Joanne Wang

The placenta serves a critical role in nutrient uptake and waste elimination for the developing fetus. The placenta is also responsible for the uptake and/or exchange of xenobiotics, including medications, between the maternal and fetal bloodstreams. An estimated 40–80% of women take medications or drugs during pregnancy. Very little is understood about fetal drug and nutrient exposure during pregnancy and how it may change over the course of fetal development. This study aimed to characterize the abundance of transport proteins in placental tissue, which are important in modulating fetal nutrient and drug exposure, over the duration of pregnancy. Using a mass spectrometry‐based global proteomic approach, the relative abundance of proteins in placental tissue samples was profiled from first trimester, second trimester, and term placenta samples. Our analysis detected a total of 6331 proteins which included a total of 139 solute carrier and ATP‐binding cassette transport proteins that were detected across the dataset, and 80 that were quantified in every sample. In general, membrane‐associated proteins, including solute carrier and ATP‐binding cassette transport proteins, were significantly enriched in placental tissue collected during early gestation compared to term placental tissue. Overall, this study presents a comprehensive profiling of membrane‐associated proteomic changes during gestation and identifies significant gestational age associated abundance changes at the protein level in several transport protein families. These data provide critical information to support future studies into the understanding of fetal exposure to xenobiotics and nutrients circulating in the maternal bloodstream.

Human gestation is characterized by dynamic changes in maternal physiology and maturation of tissue functions before and after birth. The fetus and neonate are highly dependent upon transfer of maternal nutrients via the placenta and lactating mammary gland, which frequently involve selective membrane transporter proteins. At the same time, developmental changes in fetal and infant transport mechanisms critical for regulating absorption at the intestinal and blood–brain barriers are occurring. Coordinate regulation of cellular entry and efflux of nutrients, supplements, and xenobiotics, including drugs and environmental chemicals, must occur during this critical developmental period. In 2023, the National Institutes of Health launched the Transporter Elucidation Network (TEN) across four centers. This initiative applies experimental, computational, and translational approaches to (1) identify S o L ute C arrier (SLC) superfamily and A TP‐ b inding c assette (ABC) transporters in the human placenta, mammary gland, as well as the pediatric brain and intestinal barriers and, (2) characterize their localization, expression, function, and regulation. The Network seeks to identify functional determinants of transporters as gatekeepers of entry to placenta, mammary gland and brain, and applies structural biology, proteomics, metabolomics, organoids, liquid biopsies, and microphysiological systems to this end. Computational strategies, such as machine learning, virtual screening, physiologically based pharmacokinetics, and perinatal epidemiology complement our experimental approaches. This paper highlights the TEN's multidisciplinary strategies to address current challenges in perinatal and pediatric transporter research and its commitment to advancing state‐of‐the‐art tools and new approach methodologies to fill critical gaps in our understanding of nutrient and xenobiotic exposure during early human development.

Solute carriers ( SLC ) and ATP‐binding cassette ( ABC ) transporters are essential for placental solute exchange and fetal protection, yet their transcriptomic profiles in the human placenta remain poorly characterized. Although fetal sex influences placental development and function, its impact on transporter expression is unclear. Using RNA sequencing, we profiled SLC and ABC transporter expression in two anatomical regions of term human placentas ( N = 10) with balanced fetal sex distribution and controlled clinical features. In the intervillous region, 276 SLC s were detected, with 60% expressed at low levels (CPM < 25) and 110 (40%) expressed at higher levels (CPM 25–1650). Four transporters— SLC2A1 (GLUT1), SLC44A2 (CTL2), SLC38A2 (SNAT2), and SLC38A1 (SNAT1)—showed the highest expression (CPM > 500). Functional annotation of 110 SLC s revealed transporters for amino acids, metals, and inorganic ions each accounted for ~9%. Other well‐represented SLC s included xenobiotic and vitamin transporters. Mitochondrial, lysosomal, and endoplasmic reticulum and Golgi transporters together accounted for ~37%, while 11% were orphan transporters. Thirty‐seven ABC transporter transcripts were detected, primarily associated with xenobiotic and lipid transport. Compared with the intervillous region, 1676 genes—including 32 SLC and 2 ABC transporters—were differentially expressed in the decidual region. Although 117 genes in the intervillous region and 79 genes in the decidual region exhibited sexually dimorphic expression, none were SLC s. Notably, male placentas showed higher expression of ABCB1 (P‐gp), confirmed by qPCR and western blot. Our findings provide a transcriptomic map of placental transporters and highlight a potential sex‐specific difference on ABCB1 expression and xenobiotic protection.

Medication use is highly prevalent in breastfeeding persons, posing potential risks for drug exposure to nursing infants. Transporters in the lactating mammary gland carry pharmacological and toxicological significance, as they can mediate the active transfer of drugs and nutrients into breastmilk. In this narrative review, we searched and compiled current knowledge on the transport of drugs in the human mammary gland from literature indexed in PubMed (current as of 25 October 2024), and clinical evidence demonstrating active transport of drugs into milk is provided. In vitro and in vivo models of the mammary gland are outlined in brief and known drug transporters at the blood-milk barrier and their potential relevance to drug concentrations in milk are described in detail. Although clinical data show that membrane transporters mediate the transfer of multiple drugs into breastmilk, our ability to predict milk concentrations for these drugs is limited. Improving our understanding of the transporter biology and pharmacology in the mammary gland is crucial for developing models to predict drug concentrations in human milk, which will support clinicians and lactating individuals in making rational decisions to balance the benefits of breastfeeding and the risks of drug exposure to infants.

Heart failure (HF) is a chronic disease affecting 1-2% of the global population. ¹²³I-labeled meta-iodobenzylguanidine (mIBG) is FDA-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3^+/+ and Oct3^-/-mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3^+/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, adrenal gland). No difference was observed in overall plasma exposure between Oct3^+/+ and Oct3^-/- mice. Strikingly, cardiac mIBG was depleted in Oct3^-/- mice, resulting in 83% reduction in overall cardiac exposure (AUC_{0-24 hrs}: 12.7 versus 2.1 µghr/g). mIBG tissue exposure (AUC_{0-24 hrs}) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung respectively in Oct3^-/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo; and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. Significance Statement¹²³I-mIBG is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that mIBG tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, we have demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Our findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG imaging and further suggest OCT3 as a risk factor for disease progression in heart failure patients.

Accurate predictions of renal drug-drug interactions (DDIs) mediated by the human organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins (hMATEs) remain challenging. Current DDI evaluation using plasma maximal unbound inhibitor concentrations (I_max,u) and IC₅₀ values determined in single transporter-transfected cells frequently leads to false or overprediction especially for hMATE1. Emerging evidence suggests intracellular unbound inhibitor concentration may be more relevant for hMATE1 inhibition in vivo. However, determination of intrarenal inhibitor concentrations is impractical. Here we explored the use of hOCT2/hMATE1 double-transfected MDCK cells as a new in vitro tool for DDI risk assessment. Our results showed that potent in vitro hMATE1 inhibitors (hydroxychloroquine, brigatinib and famotidine) failed to inhibit metformin B-to-A flux in the double-transfected system. On the other side, the classical hOCT2/hMATE1 inhibitors, pyrimethamine and cimetidine, dose-dependently inhibited metformin apparent B-to-A permeability (P_app). The different behaviors of these hMATE1 inhibitors in the double-transfected system can be explained by their different ability to gain intracellular access either via passive diffusion or transporter-mediated uptake. A new parameter (IC_50,flux) was proposed reflecting the inhibitor9s potency on overall hOCT2/hMATE1-mediated tubular secretion. The IC_50,flux values significantly differ from the IC₅₀ values determined in single transporter-transfected cells. Importantly, the IC_50,flux accurately predicted in vivo DDIs (within 2-fold) when used in a static model. Our data demonstrated that the IC_50,flux approach circumvents the need to measure intracellular inhibitor concentrations and more accurately predicted hOCT2/hMATE1-mediated renal DDIs. This system represents a new approach that could be used for improved DDI assessment during drug development. Significance Statement This study demonstrated that flux studies in double-transfected MDCK cells and the IC_50,flux represents a better approach to assess in vivo DDI potential for the renal organic cation secretion system. Our study highlights the importance of inhibitor intracellular accessibility for accurate prediction of hMATE1-mediated renal DDIs. This approach has the potential to identify in vitro hMATE1 inhibitors that are unlikely to result in in vivo DDIs, thus reducing the burden of unnecessary and costly clinical DDI investigations.

Humans are chronically exposed to benzalkonium chlorides (BACs) from environmental sources. The FDA has recently called for additional BAC safety data, as these compounds are cytotoxic and with great potential for biochemical interactions. Biodistribution studies revealed that BACs extensively distribute to many tissues and accumulate at high levels, especially in the kidneys, but the underlying mechanisms are unclear. In this study, we characterized the interactions of BACs of varying alkyl chain length (C8 to C14) with the human organic cation transporters (hOCT1-3) and multidrug and toxin extrusion proteins (hMATE1/2K) with the goal to identify transporters that could be involved in BAC disposition. Using transporter-expressing cell lines, we showed that all BACs are inhibitors of hOCT1-3 and hMATE1/2K (IC₅₀ ranging 0.83-25.8 µM). Further, the short-chain BACs (C8 and C10) were identified as substrates of these transporters. Interestingly, while BAC C8 displayed typical Michaelis-Menten kinetics, C10 demonstrated a more complex substrate-inhibition profile. Transwell studies with transfected Madin-Darby canine kidney cells revealed that intracellular accumulation of basally applied BAC C8 and C10 was substantially higher (8.2- and 3.7-fold, respectively) in hOCT2/hMATE1 double-transfected cells in comparison to vector-transfected cells, supporting a role of these transporters in mediating renal accumulation of these compounds in vivo. Together, our results suggest that BACs interact with hOCT1-3 and hMATE1/2K as both inhibitors and substrates, and that these transporters may play important roles in tissue-specific accumulation and potential toxicity of short-chain BACs. Our findings have important implications for understanding human exposure and susceptibility to BACs due to environmental exposure. Significance Statement Humans are systemically exposed to BACs. These compounds broadly distribute through tissues, and their safety has been questioned by the FDA. Our results demonstrate that hOCT2 and hMATE1 contribute to the renal accumulation of BAC C8 and C10, and that hOCT1 and hOCT3 may be involved in the tissue distribution of these compounds. These findings can improve our understanding of BAC disposition and toxicology in humans, as their accumulation could lead to biochemical interactions and deleterious effects.

Purpose The brain is protected from circulating metabolites and xenobiotics by the blood–brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Previous studies report that P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are expressed apically or subapically at the blood-CSF barrier (BCSFB), implying a paradoxical function to mediate blood-to-CSF transport of xenobiotics. As evidence of P-gp and Bcrp activity at the BCSFB is limited, the goal of this study is to investigate functional activity of P-gp and Bcrp at the murine BCSFB using a live tissue imaging approach. Methods The choroid plexuses (CP) forming the BCSFB were freshly isolated from mouse brain ventricles and incubated with fluorescent probes calcein-AM and BODIPY FL-Prazosin. Using quantitative fluorescence microscopy, the functional contributions of Bcrp and P-gp were examined using inhibitors and mice with targeted deletion of the Abcb1a/b or Abcg2 gene. Results Apical transport of calcein-AM in choroid plexus epithelial (CPE) cells is sensitive to inhibition by elacridar and Ko143 but is unaffected by P-gp deletion. In wild-type mice, elacridar increased CPE accumulation of BODIPY FL-Prazosin by 220% whereas deletion of Bcrp increased BODIPY FL-Prazosin accumulation by 43%. There was no change in Mdr1a/1b mRNA expression in CP tissues from the Bcrp^−/− mice. Conclusions This study demonstrated functional activity of Bcrp at the BCSFB apical membrane and provided evidence supporting an additional contribution by P-gp. These findings contribute to the understanding of transport mechanisms that regulate CSF drug concentrations, which may benefit future predictions of CNS drug disposition, efficacy, and toxicity.

Small molecules targeting aberrant anaplastic lymphoma kinase (ALK) are active against ALK-positive non-small-cell lung cancers and neuroblastoma. Several targeted tyrosine kinase inhibitors (TKIs) have been shown to interact with polyspecific organic cation transporters (pOCTs), raising concerns about potential drug–drug interactions (DDIs). The purpose of this study was to assess the interaction of ALK inhibitors with pOCTs and the impact of substrate-dependent inhibition on the prediction of DDIs. Inhibition assays were conducted in transporter-overexpressing cells using meta-iodobenzylguanidine (mIBG), metformin, or 1-methyl-4-phenylpyridinium (MPP+) as the substrate. The half-maximal inhibitory concentrations (IC₅₀) of brigatinib and crizotinib for the substrates tested were used to predict their potential for in vivo transporter mediated DDIs. Here, we show that the inhibition potencies of brigatinib and crizotinib on pOCTs are isoform- and substrate-dependent. Human OCT3 (hOCT3) and multidrug and toxin extrusion protein 1 (hMATE1) were highly sensitive to inhibition by brigatinib and crizotinib for all three tested substrates. Apart from hMATE1, substrate-dependent inhibition was observed for all other transporters with varying degrees of dependency; hOCT1 inhibition showed the greatest substrate dependency, with differences in IC₅₀ values of up to 22-fold across the tested substrates, followed by hOCT2 and hMATE2-K, with differences in IC₅₀ values of up to 16- and 12-fold, respectively. Conversely, hOCT3 inhibition only showed a moderate substrate dependency (IC₅₀ variance < 4.8). Among the substrates used, metformin was consistently shown to be the most sensitive substrate, followed by mIBG and MPP+. Pre-incubation of ALK inhibitors had little impact on their potencies toward hOCT2 and hMATE1. Our results underscore the complexity of the interactions between substrates and the inhibitors of pOCTs and have important implications for the clinical use of ALK inhibitors and their DDI predictions.