Digestion of surfactants does not affect their ability to inhibit P-gp-mediated transport in vitro.

While various non-ionic surfactants at low concentrations have been shown to increase the transport of P-gp substrates in vitro, in vivo studies in rats have shown that a higher surfactant concentration is needed to increase the oral absorption of e.g. the P-gp substrates digoxin and etoposide. The aim of the present study was to investigate if intestinal digestion of surfactants could be the reason for this deviation between in vitro and in vivo data. Therefore, Kolliphor EL, Brij-L23, Labrasol and polysorbate 20 were investigated for their ability to inhibit P-gp and increase digoxin absorption in vitro. Transport studies were performed in Caco-2 cells, while P-gp inhibition and cell viability assays were performed in MDCKII-MDR1 cells. Polysorbate 20, Kolliphor EL and Brij-L23 increased absorptive transport and decreased secretory digoxin transport in Caco-2 cells, whereas only polysorbate 20 and Brij-L23 showed P-gp inhibiting properties in the MDCKII-MDR1 cells. Polysorbate 20 and Brij-L23 were chosen for in vitro digestion prior to transport- or P-gp inhibiting assays. Brij-L23 was not digestible, whereas polysorbate 20 reached a degree of digestion around 40%. Neither of the two surfactants showed any significant difference in their ability to affect absorptive or secretory transport of digoxin after pre-digestion. Furthermore, the P-gp inhibiting effects of polysorbate 20 were not decreased significantly. In conclusion, the mechanism behind the non-ionic surfactant mediated in vitro P-gp inhibition seemed independent of the intestinal digestion and the results presented here did not suggest it to be the cause of the observed discrepancy between in vitro and in vivo.

Co-release of paclitaxel and encequidar from amorphous solid dispersions increase oral paclitaxel bioavailability in rats.

The oral bioavailability of paclitaxel is limited due to low solubility and high affinity for the P-glycoprotein (P-gp) efflux transporter. Here we hypothesized that maximizing the intestinal paclitaxel levels through apparent solubility enhancement and controlling thesimultaneous release of both paclitaxel and the P-gp inhibitor encequidar from amorphous solid dispersions (ASDs) would increase the oral bioavailability of paclitaxel. ASDs of paclitaxel and encequidar in polyvinylpyrrolidone K30 (PVP-K30), hydroxypropylmethylcellulose 5 (HPMC-5), and hydroxypropylmethylcellulose 4 K (HPMC-4K) were hence prepared by freeze-drying. In vitro dissolution studies showed that both compounds were released fastest from PVP-K30, then from HPMC-5, and slowest from HPMC-4K ASDs. The dissolution of paclitaxel from all polymers resulted in stable concentration levels above the apparent solubility. The pharmacokinetics of paclitaxel after oral administration to male Sprague-Dawley rats was investigated with or without 1 mg/kg encequidar, as amorphous solids or polymer-based ASDs. The bioavailability of paclitaxel increased 3- to 4-fold when administered as polymer-based ASDs relative to solid amorphous paclitaxel. However, when amorphous paclitaxel was co-administered with encequidar, either as an amorphous powder or as a polymer-based ASD, the bioavailability increased 2- to 4-fold, respectively. Interestingly, a noticeable increase in paclitaxel bioavailability of 24-fold was observed when paclitaxel and encequidar were co-administered as HPMC-5-based ASDs. We, therefore, suggest that controlling the dissolution rate of paclitaxel and encequidar in order to obtain simultaneous and timed release from polymer-based ASDs is a strategy to increase oral paclitaxel bioavailability.

Exploring Amino Acid Transporters as Therapeutic Targets for Cancer: An Examination of Inhibitor Structures, Selectivity Issues, and Discovery Approaches.

Amino acid transporters are abundant amongst the solute carrier family and have an important role in facilitating the transfer of amino acids across cell membranes. Because of their impact on cell nutrient distribution, they also appear to have an important role in the growth and development of cancer. Naturally, this has made amino acid transporters a novel target of interest for the development of new anticancer drugs. Many attempts have been made to develop inhibitors of amino acid transporters to slow down cancer cell growth, and some have even reached clinical trials. The purpose of this review is to help organize the available information on the efforts to discover amino acid transporter inhibitors by focusing on the amino acid transporters ASCT2 (SLC1A5), LAT1 (SLC7A5), xCT (SLC7A11), SNAT1 (SLC38A1), SNAT2 (SLC38A2), and PAT1 (SLC36A1). We discuss the function of the transporters, their implication in cancer, their known inhibitors, issues regarding selective inhibitors, and the efforts and strategies of discovering inhibitors. The goal is to encourage researchers to continue the search and development within the field of cancer treatment research targeting amino acid transporters.

Increased bioavailability of a P-gp substrate: Co-release of etoposide and zosuquidar from amorphous solid dispersions.

P-glycoprotein (P-gp) inhibitors, like zosuquidar, partly increase oral bioavailability of P-gp substrates, such as etoposide. Here, it was hypothesised that co-release of etoposide and zosuquidar from amorphous solid dispersions (ASDs) may further increase oral etoposide bioavailability. This was envisioned through simultaneous co-release and subsequent spatiotemporal association of etoposide and zosuquidar in the small intestinal lumen. To further achieve this, ASDs of etoposide and zosuquidar in polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMC) 5, and HPMC 4 k were prepared by freeze-drying. From these ASDs, etoposide release was fastest from PVP, then HPMC 5 and slowest from HPMC 4. Release from PVP and HPMC5 resulted in stable supersaturations of etoposide. In transcellular permeability studies across MDCKII-MDR1 cell monolayers, the accumulated amount of etoposide increased 3.7-4.9-fold from amorphous etoposide or when incorporated into PVP- or HPMC 5-based ASDs, compared to crystalline etoposide. In vivo, the oral bioavailability in Sprague Dawley rats increased from 1.0 to 2.4-3.4 %, when etoposide was administered as amorphous drug or in ASDs. However, when etoposide and zosuquidar were co-administered, the oral bioavailability increased further to 8.2-18 %. Interestingly, a distinct increase in oral etoposide bioavailability to 26 % was observed when etoposide and zosuquidar were co-administration in HPMC5-based ASDs. The supersaturation of etoposide as well as the simultaneous co-release of etoposide and zosuquidar in the small intestinal lumen may explain the observed bioavailability increase. Overall, this study suggested that simultaneous co-release of an amorphous P-gp substrate and inhibitor may be a novel and viable formulation strategy to increase the bioavailability P-gp substrates.

Combinational Inhibition of P-Glycoprotein-Mediated Etoposide Transport by Zosuquidar and Polysorbate 20.

P-glycoprotein (P-gp) limits the oral absorption of drug substances. Potent small molecule P-gp inhibitors (e.g., zosuquidar) and nonionic surfactants (e.g., polysorbate 20) inhibit P-gp by proposedly different mechanisms. Therefore, it was hypothesised that a combination of zosuquidar and polysorbate 20 may potentiate inhibition of P-gp-mediated efflux. P-gp inhibition by zosuquidar and polysorbate 20 in combination was assessed in a calcein-AM assay and in a transcellular etoposide permeability study in MDCKII-MDR1 and Caco-2 cells. Furthermore, solutions of etoposide, zosuquidar, and polysorbate 20 were orally administered to Sprague Dawley rats. Zosuquidar elicited a high level of nonspecific adsorption to various labware, which significantly affected the outcomes of the in vitro studies. Still, at certain zosuquidar and polysorbate 20 concentrations, additive P-gp inhibition was observed in vitro. In vivo, however, oral etoposide bioavailability decreased by coadministration of both zosuquidar and polysorbate 20 when compared to coadministration of etoposide with zosuquidar alone. For future formulation development, the present study provided important and novel knowledge about nonspecific zosuquidar adsorption, as well as insights into combinational P-gp inhibition by a third-generation P-gp inhibitor and a P-gp-inhibiting nonionic surfactant.

Investigations of potential non-amino acid SNAT2 inhibitors.

The sodium-coupled neutral amino acid transporter 2 (SNAT2, SLC38A2) has been implicated in cancer for its ability to supply cancer cells with glutamine and sarcosine. A recent high-throughput screen published by Gauthier-Coles et al. identified the non-amino acid 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide (MMTC or 57E) as a potent and selective SNAT2 inhibitor. Here we have investigated the ability of MMTC and four other compounds selected from the screen by Gauthier-Coles et al. to decrease 3H-Gly uptake in hyperosmotically treated human prostate cancer PC-3 cells. In these cells, SNAT2 is highly upregulated when the cells are hyperosmotically stressed for 24 h and is the primary contributor to glycine uptake. The five compounds were investigated at concentrations of 1-50 µM based on their equilibrium solubility. At 37°C the equilibrium solubility in HEPES buffered HBSS at pH 7.4 was measured to be 24.9 (53B), 56.1 (54F), 13.3 (55B), and 27.5 (57B) µM, respectively. The equilibrium solubility of MMTC was below the detection limit of the HPLC-UV method, thus less than 1.8 µM. However, a kinetic solubility of approximately 2.5-10 µM could be achieved during the course of the uptake study. In contrast to the previous publication, MMTC showed no inhibition of SNAT2-mediated 3H-Gly uptake in PC-3 cells at a concentration of 1 or 5 µM, despite a published IC50 of 0.8 µM. Similarly, 53B, 55B, and 57B showed no inhibition at soluble conditions, whereas 54F showed approximately 20% inhibition at 50 µM. In our experimental setup, the investigated compounds showed limited potential as SNAT2 inhibitors.

SNAT2 is responsible for hyperosmotic induced sarcosine and glycine uptake in human prostate PC-3 cells.

Solute carriers (SLC) are important membrane transport proteins in normal and pathophysiological cells. The aim was to identify amino acid SLC(s) responsible for uptake of sarcosine and glycine in prostate cancer cells and investigate the impact hereon of hyperosmotic stress. Uptake of 14C-sarcosine and 3H-glycine was measured in human prostate cancer (PC-3) cells cultured under isosmotic (300 mOsm/kg) and hyperosmotic (500 mOsm/kg) conditions for 24 h. Hyperosmotic culture medium was obtained by supplementing the medium with 200 mM of the trisaccharide raffinose. Amino acid SLC expression was studied using RT-PCR, real-time PCR, and western blotting. siRNA knockdown of SNAT2 was performed. Experiments were conducted in at least 3 independent cell passages. The uptake of Sar and Gly was increased approximately 8-ninefold in PC-3 cells after 24 h hyperosmotic culture. PAT1 mRNA and protein could not be detected, while SNAT2 was upregulated at the mRNA and protein level. Transfection with SNAT2-specific siRNA reduced Vmax of Sar uptake from 2653 ± 38 to 513 ± 38 nmol mg protein-1 min-1, without altering the Km value (3.19 ± 0.13 vs. 3.42 ± 0.71 mM), indicating that SNAT2 is responsible for at least 80% of Sar uptake in hyperosmotic cultured PC-3 cells. SNAT2 is upregulated in hyperosmotic stressed prostate cancer cells and SNAT2 is responsible for cellular sarcosine and glycine uptake in hyperosmotic cultured PC-3 cells. Sar is identified as a substrate for SNAT2, and this has physiological implications for understanding cellular solute transport in prostate cancer cells.

Mechanism behind Polysorbates' Inhibitory Effect on P-Glycoprotein.

Much effort has been invested in the search for modulators of membrane transport proteins such as P-glycoprotein (P-gp) to improve drug bioavailability and reverse multidrug resistance in cancer. Nonionic surfactants, a class of pharmaceutical excipients, are known to inhibit such proteins, but knowledge about the exact mechanism of this inhibition is scarce. Here, we perform multiscale molecular dynamics simulations of one of these surfactants, polysorbate 20 (PS20), to reveal the behavior of such compounds on the molecular level and thereby discover the molecular mechanism of the P-gp inhibition. We show that the amphiphilic headgroup of PS20 is too hydrophobic to partition in the water phase, which drives the binding of PS20 to the amphiphilic drug-binding domain of P-gp and thereby causes the inhibition of the protein. Based on our findings, we conclude that PS20 primarily inhibits P-gp through direct binding to the drug-binding domain (DBD) from the extracellular leaflet.

Exploration of in vitro drug release testing methods for saquinavir microenvironmental pH modifying buccal films.

Buccal films containing a pH modifying excipient may be able to increase bioavailability of drugs with pH-dependent solubility such as saquinavir. Access to suitable in vitro drug release testing methods may facilitate buccal formulation development. This study aimed to explore two release testing methods for characterising buccal films and to elucidate the relationship between microenvironmental pH (pHM, i.e. the pH around the swelling films) and saquinavir release. The Franz diffusion cell method was applicable to investigate the effect of hydroxypropyl methylcellulose (HPMC) grade on saquinavir release. Films containing HPMC K3 LV had a faster saquinavir release than films containing HPMC K100 LV. A UV/Vis imaging method was developed to visualise saquinavir release and pHM changes during the initial dissolution. Within 5 min, the pHM decreased from 6.8 to around 5.4 for HPMC K100 LV-based films containing 11.1 % or 16.6 % (w/w) malic acid. Subsequently, the pHM increased due to increasing concentrations of saquinavir. An increase in malic acid content led to a faster saquinavir release. The combination of methods may be broadly applicable for excipient screening in development of buccal formulations. The imaging approach holds promise for characterizing other pH modifying formulation principles.

Inhibitory Effects of 17-α-Ethinyl-Estradiol and 17-ß-Estradiol on Transport Via the Intestinal Proton-Coupled Amino Acid Transporter (PAT1) Investigated In Vitro and In Vivo.

The proton-coupled amino acid transporter, PAT1, is known to be responsible for intestinal absorption drug substances such as gaboxadol and vigabatrin. The aim of the present study was to investigate, if 17-α-ethinyl-estradiol (E-E2) and 17-ß-estradiol (E) inhibit PAT1-mediated intestinal absorption of proline and taurine in vitro in Caco-2 cells and in vivo using Sprague-Dawley rats to assess the potential for taurine-drug interactions. E and E-E2 inhibited the PAT1-mediated uptake of proline and taurine in Caco-2 cells with IC50 values of 10.0-50.0 µM without major effect on other solute carriers such as the taurine transporter (TauT), di/tri-peptide transporter (PEPT1), and serotonin transporter (SERT1). In PAT1-expressing oocytes E and E-E2 were non-translocated inhibitors. In Caco-2 cells, E and E-E2 lowered the maximal uptake capacity of PAT1 in a non-competitive manner. Likewise, the transepithelial permeability of proline and taurine was reduced in presence of E and E-E2. In male Sprague Dawley rats pre-dosed with E-E2 a decreased maximal plasma concentration (Cmax) of taurine and increased the time (tmax) to reach this was indicated, suggesting the possibility for an in vivo effect on the absorption of PAT1 substrates. In conclusion, 17-α-ethinyl-estradiol and 17-ß-estradiol were identified as non-translocated and non-competitive inhibitors of PAT1.

Oral etoposide and zosuquidar bioavailability in rats: Effect of co-administration and in vitro-in vivo correlation of P-glycoprotein inhibition.

P-glycoprotein inhibitors, like zosuquidar, have widely been used to study the role of P-glycoprotein in oral absorption. Still, systematic studies on the inhibitor dose-response relationship on intestinal drug permeation are lacking. In the present study, we investigated the effect of 0.79 nM-2.5 µM zosuquidar on etoposide permeability across Caco-2 cell monolayers. We also investigated etoposide pharmacokinetics after oral or IV administration to Sprague Dawley rats with co-administration of 0.063-63 mg/kg zosuquidar, as well as the pharmacokinetics of zosuquidar itself. Oral zosuquidar bioavailability was 2.6-4.2%, while oral etoposide bioavailability was 5.5 ± 0.9%, which increased with increasing zosuquidar doses to 35 ± 5%. The intestinal zosuquidar concentration required to induce a half-maximal increase in bioavailability was estimated to 180 µM. In contrast, the IC50 of zosuquidar on etoposide permeability in vitro was only 5-10 nM, and a substantial in vitro-in vivo discrepancy of at least four orders of magnitude was thereby identified. Overall, the present study provides valuable insights for future formulation development that applies fixed dose combinations of P-glycoprotein inhibitors to increase the absorption of poorly permeable P-glycoprotein substrate drugs.

BeerMIMS: Exploring the Use of Membrane-Inlet Mass Spectrometry (MIMS) Coupled to KNIME for the Characterization of Danish Beers.

Beer is a complex mix of more than 7700 compounds, around 800 of which are volatile. While GC-MS has been actively employed in the analysis of the volatome of beer, this method is challenged by the complex nature of the sample. Herein, we explored the possible of using membrane-inlet mass spectrometry (MIMS) coupled to KNIME to characterize local Danish beers. KNIME stands for Konstanz Information Miner and is a free open-source data processing software which comes with several prebuilt nodes, that, when organized, result in data processing workflows allowing swift analysis of data with outputs that can be visualized in the desired format. KNIME has been shown to be promising in automation of large datasets and requires very little computing power. In fact, most of the computations can be carried out on a regular PC. Herein, we have utilized a KNIME workflow for data visualization of MIMS data to understand the global volatome of beers. Feature identification was not possible as of now but with a combination of MIMS and a KNIME workflow, we were able to distinguish beers from different micro-breweries located in Denmark, laying the foundation for the use of MIMS in future analysis of the beer volatome.

Microenvironmental pH modifying films for buccal delivery of saquinavir: Effects of organic acids on pH and drug release in vitro.

Buccal delivery of saquinavir has the advantage to bypass the gastrointestinal enzymatic degradation and the hepatic first-pass metabolism. Saquinavir has a pH-dependent solubility and is poorly soluble in human saliva at the physiological pH. Decreasing microenvironmental pH (pHM) in saliva may increase saquinavir release from buccal formulations. The present study aimed to investigate the effects of organic acids on the pHM, saquinavir release in vitro and the solid-state form of saquinavir. An UV/Vis imaging method was used to measure pHM. After 5 min of swelling of the buccal films containing malic acid, pHM was reduced from 6.8 to 5.4. The films containing malic acid were more efficient in maintaining low pHM than films containing citric acid and succinic acid. Addition of organic acids in the buccal films resulted in a faster drug release than films without acids due to the reduced pHM. However, the enhancement of saquinavir release was limited by the fast release of organic acids. Addition of malic acid and citric acid suppressed the crystallization of saquinavir during 3 months storage at the elevated temperature (40 °C) and humidity (RH 75%) respectively. These results suggest that pHM modifying film is a potential formulation strategy for buccal delivery of saquinavir.

High-dose etoposide formulations do not saturate intestinal P-glycoprotein: Development, stability, and pharmacokinetics in Sprague-Dawley rats.

It has been suggested that oral absorption of low-permeable P-glycoprotein (P-gp) substrates can be increased through saturation of P-gp. For BCS class IV drug substances, saturating P-gp is challenging due to low aqueous solubility. The present study investigated if the BCS IV drug substance etoposide could be solubilized to a concentration saturating P-gp after oral administration. A formulation consisting of 10% (w/v) of pluronic® F-127 and polyvinylpyrrolidone/vinyl acetate (PVP/VA), and 57% (v/v) ethanol enhanced etoposide's solubility approximately 100 times (16 mg mL-1) compared to its aqueous solubility. In vitro, this formulation was stable upon dilution in simulated intestinal fluid. In male Sprague-Dawley rats, oral administration of increasing solubilized etoposide doses using the formulation matrix increased the AUC0-∞ of etoposide dose-proportionally but resulted in a lower absolute oral bioavailability (F) and rate of absorption as compared to control. At the highest investigated dose (100 mg kg-1), AUC0-∞ and Cmax were significantly increased by 2.9- and 1.4-fold, respectively, compared to control dosed at 20 mg kg-1. A single oral dose of 20 mg kg-1 zosuquidar followed by 20 mg kg-1 oral etoposide increased F 8.6-fold. In conclusion, a stable formulation with improved etoposide solubility was developed, yet the formulation did not result in increased oral bioavailability of etoposide.

Acamprosate Is a Substrate of the Human Organic Anion Transporter (OAT) 1 without OAT3 Inhibitory Properties: Implications for Renal Acamprosate Secretion and Drug-Drug Interactions.

Acamprosate is an anionic drug substance widely used in treating symptoms of alcohol withdrawal. It was recently shown that oral acamprosate absorption is likely due to paracellular transport. In contrast, little is known about the eliminating mechanism clearing acamprosate from the blood in the kidneys, despite the fact that studies have shown renal secretion of acamprosate. The hypothesis of the present study was therefore that renal organic anion transporters (OATs) facilitate the renal excretion of acamprosate in humans. The aim of the present study was to establish and apply OAT1 (gene product of SLC22A6) and OAT3 (gene product of SLC22A8) expressing cell lines to investigate whether acamprosate is a substrate or inhibitor of OAT1 and/or OAT3. The studies were performed in HEK293-Flp-In cells stably transfected with SLC22A6 or SLC22A8. Protein and functional data showed that the established cell lines are useful for studying OAT1- and OAT3-mediated transport in bi-laboratory studies. Acamprosate inhibited OAT1-mediated p-aminohippuric acid (PAH) uptake but did not inhibit substrate uptake via OAT3 expressing cells, neither when applied concomitantly nor after a 3 h preincubation with acamprosate. The uptake of PAH via OAT1 was inhibited in a competitive manner by acamprosate and cellular uptake studies showed that acamprosate is a substrate for OAT1 with a Km-value of approximately 700 µM. Probenecid inhibited OAT1-mediated acamprosate uptake with a Ki-value of approximately 13 µM, which may translate into an estimated clinically significant DDI index. In conclusion, acamprosate was identified as a substrate of OAT1 but not OAT3.

Montmorillonite-surfactant hybrid particles for modulating intestinal P-glycoprotein-mediated transport.

In the small intestine, P-glycoprotein (P-gp) may limit the permeability of its substrates, which lead to reduced oral absorption. To circumvent the effect of P-gp, a nanocomposite material termed montmorillonite-surfactant hybrid particles was developed. The particles consisted of montmorillonite, the P-gp-inhibiting, nonionic surfactant, polysorbate 20, and the P-gp substrate, digoxin. The present study aimed to investigate if montmorillonite-surfactant hybrid particles could modulate the absorption of digoxin in vivo. Montmorillonite-surfactant hybrid particles were prepared by lyophilising an aqueous suspension of the constituents. Scanning electron microscopy, thermogravimetric analysis, and powder X-ray diffraction revealed an altered surface morphology, decreased water content, and intercalation of polysorbate 20 between montmorillonite layers. The particles were administered orally to Sprague Dawley rats, and digoxin was quantified by liquid chromatography-tandem mass spectrometry. Control digoxin-containing montmorillonite decreased the exposure of digoxin. In contrast, montmorillonite-surfactant hybrid particles increased AUC and Cmax by 31 and 91%, respectively, compared to digoxin in solution. It was hypothesised that montmorillonite-surfactant hybrid particles increased digoxin exposure by forming mucosa-localised elevated concentrations of polysorbate 20 and digoxin, which enhanced the inhibitory effect of polysorbate 20 on P-gp.

The Permeation of Acamprosate Is Predominantly Caused by Paracellular Diffusion across Caco-2 Cell Monolayers: A Paracellular Modeling Approach.

In drug development, estimating fraction absorbed (Fa) in man for permeability-limited compounds is important but challenging. To model Fa of such compounds from apparent permeabilities (Papp) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the Papp across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The Papp values of acamprosate and of several paracellular marker molecules were measured. These Papp values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (Pmodeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate Papp across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate Papp, 1.56 ± 0.28 × 10-7 cm·s-1, is similar to the Papp of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10-7 cm·s-1), lucifer yellow (1.80 ± 0.35 × 10-7 cm·s-1), and fluorescein (2.10 ± 0.28 × 10-7 cm·s-1), and lower than that of atenolol (7.32 ± 0.60 × 10-7 cm·s-1; mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Qmonolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the Papp of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The Pmodeled for all the paracellular markers and acamprosate was dominated by the Ppara component and matched the experimentally obtained Papp. Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B0,+AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the Pmodeled estimated well Ppara, and the paracellular route appears to be the predominant mechanism for acamprosate Papp across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.

Nfat5 is involved in the hyperosmotic regulation of Tmem184b: a putative modulator of ibuprofen transport in renal MDCK I cells.

Nuclear factor of activated T cells 5 (NFAT5) is a transcription factor involved in the regulation of several genes involved in the response to extracellular hyperosmolality. Recently, the uptake of ibuprofen by an as yet unknown carrier was suggested in Madin-Darby canine kidney (MDCK) I cells exposed to hyperosmolality. We therefore speculated that Nfat5 could be involved in the regulation of this ibuprofen carrier. Reverse transfection with siRNA against Nfat5 was used to knock down Nfat5 in MDCK I cells. The uptake of both radiolabelled taurine and ibuprofen was measured in MDCK I cells, first treated with siRNA against Nfat5 and afterwards cultivated with raffinose-supplemented normal growth medium (500 mOsm) for 24 h. The siRNA transfection resulted in knockdown of Nfat5, and uptake of both taurine and ibuprofen was significantly decreased in transfected MDCK I cells. The decrease in ibuprofen uptake indicates that Nfat5 is involved in upregulation of the ibuprofen carrier. A transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 revealed 989 genes upregulated by Nfat5 during hyperosmotic exposure. From these genes, the gene product transmembrane protein 184b was found to be regulated by Nfat5, and Tmem184b was the only potential gene product involved in the uptake of ibuprofen in MDCK I cells. DATASET: The RNA sequencing dataset is available from the NCBI Gene Expression 452 Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE122074.

MRP2-mediated transport of etoposide in MDCKII MRP2 cells is unaffected by commonly used non-ionic surfactants.

The aim of the present study was to investigate the ability of non-ionic surfactants to inhibit MRP2-mediated transport in vitro in MDCKII MRP2 cells. Transport studies across MDCKII MRP2 cell monolayers were performed using 3H-etoposide and 3H-digoxin. 19 different non-ionic surfactants, including several polysorbates (PS), cremophor EL, vitamin E-TPGS, and n-nonyl ß-D-glucopyranoside (NG), were investigated. Barrier function of the cells was investigated measuring TEER and transport of 14C-glycine. The amount of isotope was quantified using liquid scintillation counting. In MDCKII MRP2 cells a polarized transport of etoposide and digoxin in the secretory (basolateral to apical) direction with efflux ratios of 5.5 ±â€¯0.7 and 18.5 ±â€¯4.2, respectively, was measured. P-gp inhibitors such as valspodar and zosuquidar did not affect etoposide transport, and furthermore PS20 decreased secretory transport of digoxin, but not of etoposide. Transport of etoposide was therefore mainly MRP2-mediated and used as a probe to investigate pharmaceutical excipients. Non-ionic surfactants did not modulate etoposide transport across intact cell monolayers of MRP2 overexpressing MDCKII cells, although preliminary studies suggest that most were able to alter MRP2-mediated efflux of the fluorescent 5-chloromethylfluorescein (CMF). In conclusion, etoposide transport across MDCKII MRP2 cells was modulated by cyclosporin A, an inhibitor of MRP2 and P-gp, but not by specific P-gp inhibitors (valspodar and zosuquidar), which suggests that etoposide transport is primarily influenced by MRP2. In addition, commonly used non-ionic surfactants did not decrease MRP2-mediated etoposide transport in MDCKII MRP2 cells. These results suggest that etoposide transport in MDCKII MRP2 cells is a model system to investigate MRP2 interactions, and that surfactants may not have a large potential for increasing oral bioavailability of drugs through inhibition of MRP2 transport activity.

Nonionic surfactants modulate the transport activity of ATP-binding cassette (ABC) transporters and solute carriers (SLC): Relevance to oral drug absorption.

Recently, it has become evident that pharmaceutical excipients may interfere with the activity of ATP-binding cassette (ABC) transporters and solute carriers (SLC). The present review aims to provide an overview of surfactants shown to modulate substrate transport via SLCs and ABCs, and to discuss the relevance for oral drug absorption. In vitro, more than hundred surfactants have been suggested to decrease the efflux activity of P-glycoprotein (P-gp, ABCB1), and many of these surfactants also inhibit the breast cancer resistance protein (BCPR, ABCG2), while conflicting results have been reported for multidrug resistance-associated protein 2 (MRP2, ABCC2). In animals, surfactants such as pluronic® P85 and polysorbate 20 have been shown to enhance the oral absorption of P-gp and BCRP substrates. Many surfactants, including cremophor® EL and Solutol® HS 15 inhibiting ABC transporters, were also found to inhibit SLCs in cell cultures. These carriers were SLC16A1, SLC21A3, SLC21A9, SLC15A1-2, and SLC22A1-3. This overlap in specificity of surfactants that inhibit both transporters and carriers might influence the oral absorption of various drug substances, nutrients, and vitamins. Such biopharmaceutical elements may be relevant for future drug formulation design.