM) on the 1 min carnitine uptake in Na+ conditions. (B) Time course studies of L-carnitine uptake into human placental MVM vesicles in the presence and absence of Na+ revealing dominant OCTN2-mediated uptake. (C) Carnitine uptake upon exposition to 1 μM concentration of antiretroviral drugs. The data are presented as mean + S.D. (n ≥ 4 donors). Significance to control was evaluated by paired t-tests and are labeled * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001).

Fig. 4. [3H]-L-carnitine uptake studies into fresh villous fragments isolated from human term placentas. Time course confirming Na+ dependency and saturation of the transport system by 1 mM carnitine (A). Subsequent exposure to the ARVs of highest inhibition potential obtained from previous models reveal potent inhibitory effect caused by ritonavir and saquinavir in 15 min carnitine uptake (B). The data are presented as mean ± SD (n ≥ 3 donors). Significance was evaluated by paired t-tests; significant differences relative to the control are labeled * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001).

evaluated ex vivo at 10 times lower, 1 μM concentrations. For most of the ARV drugs this helps to address the relevance of their inhibitory effect in clinical settings, since their reported plasma concentrations range between 1 and 10 μM (Roustit et al., 2008; Mirochnick and Capparelli, 2004; Tran et al., 2016; Ripamonti et al., 2007; Mulligan et al., 2016; 2018; Gilbert et al., 2015; Colbers et al., 2015; Cressey et al., 2012; Watts & Stek, 2014). The only exception is represented by

tipranavir and lopinavir with reported maximal concentrations above 10 μM (King and Acosta, 2006; Best et al., 2010). In the studied ARV drugs lacking pharmacokinetic data and average/maximum plasma concentration in pregnant women, plasma concentrations reported for non-pregnant patients were considered. When applied at the lower 1 μM concentration, significant inhibitory effect on transport of L-carnitine across the apical (maternal-blood facing) MVM was confirmed in 23

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Funding information

the case of ritonavir, saquinavir and elvitegravir (Fig. 3C). Subsequent experiments in fresh villous fragments were performed only with those drugs that showed inhibition potential on carnitine uptake in both BeWo cells and MVM vesicles at their clinically achievable plasma concentrations. Significant inhibition of carnitine uptake into the fresh villous placental fragments by 44.1 and 41.7% was observed with ritonavir and saquinavir, both applied at 10 μM concentrations. In the case of ritonavir, such concentrations are only achievable upon full therapeutic dose administration (Gatti et al., 1999). Nevertheless, due to poor tolerance and toxic events, this drug is nowadays used only as a pharmacokinetic enhancer (booster), reaching concentrations around 1.1 μM (von Hentig et al., 2008). On the other hand, maximal plasma concentrations reported clinically for saquinavir vary around 3.2 μM (von Hentig et al., 2008). There are no guidelines regarding relevance of in vitro observed interactions on OCTN transporters for in vivo effects so far. Nevertheless, in the case of P-glycoprotein or Breast Cancer Resistance Protein, the International Transporter Consortium recommends further investigation for drugs, which at their highest clinical dose, show in vitro half-maximum inhibitory effect at concentrations 10 times exceeding the steady-state total plasma concentrations (The International Transporter C, 2010). Analogically, we assume that the reported interaction of ritonavir and saquinavir might possess clinical significance and is worth further evaluations. Indeed for both the protease inhibitors, dyslipidemias and suppressions in fatty acid handling and oxidation have been reported and are suggested to be related to decreases in cytosolic- and mitochondrialassociated fatty acid transporters (Li et al., 2018; Richmond et al., 2010; Claessens et al., 2000). Here we show, that ritonavir and saquinavir might also affect placental L-carnitine transport mediated by OCTN2. Impairments in carnitine homeostasis might contribute to the cluster of metabolic complications observed with these drugs and may at least partially explain also the musculoskeletal defects or the decreased myocardial contractility observed in children exposed to ritonavir or ritonavir-boosted lopinavir therapies during the prenatal period (Van Dyke et al., 2016). In conclusion, we show potential of several antiretrovirals, mainly protease inhibitors to affect L-carnitine uptake in the placenta, which might have consequences for mitochondrial metabolism and FAO and result in adverse effects in neonate when used in pregnancy. On the contrary, zidovudine and other nucleoside reverse transcriptase inhibitors, which are currently recommended for prevention of motherto-child transmission in HIV-infected pregnant women, lack this inhibitory effect, further confirming their safety in terms of carnitine delivery to the fetus. The cardio- and skeletal myopathies observed in zidovudine, may be a result of impaired FAO due to defects on other steps of the cycle than carnitine supply. Further studies, in particular after birth measurement of plasma L-carnitine levels of neonates born to mothers receiving ritonavir or saquinavir during pregnancy would be required to confirm the significance of OCTN2 inhibition at a clinical level. Early detection of carnitine deficiency is crucial as these patients respond well to carnitine supplementation before any organ damage occurs (Longo et al., 2006).

The study was supported by the Grant Agency of Charles University (GAUK 1574217/C/2017, SVV 2018/260414), the Czech Science Foundation (GACR 17-16169S) and EFSA-CDN (No. CZ.02.1.01/0.0/ 0.0/16_019/0000841) co-funded by ERDF. References Atkinson, D.E., Sibley, C.P., Fairbairn, L.J., Greenwood, S.L., 2006. MDR1 P-gp expression and activity in intact human placental tissue; upregulation by retroviral transduction. Placenta 27 (6–7), 707–714. Best, B.M., Stek, A.M., Mirochnick, M., Hu, C., Li, H., Burchett, S.K., et al., 2010. Lopinavir tablet pharmacokinetics with an increased dose during pregnancy. J. Acquir. Immune Defic. Syndr. (1999) 54 (4), 381–388. Blanche, S., Tardieu, M., Benhammou, V., Warszawski, J., Rustin, P., 2006. Mitochondrial dysfunction following perinatal exposure to nucleoside analogues. AIDS (London, England) 20 (13), 1685–1690. Ceckova, M., Libra, A., Pavek, P., Nachtigal, P., Brabec, M., Fuchs, R., et al., 2006. 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Acknowledgement We would like to thank Dr. Marian Kacerovsky (Department of Obstetrics and Gynecology, University Hospital in Hradec Kralove) for providing us with human placentas, Martina Hudeckova for her help with the human placenta collection and Dana Souckova for her skillful technical assistance with placental villous fragment isolations. Assistance on ddPCR experiments by Ing. Katerina Hrochova and Mgr. Lucie Petrova, Ph.D. (Institute of Molecular Biology, University Hospital in Hradec Kralove) is further gratefully acknowledged.

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