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This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L -1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A new design of permeation module based on 3D printing was developed to monitor the interaction of exogenic compounds with cell membrane transporters in real-time. The fluorescent marker Rhodamine 123 (Rho123) was applied as a substrate to study the activity of the P-glycoprotein membrane transporter using the MDCKII-MDR1 genetically modified cell line. In addition, the inhibitory effect of verapamil (Ver), a prototype P-glycoprotein inhibitor, was examined in the module, demonstrating an enhanced Rho123 transfer and accumulation into cells as well as the applicability of the module for P-glycoprotein inhibitor testing. Inhibition was demonstrated for different ratios of Rho123 and Ver, and their competition in terms of interaction with the P-glycoprotein transporter was monitored in real-time. Employing the 3D-printed module, permeation testing was shortened from 8 h in the conventional module to 2 h and evaluation based on kinetic profiles in every 10 min was possible in both donor and acceptor compartments. We also show that monitoring Rho123 levels in both compartments enables calculate the amount of Rho123 accumulated inside cells without the need of cell lysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Members of acyclic nucleoside phosphonates (ANPs) possess antiviral and antiproliferative activities. However, several clinically important ANPs may cause renal injury, most likely due to their active accumulation in the renal tubular cells. The goal of this study was to investigate in vitro relationships between the affinity of several structurally related potent ANPs to selected human transporters and their cytotoxicity. SLC (solute carrier family) transporters (hOAT1, hOCT2, hCNT2, hCNT3) and ABC (ATP-binding cassette) transporters (MDR1, BCRP), which are typically expressed in the kidney, were included in the study. The transport and toxic parameters of the tested compounds were compared to those of two clinically approved ANPs, adefovir and tenofovir. Transport studies with transiently transfected cells were used as the main method in the experiments. Most of the ANPs studied showed the potency to interact with hOAT1. GS-9191, a double prodrug of PMEG, displayed an affinity for hOAT1 comparable with that of adefovir and tenofovir. No significant interaction of the tested ANPs with hOCT2, hCNT2 and hCNT3 was observed. Only GS-9191 was found to be a strong inhibitor for both MDR1 and BCRP. PMEO-DAPy showed the potency to interact with MDR1. Most of the tested substances caused a significant decrease in cellular viability in the cells transfected with hOAT1. Only with the exclusion of GS-9191, a relatively lipophilic compound, did the in vitro cytotoxicity of the ANPs closely correspond to their potential to interact with hOAT1. The increased cytotoxicity of the studied ANPs found in OAT1 transfected cells was effectively reduced by OAT inhibitors probenecid and quercetin. The higher cytotoxicity of the compounds with affinity to hOAT1 proved in the inhibitory experiments evidences that ANPs are not only inhibitors but also substrates of hOAT1. Any clear relationship between the potency of ANPs to inhibit the studied efflux transporters and their cytotoxicity was not demonstrated. In conclusion, the study documented that among the studied transporters hOAT1 seems to be the decisive determinant for renal handling in most of the tested ANPs. This transporter may also play an important role in the mechanism of their potential cytotoxic effects. These facts are in good accordance with previous findings in the clinically used ANPs., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Although rat is the most widely used model of glucocorticoid programming of the fetus, the role of rat placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in the transplacental pharmacokinetics of the naturally occurring glucocorticoid, corticosterone, has not yet been fully elucidated. In this study, expression of 11beta-HSD2 in the rat placenta on two different gestation days (16 and 22) was examined using quantitative RT-PCR and Western blotting, and dually perfused rat term placenta was employed to evaluate its functional capacity to transfer and metabolize corticosterone. Marked decrease in placental expression of 11beta-HSD2 toward term was observed on both mRNA and protein levels. In perfusion studies, increasing maternal corticosterone concentration from 3 to 200 nM resulted in the fall of 11beta-HSD2 conversion capacity from 64.3 to 16.3%, respectively. Enzyme saturation occurred at about 50 nM substrate concentration. When delivering corticosterone (3 or 100 nM) from the fetal side, a similar decline of 11beta-HSD2 conversion capacity was observed (66.5% and 48.5%, respectively). Addition of carbenoxolone (10 or 100 microM), a non-specific 11beta-HSD inhibitor, to maternal perfusate decreased conversion capacity from 66.7 to 12.6 or 8.1%, respectively. Similarly potent inhibitory effect was observed in feto-maternal studies. Neither saturation nor inhibition of 11beta-HSD2 was associated with transformation of corticosterone in metabolites other than 11-dehydrocorticosterone. These data suggest that 11beta-HSD2 is the principal enzyme controlling transplacental passage of corticosterone in rats and is able to eliminate corticosterone in both maternal and fetal circulations.

The sequential injection (SIA) technique was applied in pharmacokinetic studies of the transporter-mediated passage of a model substrate, rhodamine 123 (Rho123), through the dually perfused rat term placenta. The method described was used for real-time monitoring of Rho123 concentrations in both the maternal and fetal compartments. Determination was processed by fluorescence detection (lambda(ex)=480 nm, lambda(em)580 nm); calibration curve was linear over the range 0.01-50 mumoll(-1) (r=0.99965), detection limit was 10 nmoll(-1) (3sigma) and RSD2% (10 readings). Transport of Rho123 was scanned under various conditions (ATP-synthesis inhibition) and several inhibitors of P-glycoprotein transporter were tested (e.g. quinidine). The advantages of the modern SIA method-an automated analytical tool providing both fast and precise analysis-were successfully demonstrated for examination of transport profiles to investigate the effect of P-glycoprotein on the placental transfer of Rho123.

The transfer kinetics of cyclosporine across the dually perfused rat placenta in the maternal to fetal direction and a possible involvement of P-glycoprotein were investigated. The transplacental clearance of cyclosporine in the materno-fetal direction was found to be dependent on the maternal inflow concentration of cyclosporine. Coadministration of cyclosporine with an excess of quinidine or chlorpromazine into the maternal compartment revealed 1.7- and 1.9-fold increase in cyclosporine concentration in the fetal compartment. In the experiments where quinidine was present both in the maternal and fetal compartments, cyclosporine appeared in the fetal compartment significantly faster, and its amount was three times higher when compared with controls. Conversely, quinidine or chlorpromazine did not affect the transplacental passage of L-[(3)H]-glucose. The interference of quinidine with the metabolism of cyclosporine in the placenta was excluded because only traces of M-1 and M-17 metabolites were found in the fetal solutions. Sodium azide, a mitochondrial respiratory inhibitor, was found to double the rate of cyclosporine, but not L-[(3)H]-glucose, passage across the placenta. Our findings indicate that P-glycoprotein pumps cyclosporine out of the trophoblast cells of the rat placenta in the ATP-dependent manner and restricts the passage of cyclosporine across the placental barrier., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1583-1592, 2001)