Carrier-mediated Transport Mechanism Research Articles

The Active Glucuronide Metabolite of the Brain Protectant IMM-H004 with Poor Blood-Brain Barrier Permeability Demonstrates a High Partition in the Rat Brain via Multiple Mechanisms.

Glucuronidation is an essential metabolic pathway for a variety of drugs. IMM-H004 is a novel neuroprotective agent against ischemic stroke, and its glucuronide metabolite IMM-H004G exhibits similar pharmacological activity. Despite possessing a higher molecular weight and polarity, brain exposure of IMM-H004G is much higher than that of IMM-H004. This study aimed to investigate the brain metabolism and transport mechanisms of IMM-H004 and IMM-H004G. First, the possibility of IMM-H004 glucuronidation in the brain was evaluated in several human brain cell lines and rat homogenate. Subsequently, the blood-brain barrier carrier-mediated transport mechanism of IMM-H004 and IMM-H004G was studied using overexpression cell models. In addition, intracerebroventricular injection, in situ brain perfusion model, and microdialysis/microinjection techniques were performed to study the distribution profiles of IMM-H004 and IMM-H004G. IMM-H004 could be metabolized to IMM-H004G in both rat brain and HEB cells mediated by UGT1A7. However, IMM-H004G could not be hydrolyzed back into IMM-H004. Furthermore, the entry and efflux of IMM-H004 in the brain were mediated by the pyrilamine-sensitive H+/OC antiporter and P-gp, respectively, while the transport of IMM-H004G from the blood to the brain was facilitated by OATP1A2 and OATP2B1. Ultimately, stronger concentration gradients and OATP-mediated uptake played a critical role in promoting greater brain exposure of IMM-H004G. The active glucuronide metabolite of the brain protectant IMM-H004 with poor blood-brain barrier permeability demonstrates a high partition in the rat brain via multiple mechanisms, and our findings deepen the understanding of the mechanisms underlying the blood-brain barrier metabolism and transport of active glucuronide conjugates.

Integration of In Silico, In Vitro and In Situ Tools for the Preformulation and Characterization of a Novel Cardio-Neuroprotective Compound during the Early Stages of Drug Development.

The main aim of this work is the biopharmaceutical characterization of a new hybrid benzodiazepine-dihydropyridine derivative, JM-20, derived with potent anti-ischemic and neuroprotective effects. In this study, the pKa and the pH-solubility profile were experimentally determined. Additionally, effective intestinal permeability was measured using three in vitro epithelial cell lines (MDCK, MDCK-MDR1 and Caco-2) and an in situ closed-loop intestinal perfusion technique. The results indicate that JM-20 is more soluble at acidic pH (9.18 ± 0.16); however, the Dose number (Do) was greater than 1, suggesting that it is a low-solubility compound. The permeability values obtained with in vitro cell lines as well as with the in situ perfusion method show that JM-20 is a highly permeable compound (Caco-2 value 3.8 × 10−5). The presence of an absorption carrier-mediated transport mechanism was also demonstrated, as well as the efflux effect of P-glycoprotein on the permeability values. Finally, JM-20 was provisionally classified as class 2 according to the biopharmaceutical classification system (BCS) due to its high intestinal permeability and low solubility. The potential good oral absorption of this compound could be limited by its solubility.

Feasibility of Adsorption Kinetic Models to Study Carrier-Mediated Transport of Heavy Metal Ions in Emulsion Liquid Membranes.

Emulsion liquid membranes have been successfully used for the removal of different types of organic and inorganic pollutants by means of carrier-mediated transport mechanisms. However, the models that describe the kinetics and transport of such mechanisms are very complex due to the high number of model parameters. Starting from an analysis of the similarity between the elemental mechanisms of carrier-mediated transport in liquid membranes and of transport in adsorption processes, this paper presents an experimental analysis of the possibility of applying kinetic and mechanistic models developed for adsorption to carrier-mediated transport in emulsion liquid membranes. We study the removal of a target species, in this case, Cu(II), by emulsion liquid membranes containing membrane phase solutions of benzoylacetone (carrier agent), Span 80 (emulsifying agent) and kerosene (diluent), and hydrochloric acid as a stripping agent in the product phase. The experimental results fit the pseudo-second-order adsorption kinetic model, showing good relationships between the experimental and model parameters. Although both Cu(II) diffusion through the feed/membrane interface boundary layer and complex Cu-benzoylacetone diffusion through the membrane phase controls Cu(II) transport, it is the former step that mainly controls the transport process.

Characterization of the segmental transport mechanisms of DL-methionine hydroxy analogue along the intestinal tract of rainbow trout with an additional comparison to DL-methionine

The aim of this study was to identify the unknown transport mechanism of the extensively used monocarboxylate methionine feed supplement DL-methionine hydroxy analogue (DL-MHA) in rainbow trout intestine. Transport across the pyloric caeca (PC), midgut (MG), and hindgut (HG) regions were kinetically studied in Na+- and H+-dependent manners. Gene expression of monocarboxylate (MCTs) and sodium monocarboxylate transporters (SMCTs) were assessed. Results demonstrated that DL-MHA transport from 0.2‐20 mM was Na+-dependent and obeyed Michaelis-Menten kinetics with low affinity in PC & MG in apical/basal pH of 7.7/7.7. Changes in apical/basal pH (6.0/6.0, 6.0/7.7, and 7.7/8.7) had insignificant effects on kinetics. In contrast, HG flux kinetics were only obtained in pH 7.7/8.7 or in the presence of lactate with medium affinity. Additionally, DL-MHA transport from 0‐150 μM demonstrated the presence of a Na+-dependent high-affinity transporter in PC & MG. Conclusively, two distinct carrier-mediated DL-MHA transport mechanisms along the trout gut were found: 1) in PC & MG: apical transport was regulated by Na+-requiring systems that possibly contained low- and high-affinity transporters, and basolateral transport was primarily achieved through a H+-independent transporter; 2) in HG: uptake was apically mediated by a Na+-dependent transporter with medium affinity, and basolateral exit was largely controlled by an H+-dependent transporter. Finally, two major methionine feed supplements, DL-MHA and DL-methionine (DL-Met) were compared to understand the differences in their bioefficacy. Flux rates of DL-MHA were only about 42.2–66.0% in PC and MG compared to DL-Met, suggesting intestinal transport of DL-MHA was lower than DL-Met.

Thyroxine (T4) Transfer from Blood to Cerebrospinal Fluid in Sheep Isolated Perfused Choroid Plexus: Role of Multidrug Resistance-Associated Proteins and Organic Anion Transporting Polypeptides.

Thyroxine (T4) enters the brain either directly across the blood–brain barrier (BBB) or indirectly via the choroid plexus (CP), which forms the blood–cerebrospinal fluid barrier (B-CSF-B). In this study, using isolated perfused CP of the sheep by single-circulation paired tracer and steady-state techniques, T4 transport mechanisms from blood into lateral ventricle CP has been characterized as the first step in the transfer across the B-CSF-B. After removal of sheep brain, the CPs were perfused with 125I-T4 and 14C-mannitol. Unlabeled T4 was applied during single tracer technique to assess the mode of maximum uptake (Umax) and the net uptake (Unet) on the blood side of the CP. On the other hand, in order to characterize T4 protein transporters, steady-state extraction of 125I-T4 was measured in presence of different inhibitors such as probenecid, verapamil, BCH, or indomethacin. Increasing the concentration of unlabeled-T4 resulted in a significant reduction in Umax%, which was reflected by a complete inhibition of T4 uptake into CP. In fact, the obtained Unet% decreased as the concentration of unlabeled-T4 increased. The addition of probenecid caused a significant inhibition of T4 transport, in comparison to control, reflecting the presence of a carrier mediated process at the basolateral side of the CP and the involvement of multidrug resistance-associated proteins (MRPs: MRP1 and MRP4) and organic anion transporting polypeptides (Oatp1, Oatp2, and Oatp14). Moreover, verapamil, the P-glycoprotein (P-gp) substrate, resulted in ~34% decrease in the net extraction of T4, indicating that MDR1 contributes to T4 entry into CSF. Finally, inhibition in the net extraction of T4 caused by BCH or indomethacin suggests, respectively, a role for amino acid “L” system and MRP1/Oatp1 in mediating T4 transfer. The presence of a carrier-mediated transport mechanism for cellular uptake on the basolateral membrane of the CP, mainly P-gp and Oatp2, would account for the efficient T4 transport from blood to CSF. The current study highlights a carrier-mediated transport mechanism for T4 movement from blood to brain at the basolateral side of B-CSF-B/CP, as an alternative route to BBB.

Involvement of a proton-coupled organic cation antiporter in the blood-brain barrier transport of amantadine.

The blood-to-brain transport of amantadine, a weak N-methyl-d-aspartate (NMDA) antagonist, has been shown previously to participate in the cationic drug-sensitive transport system across the mouse blood-brain barrier (BBB). The purpose of the present study was to characterize the influx transport system by means of both an in situ mouse brain perfusion technique and in vitro studies using rat immortalized brain capillary endothelial cells (GPNT). The observed concentration-dependent initial uptake rate of [(3) H]amantadine suggested the involvement of a carrier-mediated transport mechanism. The normal uptake at physiological pH7.4 was decreased by 72.9% in acidic perfusate, while it was increased by 35.3% in alkaline perfusate. These results suggest that pH-dependent transport is regulated by utilizing an oppositely directed proton gradient as a driving force. In addition, the [(3) H]amantadine uptake was moderately inhibited by the adamantane structural analogs (rimantadine and memantine) and other cationic drugs (pyrilamine, clonidine, nicotine, etc.), but not by substrates or inhibitors of the well-characterized organic cation transporters (tetraethylammonium, l-carnitine and choline). A similar inhibition pattern was observed between the in vivo studies and the in vitro experiments. These results indicate that the influx transport for amantadine across the BBB involves a proton-coupled organic cation antiporter. Copyright © 2016 John Wiley & Sons, Ltd.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport.

Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. Not applicable. This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Strategies to target drugs to gliomas and CNS metastases of solid tumors.

The treatment for central nervous system metastases of solid tumors and gliomas is limited as the blood-brain barrier (BBB) is an obstacle to systemic therapy. Here, we review the physiochemical properties of the BBB and both current and new drug strategies to penetrate brain tumors. We focus on targeting receptor- or carrier-mediated transport mechanisms over the BBB used by drug conjugates, nanoparticles, polymer-based nanocarriers, siRNA, and antibodies.

Facilitated transport of small molecules and ions for energy-efficient membranes.

In nature, the biological membrane can selectively transport essential small molecules/ions through facilitated diffusion via carrier proteins. Intrigued by this phenomenon and principle, membrane researchers have successfully employed synthetic carriers and carrier-mediated reversible reactions to enhance the separation performance of synthetic membranes. However, the existing facilitated transport membranes as well as the relevant facilitated transport theories have scarcely been comprehensively reviewed in the literature. This tutorial review primarily covers the two aspects of facilitated transport theories: carrier-mediated transport mechanisms and facilitated transport chemistries, including the design and fabrication of facilitated transport membranes. The applications of facilitated transport membranes in energy-intensive membrane processes (gas separation, pervaporation, and proton exchange membrane fuel cells) have also been discussed. Hopefully, this review will provide guidelines for the future research and development of facilitated transport membranes with high energy efficiency.

Transendothelial Transport and Its Role in Therapeutics.

Present review paper highlights role of BBB in endothelial transport of various substances into the brain. More specifically, permeability functions of BBB in transendothelial transport of various substances such as metabolic fuels, ethanol, amino acids, proteins, peptides, lipids, vitamins, neurotransmitters, monocarbxylic acids, gases, water, and minerals in the peripheral circulation and into the brain have been widely explained. In addition, roles of various receptors, ATP powered pumps, channels, and transporters in transport of vital molecules in maintenance of homeostasis and normal body functions have been described in detail. Major role of integral membrane proteins, carriers, or transporters in drug transport is highlighted. Both diffusion and carrier mediated transport mechanisms which facilitate molecular trafficking through transcellular route to maintain influx and outflux of important nutrients and metabolic substances are elucidated. Present review paper aims to emphasize role of important transport systems with their recent advancements in CNS protection mainly for providing a rapid clinical aid to patients. This review also suggests requirement of new well-designed therapeutic strategies mainly potential techniques, appropriate drug formulations, and new transport systems for quick, easy, and safe delivery of drugs across blood brain barrier to save the life of tumor and virus infected patients.

Intestinal transport of TRH analogs through PepT1: the role of in silico and in vitro modeling.

The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco-2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1-mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco-2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly-Sar, a typical PepT1 substrate, to confirm the PepT1-mediated transport mechanism of TRH analogs. Papp of the four analogs follows the order: NP-1894 < NP-2378 < NP-1896 < NP-1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier-mediated transport mechanism. Further, the significant inhibition of the uptake of Gly-Sar by TRH analogs confirmed the PepT1-mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1-mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N-histidyl moiety of TRH analogs.

Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane.

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush-border membrane vesicles. The uptake of nicotinic acid by the membrane vesicles showed an overshoot phenomenon in the presence of an outwardly directed bicarbonate gradient or an inwardly directed proton gradient and the uptakes were two times and six times greater, respectively, than that in the absence of any ion gradient. The bicarbonate-dependent initial uptake of nicotinic acid was increased at acidic pH, showing pH-dependent transport activity. An inhibitor of anion transport, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, specifically reduced bicarbonate-dependent transport of nicotinic acid. The initial uptakes of nicotinic acid via the anion antiporter and the proton cotransporter were specifically inhibited by monocarboxylic acids such as acetic acid, benzoic acid, D- and L-lactic acid, pravastatin and valproic acid, but not by di- or tricarboxylic acids, bile acids or amino acids. Nicotinic acid uptake activity was, furthermore, expressed in a Xenopus laevis oocyte system after injection of messenger RNA (mRNA) derived from rabbit intestinal epithelial cells. These observations demonstrate that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine, i.e. a proton cotransporter and an anion antiporter. The pH-dependence observed in the intestinal absorption of nicotinic acid might, therefore, be ascribed partly to pH-sensitive and partly to carrier-mediated transport mechanisms in the brush-border membrane.

PH-dependent and carrier-mediated transport of salicylic acid across Caco-2 cells.

The transport of monocarboxylic acid drugs such as salicylic acid was examined in the human colon adenocarcinoma cell line, Caco-2 cells that possess intestinal epithelia-like properties. [14C]Salicylic acid transport was pH-dependent and appeared to follow the pH-partition hypothesis. However, 10 mM unlabelled salicylic acid significantly reduced the permeability coefficient of [14C]salicylic acid. Kinetic analysis of the concentration dependence of the permeation rate of salicylic acid across Caco-2 cells showed both saturable (Kt = 5.28 +/- 0.72 mM Jmax = 36.6 +/- 3.54 nmol min-1 (mg protein)-1) and nonsaturable (kd = 0.37 +/- 0.08 microL min-1 (mg protein)-1) processes. The permeation rate of [14C]salicylic acid was competitively inhibited by both acetic acid and benzoic acid, which were demonstrated in our previous studies to be transported in the carrier-mediated-transport mechanism which is responsible for monocarboxylic acids. Furthermore, certain monocarboxylic acids significantly inhibited [14C]salicylic acid transport, whereas salicylamide and dicarboxylic acids such as succinic acid did not. From these results, it was concluded that the transcellular transport of [14C]salicylic acid across Caco-2 cells is by the pH-dependent and carrier-mediated transport mechanism specific for monocarboxylic acids.

Intestinal Absorption Mechanism of Tebipenem Pivoxil, a Novel Oral Carbapenem: Involvement of Human OATP Family in Apical Membrane Transport

Tebipenem pivoxil (TBPM-PI) is an oral carbapenem antibiotic for treating otolaryngologic and respiratory infections in pediatric patients. This agent is a prodrug to improve intestinal absorption of TBPM, an active form, and an absorption rate of TBPM-PI is higher than those of other prodrug-type β-lactam antibiotics. In the present study, we hypothesized that a certain mechanism other than simple diffusion is involved in the process of improved intestinal absorption of TBPM-PI and examined the mechanism. TBPM-PI uptake by Caco-2 cells was decreased by ATP-depletion and lowering the temperature to 4 °C, suggesting the contribution of carrier-mediated transport mechanisms. This uptake was partially decreased by ACE inhibitors, and the reduction of the absorption by captopril was observed by in vivo study and in situ single-pass intestinal perfusion study in rat, supporting the contribution of influx transporters. Since some ACE inhibitors and β-lactam antibiotics are reported to be substrates of PEPT and OATP families, we measured transporting activity of TBPM-PI by intestinally expressed transporters, PEPT1, OATP1A2, and OATP2B1. As a result, significant transport activities were observed by both OATP1A2 and OATP2B1 but not by PEPT1. Interestingly, pH dependence of TBPM-PI transports was different between OATP1A2 and OATP2B1, showing highest activity by OATP1A2 at pH 6.5, while OATP2B1-mediated uptake was higher at neutral and weak alkaline pH. OATP1A2 exhibited higher affinity for TBPM-PI (K(m) = 41.1 μM) than OATP2B1 (K(m) > 1 mM) for this agent. These results suggested that TBPM-PI has high intestinal apical membrane permeability due to plural intestinal transport routes, including the uptake transporters such as OATP1A2 and OATP2B1 as well as simple diffusion.

Involvement of an influx transporter in the blood–brain barrier transport of naloxone

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood-brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [(3)H]naloxone exhibited saturability in a concentration-dependent manner (concentration range 0.5 microM to 15 mM) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier-mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K(t), was 2.99+/-0.71 mM; the maximum uptake rate, J(max), was 0.477+/-0.083 micromol/min/g brain; and the nonsaturable first-order rate constant, K(d), was 0.160+/-0.044 ml/min/g brain. The uptake of [(3)H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H(1)-antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [(3)H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L-carnitine or substrates for organic anion transport systems such as p-aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH-dependent and saturable influx transport system that is a selective transporter for cationic H(1)-antagonists is involved in the BBB transport of naloxone in the rat.

Functional characterization of active transport of progesterone to adrenal cells.

The characterization of the transport mechanism of progesterone, which is one of the neutral steroids in the adrenal cells, has been studied by the examination of progesterone uptake into the monolayers of SW-13 cells (a human adrenal adenocarcinoma cell line). The uptake of [(3)H]progesterone at a tracer concentration (1 nM) exhibited temperature, pH and sodium dependency. According to kinetic analysis of the concentration dependence, the uptake of progesterone involves saturable and non-saturable processes. The uptake for the saturable process, which gave K(t) values (half-saturation concentration) of 4.7 +/- 8.7 microM, was inhibited by metabolic inhibitors and amino-acid modifiers but not by endocytosis inhibitors or substrates for known transporters. The uptake of progesterone was also inhibited by several neutral steroids but not by anionic steroids. The inhibition by both beta-estradiol and estriol was competitive. The uptake of progesterone by the adrenal cells might be at least partially accounted for by a specific carrier-mediated transport mechanism generated by sodium ions and an electrochemical mechanism.

Nonlinear absorption of methylprednisolone by absorptive and secretory transporters

The intestinal absorption rate constant of methylprednisolone (MP) evaluated by the loop method increased significantly with increasingly higher concentrations of the drug up to 500 μM in a nonlinear fashion but did not increase further at higher concentrations. Mucosal-to-serosal directed permeation of MP across rat jejunal sheets also increased in a nonlinear fashion in a low concentration range (100–150 μM), followed by a decrease as the concentration increased further, whereas serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. Vectorial transport of MP across Caco-2 cell monolayers was observed, with greater transport in the basolateral-to-apical direction at 37 °C. These observations suggest that MP is taken up in the intestinal epithelial cells by a carrier-mediated transport mechanism. The absorptive and secretory clearance of MP increased and decreased with P-glycoprotein (P-gp) inhibitors, respectively. These results strongly suggest that MP is secreted into the intestinal lumen predominantly by P-gp. We conclude that intestinal transport of MP involves P-gp or some other transporters in both the absorptive and secretory directions, and complex nonlinear intestinal absorption characteristics can be ascribed to the existence of multiple transport mechanisms.

RT-PCR analysis of ABC, SLC and SLCO drug transporters in human lung epithelial cell models

Carrier-mediated transport mechanisms play crucial roles in drug absorption and elimination processes, as well as in the transport of endogenous molecules affecting cellular regulation and function. In this study we used RT-PCR analysis to characterise the mRNA transcript expression of a wide range of membrane carrier transporters in several in-vitro lung epithelial cell models. Transporters studied included: 11 ATP-binding cassette (ABC) transporters, 11 solute carrier (SLC) transporters and 9 solute carrier organic anion (SLCO) transporters. The cell culture models included both established cell lines (A549, Calu-3, 16HBE14o-, BEAS-2B) and freshly isolated lung epithelial cells in primary culture (human bronchial and alveolar epithelial cells). The expression profiles of several clinically relevant drug transporters were characterised using RT-PCR analysis. Our results showed differential transporter expression in cell culture models from different regions of the lung and also highlighted disparities when comparing lung cell lines with primary cell culture models. Differences in transporter expression between cell models of pulmonary and gastrointestinal origin were also noted. The information will guide and validate the use of in-vitro lung epithelial cell lines in the study of pulmonary administered drugs and candidate molecules.

Glucose Promoiety Enables Glucose Transporter Mediated Brain Uptake of Ketoprofen and Indomethacin Prodrugs in Rats

The brain uptake of solutes is efficiently governed by the blood-brain barrier (BBB). The BBB expresses a number of carrier-mediated transport mechanisms, and new knowledge of these BBB transporters can be used in the rational targeted delivery of drug molecules for active transport. One attractive approach is to conjugate an endogenous transporter substrate to the active drug molecule to utilize the prodrug approach. In the present study, ketoprofen and indomethacin were conjugated with glucose and the brain uptake mechanism of the prodrugs was determined with the in situ rat brain perfusion technique. Two of the prodrugs were able to significantly inhibit the uptake of glucose transporter (GluT1)-mediated uptake of glucose, thereby demonstrating affinity to the transporter. Furthermore, the prodrugs were able to cross the BBB in a temperature-dependent manner, suggesting that the brain uptake of the prodrugs is carrier-mediated.

High-Density Lipoprotein-Associated .ALPHA.-Tocopherol Uptake by Human Retinal Pigment Epithelial Cells (ARPE-19 Cells): the Irrelevance of Scavenger Receptor Class B, Type I

The purpose of this study was to determine the high-density lipoprotein (HDL)-associated alpha-tocopherol (alpha-tocopherol-HDL) transport and clarify the contribution of scavenger receptor class B, type I (SR-BI) to the uptake in the human retinal pigment epithelial cell line (ARPE-19 cells). [(3)H]alpha-Tocopherol-HDL uptake into ARPE-19 cells seeded onto a transwell from both the apical (apical-to-cell) and basal compartment (basal-to-cell) exhibited a time-dependent increase for 90 min and there was a reduction at 4 degrees C. These results indicate the involvement of carrier-mediated process in alpha-tocopherol-HDL uptake in ARPE-19 cells. Immunoblot analysis revealed that SR-BI protein was expressed in ARPE-19 cells. However, the uptake of [(3)H]alpha-tocopherol from the apical or basal compartment was hardly inhibited by block lipid transport-1 (BLT-1), a specific inhibitor of the SR-BI-mediated lipid transfer. In conclusion, ARPE-19 cells have a carrier-mediated transport mechanism of [(3)H]alpha-tocopherol-HDL regardless of any SR-BI-mediated process.