Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

Current intervention strategies have not been successful in reducing the risks of adverse pregnancy complications nor maternal and fetal morbidities associated with pregnancy complications. Improving pregnancy and neonatal outcomes requires a better understanding of drug transport mechanisms at the feto-maternal interfaces, specifically the placenta and fetal membrane (FM). The role of several solute carrier uptake transporter proteins (TPs), such as the organic anion transporting polypeptide 2B1 (OATP2B1) in transporting drug across the placenta, is well-established. However, the mechanistic role of FMs in this drug transport has not yet been elucidated. We hypothesize that human FMs express OATP2B1 and functions as an alternate gatekeeper for drug transport at the feto-maternal interface. We determined the expression of OATP2B1 in term, not-in-labor, FM tissues and human FM cells [amnion epithelial cell (AEC), chorion trophoblast cell (CTC), and mesenchymal cells] using western blot analyses and their localization using immunohistochemistry. Changes in OATP2B1 expression was determined for up to 48 h after stimulation with cigarette smoke extract (CSE), an inducer of oxidative stress. The functional role of OATP2B1 was determined by flow cytometry using a zombie violet dye substrate assay. After OATP2B1 gene silencing, its functional relevance in drug transport through the feto-maternal interface was tested using a recently developed feto-maternal interface organ-on-a-chip (OOC) system that contained both FM and maternal decidual cells. Propagation of a drug (Rosuvastatin, that can be transported by OATP2B1) within the feto-maternal interface OOC system was determined by mass spectrometry. FMs express OATP2B1 in the CTC and AEC layers. In FM explants, OATP2B1 expression was not impacted by oxidative stress. Uptake of the zombie violet dye within AECs and CTCs showed OATP2B1 is functionally active. Silencing OATP2B1 in CTCs reduced Rosuvastatin propagation from the decidua to the fetal AEC layer within the feto-maternal interface-OOC model. Our data suggest that TPs in FMs may function as a drug transport system at the feto-maternal interface, a function that was previously thought to be performed exclusively by the placenta. This new knowledge will help improve drug delivery testing during pregnancy and contribute to designing drug delivery strategies to treat adverse pregnancy outcomes.

Organic Anion Transporting Polypeptide 2B1 (OATP2B1) Genetic Variants: In Vitro Functional Characterization and Association With Circulating Concentrations of Endogenous Substrates

Organic anion transporting polypeptide 2B1 (OATP2B1, gene SLCO2B1) is an uptake transporter that is thought to determine drug disposition and in particular, the oral absorption of medications. At present, the clinical relevance of SLCO2B1 genetic variation on pharmacokinetics is poorly understood. We sought to determine the functional activity of 5 of the most common missense OATP2B1 variants (c.76_84del, c.601G>A, c.917G>A, c.935G>A, and c.1457C>T) and a predicted dysfunctional variant (c.332G>A) in vitro. Furthermore, we measured the basal plasma concentrations of endogenous OATP2B1 substrates, namely estrone sulfate, dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate, coproporphyrin I (CPI), and CPIII, and assessed their relationships with SLCO2B1 genotypes in 93 healthy participants. Compared to reference OATP2B1, the transport activities of the c.332G>A, c.601G>A and c.1457C>T variants were reduced among the substrates examined (estrone sulfate, DHEAS, CPI, CPIII and rosuvastatin), although there were substrate-dependent effects. Lower transport function of OATP2B1 variants could be explained by diminished cell surface expression. Other OATP2B1 variants (c.76-84del, c.917G>A and c.935G>A) had similar activity to the reference transporter. In the clinical cohort, the SLCO2B1 c.935G>A allele was associated with both higher plasma CPI (42%) and CPIII (31%) concentrations, while SLCO2B1 c.917G>A was linked to lower plasma CPIII by 28% after accounting for the effects of age, sex, and SLCO1B1 genotypes. No association was observed between SLCO2B1 variant alleles and estrone sulfate or DHEAS plasma concentrations, however 45% higher plasma pregnenolone sulfate level was associated with SLCO2B1 c.1457C>T. Taken together, we found that the impacts of OATP2B1 variants on transport activities in vitro were not fully aligned with their associations to plasma concentrations of endogenous substrates in vivo. Additional studies are required to determine whether circulating endogenous substrates reflect OATP2B1 activity.

Development of a Rat Sandwich-Cultured Hepatocytes Model Expressing Functional Human Organic Anion Transporting Polypeptide (OATP) 1B3: A Potential Screening Tool for Liver-Targeting Compounds

Purpose: Organic anion transporting polypeptide (OATP) 1B3 transports many clinically important drugs, including statins, from blood into the liver. It exclusively expresses in human liver under normal physiological conditions. There is no rodent ortholog of human OATP1B3. Tissue targeting of therapeutic molecules mediated by transporters, including liver-targeting via liver-specific OATPs, is an emerging area in drug development. Sandwich-cultured primary hepatocytes (SCH) are a well characterized in vitro model for assessment of hepatic drug uptake and biliary excretion. The current study was designed to develop a novel rat SCH model expressing human OATP1B3 to study the hepatic disposition of OATP1B3 substrates. Methods: Primary rat hepatocytes transduced with adenoviral vectors expressing FLAG-tagged OATP1B3 (Ad-OATP1B3), a control vector Ad-LacZ, or that were non-transduced were cultured in a sandwich configuration. FLAG immunoblot and immunofluorescence-staining determined expression and localization of OATP1B3. Uptake of [3H]-cholecystokinin octapeptide (CCK-8), a specific OATP1B3 substrate, was determined. Taurocholate (TC) is a substrate routinely used in SCH to assess biliary excretion via bile canaliculi (BC) and is also a substrate of OATP1B3. [3H]-TC accumulation in cells+BC, cells, biliary excretion index (BEI) and in vitro Clbiliary were determined using B-CLEAR® technology. Results: OATP1B3 protein was extensively expressed and primarily localized on the plasma membrane in day 4 Ad-OATP1B3-transduced rat SCH. [3H]-CCK-8 accumulation in cells+BC was significantly greater (~5-13 folds, p<0.001) in day 4 SCH with vs. without Ad-OATP1B3-transduction. Expressing OATP1B3 in rat SCH significantly increased [3H]-TC accumulation in cells+BC and cells, without affecting BEI and in vitro Clbiliary. Conclusions: Rat SCH expressing human OATP1B3-is a novel in vitro model allowing simultaneous assessment of hepatic uptake, hepatocellular accumulation and biliary excretion process of a human OATP1B3 substrate. This model could be a potential tool for screening for liver-targeting compounds mediated by OATP1B3.

Essential functions of mosquito ecdysone importers in development and reproduction

ABSTRACTThe primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, 3, and 4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

Organic Anion Transporting Polypeptide 1B1 Is a Potential Reporter for Dual MR and Optical Imaging

Membrane proteins responsible for transporting magnetic resonance (MR) and fluorescent contrast agents are of particular importance because they are potential reporter proteins in noninvasive molecular imaging. Gadobenate dimeglumine (Gd-BOPTA), a liver-specific MR contrast agent, has been used globally for more than 10 years. However, the corresponding molecular transportation mechanism has not been validated. We previously reported that the organic anion transporting polypeptide (OATP) 1B3 has an uptake capability for both MR agents (Gd-EOB-DTPA) and indocyanine green (ICG), a clinically available near-infrared (NIR) fluorescent dye. This study further evaluated OATP1B1, another polypeptide of the OATP family, to determine its reporter capability. In the OATP1B1 transfected 293T transient expression model, both Gd-BOPTA and Gd-EOB-DTPA uptake were confirmed through 1.5 T MR imaging. In the constant OAPT1B1 and OATP1B3 expression model in the HT-1080 cell line, both HT-1080-OAPT1B1 and HT-1080-OATP1B3 were observed to ingest Gd-BOPTA and Gd-EOB-DTPA. Lastly, we validated the ICG uptake capability of both OATP1B1 and OATP1B3. OAPT1B3 exhibited a superior ICG uptake capability to that of OAPT1B1. We conclude that OATP1B1 is a potential reporter for dual MR and NIR fluorescent molecular imaging, especially in conjunction with Gd-BOPTA.

Gadolinium-free Magnetic Resonance Imaging of the Liver using an Oatp1-targeted Manganese(III) Porphyrin

Controversy surrounding gadolinium-based contrast agents (GBCAs) have rendered their continued utility highly contentious, but the liver-specific GBCA Gd(III) ethoxybenzyl-diethylene triamine pentaacetic acid (Gd(III)-EOB-DTPA) remains in use because it provides unique diagnostic information that could not be obtained by any other means. To address the need for an alternative liver-specific MRI contrast agent, we synthesized Mn(III) 20-(4-ethoxyphenyl) porphyrin-5,10,15-tricarboxylate (Mn(III)TriCP-PhOEt), which exhibited significantly higher r1 relaxivity than Gd(III)-EOB-DTPA, and targeted organic anion-transporting polypeptide 1 (Oatp1) channels as a biomarker of hepatocyte viability. Mn(III)TriCP-PhoEt increased the r1 relaxation rate of cells expressing rodent Oatp1a1 and human Oatp1b3, relative to control cells not expressing these liver channels. In mice, Mn(III)TriCP-PhoEt resulted in significant and specific increases in liver signal intensity on T1-weighted images, and significant decreases in liver T1 time relative to precontrast measurements. Our findings suggest that Mn(III)TriCP-PhOEt operates as a specific and sensitive MR contrast agent for in vivo liver imaging.