Drug Transport Studies Research Articles

Spatial distribution of cutaneous microvasculature and local drug clearance after drug application on the skin

We analysed quantitatively blood microvessels distribution in normal skin. We conclude that the segmental area of blood vessels peaks approximately 0.1 mm below the skin surface, where the upper cutaneous blood plexus resides. Total blood vessels area then decreases quasi-exponentially to a depth of approx. − 0.75 mm, with a decay length of ∼ 0.1 mm, which is site and skin condition dependent, but at greater depths the decrease is approx. 6-times less steep. The corresponding permeability sink exhibits a similar, but superficially steeper, depth-profile. The lateral localisation of superficial blood vessels is such that ensures maximum diffusion from and into the capillaries, which affects transdermal drug delivery: each hairpin-like loop is in the centre of a papilla that corresponds to a cluster of corneocytes surrounded by main diffusion pathways. The aggregate area of blood vessels in the skin is ≥ 2.5-fold greater than total organ surface area under normal physiological conditions. The molecules diffusing through the skin barrier are thus largely cleared in outermost 20% of the organ, which may create a drug concentration maximum in the dermis, if clearance increases significantly with time. Skin microdialysis data are therefore extremely sensitive to cutaneous blood flow (distribution) and sampling. Skin microvasculature and its distribution must consequently be considered in all topical or transdermal drug transport studies, for example, by including suitably formulated clearance term into generalised diffusion equation.

Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines

The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and contribute to the current knowledge of drug transport. The aim of the present study was to characterize three rat epithelial cell lines for transporter expression and activity. Transporter expression was assessed in intestinal IEC-6 and renal GERP and NRK-52E cells using RT-PCR and Western blot analysis. Pgp and Mrp transport activity were analyzed by measuring calcein accumulation and glutathione- S-bimane efflux, respectively. The three cell lines showed Pgp expression and Pgp-dependent transport, both decreasing with culture time after reaching confluency. Besides Pgp, cells expressed Mrp1, Mrp3, Mrp4, and Mrp5, while Mrp2 and Mrp6 were absent. In addition, they showed temperature- and Mrp-dependent efflux of glutathione- S-bimane. Exposure to a panel of different inhibitors showed that this efflux was probably mediated by Mrp4. In conclusion, the three rat epithelial cell lines investigated showed Pgp and Mrp expression and transport. Mrp dependent transport was most likely mediated by Mrp4. In future, these cell lines may be used as in vitro models to study drug transport.

SIMPLE AND INEXPENSIVE METHODS TO PREVENT BOTANICAL MISFORTUNE

Botanical dietary supplements are often targeted by the mainstream media and scientific/medical communities as being unscientific and unsafe relative to prescription pharmaceuticals and over-the counter drugs. However, the relative risk of botanical medicines is largely overestimated and causal relationships between botanical remedies and significant adverse reactions pale in comparison to the frequency and magnitude of those observed with normal use of FDA-approved pharmaceuticals. Historically, a greater premium has been placed on demonstrating the safety of health-related products as opposed to confirming therapeutic efficacy; in fact, the required standards for prescription drug efficacy were implemented in the U.S. more than 50 years after standards for purity and safety were firmly in place. An argument will be made that botanical manufacturers can support their place in the national health agenda and generate significant goodwill by voluntarily subjecting supplements to relatively simple and inexpensive preclinical studies, and even limited phase I clinical trials, employing the basic tenets of pharmacology and toxicology (i.e., dose-response relationships) to support the relative safety of specific products. Moreover, many of the unfortunate adverse events of drug-herb interactions over the last 10 years could have been prevented by relatively simple hepatic metabolism and drug transport studies that have become commonplace in even the smallest of academic laboratories, due in large part to the increased availability and lower cost of previously scarce and cumbersome biological reagents. Demonstrating the safety of semi-pharmaceutical formulations of herbal products now requires more than simple reliance on the historic folk record of safe use; real health risks and negative publicity can be successfully mitigated with a modest, prospective investment in basic preclinical toxicology testing. A model is presented for a non-profit research center that focuses on botanical quality and safety as a first step toward this goal.

Membrane transport function in primary cultures of human proximal tubular cells

To further develop primary cultures of human proximal tubular (hPT) cells for study of drug disposition, we determined kinetics and protein expression of several key transporters for organic anions and cations, peptides, and neutral amino acids. p-Aminohippurate uptake exhibited similar kinetics as published values, was inhibited by cephaloridine, cimetidine, methotrexate, and urate, consistent with function of both organic anion transporter 1 (OAT1) and OAT3. Transport rates by organic cation transporters (OCTs) were up to three-fold higher than those of OATs. Of the OCT substrates tested, triethanolamine exhibited the highest transport rates across the basolateral membrane (BLM). OCTN1 exhibited high-affinity, low-capacity BLM transport of l-carnitine. Glycylsarcosine transport by PepT2 was rapid and comparable to that of OCTs. Amino acid System L on the BLM exhibited comparable kinetic parameters for transport of l-leucine as the OATs. Efflux of verapamil across the brush-border membrane by P-glycoprotein was very rapid. Expression of carriers was generally maintained throughout 5 days of culture. Of the four OAT proteins studied (OAT1-4), expression of OAT1 and OAT3 was the most readily detected and exhibited interindividual variation. OCTN2 was the major OCT in hPT cells. Expression was also quantified for multidrug resistance-associated proteins 2 and 5 and P-glycoprotein. These results show that primary cultures of hPT cells express a diverse array of transporters for major classes of important drugs and are suitable for study of drug transport and disposition and assessment of potential drug–drug interactions in human kidney.

Porcine Alveolar Epithelial Cells in Primary Culture: Morphological, Bioelectrical and Immunocytochemical Characterization

The purpose of this study was to establish a primary culture of porcine lung epithelial cells as an alternative to the currently existing cell cultures from other species, such as e.g., rat or human. Primary porcine lung epithelial cells were isolated, cultivated and analyzed at distinct time points after isolation. The main part of the work focused on the morphology of the cells and the detection of alveolar epithelial cell markers by using electron microscopy, immunofluorescence microscopy and immunoblotting. Regarding a later use for in vitro pulmonary drug absorption studies the barrier properties of the cell monolayer were evaluated by monitoring bioelectrical parameters and by marker transport. Epithelial cells isolated from porcine lung grew to confluent monolayers with typical intercellular junctions within a few days. Maximum transepithelial resistance of about 2,000 Omega cm2 was achieved and demonstrated the formation of a tight epithelial barrier. Permeability data of sodium fluorescein recommended a minimal transepithelial resistance of 600 Omega cm2 for transport studies. The cell population changed from a heterogeneous morphology and marker distribution (caveolin-1, pro-SP-C, surface sugars) towards a monolayer consisting of two cell types resembling type I and type II pneumocytes. The porcine alveolar epithelial primary cell culture holds promise for drug transport studies, because it shares major hallmarks of the mammalian alveolar epithelium and it is easily available and scaled up for drug screening.

Electronic speckle pattern interferometry: A novel non-invasive tool for studying drug transport rate through free films

In this work, Electronic Speckle Pattern Interferometry (ESPI) is presented as a non-invasive tool to study drug transport in controlled release systems. ESPI is shown to be a feasible tool to measure drug film permeability via comparison with an ordinary diaphragm cell. A specially designed cuvette was used in the release study: the polymeric film separated the donor and the receiving chambers of the cuvette to create a diffusion cell with no mixing in the two chambers. Thus, the cuvette mimicked a coated system immersed in a stagnant bulk liquid. Concentration profile data were obtained for the two compartments. Using these data, it was possible to visually discriminate between a film subject only to diffusion and a film subject to diffusion as well as osmotic effects. Moreover, using the concentration profile data collected at different time intervals, it was possible to follow the film properties in terms of drug permeability, thus studying how drug permeability depended on drug concentration. Compared to other measuring techniques, ESPI offers the advantages that no invasive measurements are needed, and that no sampling and calibration are required. Furthermore, the permeability can be measured with no influence of mass transfer in the boundary layers.

P-Glycoprotein expression in human retinal pigment epithelium cell lines

P-Glycoprotein (P-gp), an active efflux transporter encoded by the MDR1 gene, has recently been identified in the human and pig retinal pigment epithelium (RPE) in situ. Efflux pumps such as P-gp are major barriers to drug delivery in several tissues. We wished to establish whether human RPE cell lines express P-gp under the culture conditions recommended for each cell line so as to determine their suitability as in vitro models for predicting drug transport across the outer blood–retinal barrier. Three human RPE cell lines, ARPE19, D407 and h1RPE were investigated. Reverse transcriptase–polymerase chain reaction (RT-PCR) was carried out to determine the expression of MDR1 mRNA. Immunocytochemistry using the P-gp-specific antibody C219 was undertaken to investigate the presence of P-gp protein in each cell type. Uptake of rhodamine 123, a P-gp substrate, in the presence or absence of pre-treatment with a P-gp inhibitor, verapamil, was measured in each cell line to determine functional expression of P-gp. For all experiments, MDCK cells stably transfected with the human MDR1 gene (MDCK-MDR1) were used as a positive control. ARPE19 cells were consistently negative for P-gp as assessed by RT-PCR and immunocytochemistry. By contrast, RT-PCR of D407 and h1RPE samples yielded weak bands corresponding to MDR1; P-gp protein expression, as demonstrated by C219 immunoreactivity, was also present. Rhodamine uptake after treatment with verapamil was significantly greater in D407 and MDCK-MDR1, indicating functional expression of P-gp in these two cell lines. No evidence of functional P-gp was found in ARPE19 and h1RPE. In conclusion, D407 and h1RPE cells express P-gp, though functional activity was demonstrable only in D407 cells. ARPE19 cells do not express P-gp. Of these human RPE cells lines D407 could be considered as a suitable model for in vitro drug transport studies, particularly those involving P-gp substrates, without modification of their usual culture conditions.

A High-Throughput Cell-Based Assay for Inhibitors of ABCG2 Activity

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 microM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z' 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (> or = 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 microM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.

Primary porcine proximal tubular cells as a model for transepithelial drug transport in human kidney

Background Kidney proximal tubular cells play a major role in the transport of endogenous and exogenous compounds. A multitude of different transporters are expressed starting with multidrug ABC transporters (e.g. abcb1, abcc1-6), slc22a6-8 (organic anion transporters) and slc22a1-3 (organic cation transporters). For transport studies of renal drug transport, cell lines like MDCK and LLC-PK1 are often used to overexpress and study one or two transporters, such as abcb1 or abcc1-6. However, the use is limited since under physiological conditions xenobiotics are transported through different transporters at the same time. Therefore, a primary in vitro model expressing functionally different transporters simultaneously, as it is the case in vivo, would be of great benefit. Methods Primary proximal tubular cells were isolated from porcine kidney. Cells were cultured under selective culturing conditions leading to specific growth of primary proximal tubular cells. Expression of important proximal transporters was checked at mRNA level with RT-PCR, at protein level with immunocytochemistry and functionally by transport and uptake assays. Results A model of primary proximal tubular cells was established expressing the most important transporters: abcb1, abcc1, abcc2, slc22a8, slco1a2, slc15a1, slc5a2 and slc4a4. In freshly isolated cells, slc22a1 and slc22a6 were expressed, but were down-regulated in culture. Abcb1, abcc1, abcc2 and slc4a4 were detected at protein level with immunostaining. Functional activity was confirmed for abcb1, abcc1/2, slc22a8, slc15a1/2 and slc5a1/2. The tightness of the monolayers of this model was better than in previously established in vitro models. Conclusion This primary cell culture model might be an interesting tool to investigate proximal tubular transport and to predict toxicity and drug interactions since it expresses functionally several transporters simultaneously.

Application of Drug Transport Studies to Preclinical Drug Discovery and Development: Functional Analysis of Transporters, Species Differences, and In Vitro In Vivo Correlation

Application of Drug Transport Studies to Preclinical Drug Discovery and Development: Functional Analysis of Transporters, Species Differences, and <i>In Vitro In Vivo</i> Correlation

Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood–brain barrier

In vitro models of the blood–brain barrier (BBB) play a major role in the study of BBB permeability of drug candidates. However, most established in vitro models use cells of non-human origin, which is not optimal for the prediction of brain permeability in humans. The aim of this study was to assess the human brain capillary endothelial cell line BB19 for its usefulness as an in vitro model of the human BBB. Restrictive tight junctions are a prerequisite for drug transport studies. Sucrose permeability of BB19 cells on different filters was compared to porcine brain capillary endothelial cells (BCEC). Tightness of BB19 cell monolayers still needs further optimization. Hardly any discrimination between Sucrose and Propranolol ( P app = 1.30 × 10 −5 vs. 2.18 × 10 −5 cm/s) was seen. Cells showed an improvement towards a more primary BCEC morphology with C6 conditioned medium, dexamethasone, and 1,25-dihydroxyvitamin D 3. The presence of P-glycoprotein (P-gp), MRP4 and BCRP, ABC-transporters located in the BBB, has been shown on mRNA level, by immunostaining, and western blot. MRP1, MRP2, MRP5, OAT3, and OAT4 were also detected by RT-PCR. Functional properties of the BBB were shown with uptake of propranolol, morphine, and sucrose. Uptake studies with daunomycin and the P-gp inhibitor verapamil showed functional activity of P-gp. We conclude that BB19 cells might be feasible as a human in vitro model of the BBB for drug uptake studies. However, for the assessment of transport studies, further improvements of this model are necessary.

Glucuronidation and the Transport of the Glucuronide Metabolites in LLC-PK1 Cells

Formation and transport of glucuronide metabolites were studied in LLC-PK1 cells. Glucuronidation of 17beta-estradiol, 1-naphthol, mycophenolic acid, and 4-methylumbelliferone was examined in microsomes prepared from LLC-PK1 cells, human livers, human kidneys, and human intestines. The rate of glucuronide metabolite formation observed with LLC-PK1 microsomes was comparable to rates observed with various human tissue microsomes. The fate of the glucuronide metabolite formed in the LLC-PK1 cells was studied by examining its extracellular transport using mycophenolic acid as a model substrate. After administration of mycophenolic acid, the amount of the glucuronide metabolite exiting to the extracellular compartments significantly decreased in the presence of MK-571, an inhibitor for the multidrug resistance-associated protein (MRP) transporter. However, the intracellular levels of the glucuronide metabolite did not change, suggesting that MK-571 was probably blocking metabolite efflux. In summary, these results suggest that the glucuronidating enzyme(s) expressed in the LLC-PK1 cells are capable of sufficient glucuronidation activity and that endogenous transporter(s) in LLC-PK1 cells are active and determine the distribution of the formed metabolites. Since these cells have been previously used to study drug transport, they may be a useful tool in future studies to explore the effect of drug transporters on glucuronidation.

Comparison of Human Tracheal/Bronchial Epithelial Cell Culture and Bovine Nasal Respiratory Explants for Nasal Drug Transport Studies

Ten drug compounds with varying physicochemical properties and transporter substrate specificities were investigated to compare their in vitro permeabilities across bovine nasal respiratory explants and the EpiAirway system, both established models for the assessment of nasal drug absorption. Permeability across the bovine explants and EpiAirway correlated well with the partitioning behavior of compounds whose clogDC values were greater than 0. The permeabilities of all ten compounds were well-correlated between the two tissue models, with the permeability values through the EpiAirway tissues being approximately 10-fold higher than through the bovine explants due to the thickness differences between the models. For more lipophilic compounds, the in vitro permeabilities measured with both tissue systems were also predictive of the reported in vivo nasal bioavailabilities. Deviations from these correlations were observed for compounds reported to be substrates of p-glycoprotein or OCT transporters, and differences were also seen between the permeabilities measured in the tissue models for these compounds. Both models can be used to estimate the systemic bioavailability of moderately lipophilic compounds administered intranasally, while each may have particular advantages or disadvantages in estimating the bioavailability of drug compounds that are subject to local mucosal metabolism or to carrier-mediated uptake or efflux.

Studies of intestinal drug transport using an in silico epithelio-mimetic device

We report the development and use of a synthetic, discrete event, discrete space model that functions as an epithelio-mimetic device (EMD). It is intended to facilitate the study of intestinal transport of drug-like compounds. We represent passive paracellular and transcellular transport, carrier-mediated transport and active efflux using stand-alone components. Systematic verification of the EMD over a wide physiologically realistic range is essential before we can use it to address questions regarding the details of the interacting mechanisms that are believed to influence absorption. We report details of key verification experiments. We demonstrate that this device can generate behaviors similar to those observed in the in vitro Caco-2 transwell system. To do that we used a series of hypothetical drugs and we simulated behaviors for two clinically used drugs, alfentanil and digoxin. The results support the feasibility and practicability of the EMD as a tool to expand the experimental options for better understanding the biological processes involved in intestinal transport and absorption of compounds of interest.

Identification of an anticancerous drug model in brain tissues using Adomian/Alienor methods

Purpose – In this paper, non‐linear compartment modelling is used to study drug transport of anticancerous substance across brain tissues. The aim of the work is to identify the pharmacokinetic parameters of the model created.Design/methodology/approach – A combination of the Adomian decomposition method and the Alienor reducing transformation method were used to solve the identification problem as if it were a classical one‐dimensional minimization problem.Findings – The numerical results using this methodology have shown that local therapeutic method should be preferred, when it comes to evaluate the rate (of healthy cells/cancerous cells), especially when, somehow, the drug transition into the tumour is speeded up. The combination method of Adomian and Alienor proved a successful strategy, and could take into account many of the pharmacokinetic parameters as necessary and use well‐known algorithms.Research limitations/implications – It is believed that this modest work can be considered as preliminary ...

Validation of in vitro cell models used in drug metabolism and transport studies; genotyping of cytochrome P450, phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines

Human cell lines are often used for in vitro biotransformation and transport studies of drugs. In vivo, genetic polymorphisms have been identified in drug-metabolizing enzymes and ABC-drug transporters leading to altered enzyme activity, or a change in the inducibility of these enzymes. These genetic polymorphisms could also influence the outcome of studies using human cell lines. Therefore, the aim of our study was to pharmacogenotype four cell lines frequently used in drug metabolism and transport studies, HepG2, IGROV-1, CaCo-2 and LS180, for genetic polymorphisms in biotransformation enzymes and drug transporters. The results indicate that, despite the presence of some genetic polymorphisms, no real effects influencing the activity of metabolizing enzymes or drug transporters in the investigated cell lines are expected. However, this characterization will be an aid in the interpretation of the results of biotransformation and transport studies using these in vitro cell models.

Air-Liquid Interface Culture of Serially Passaged Human Nasal Epithelial Cell Monolayer for In Vitro Drug Transport Studies

The objective of this study was to establish a drug transport study using human nasal epithelial (HNE) cell monolayers cultured by the air-liquid interface (ALI) method using serum-free medium (BEGM:DME/F12, 50:50). The cells were developed and characterized in comparison to those that have been previously cultured by the liquid-covered culture (LCC) method. The epithelial cell monolayer cultured by the ALI method resulted in a significantly higher transepithelial electrical resistance value (3,453 ± 302 ohm × cm2) that was maintained (>1,000 ohm × cm2) for up to 20 days compared with that cultured by the LCC method. Observation by scanning electron microscopy revealed mature cilia after 2 weeks in the ALI culture, while flatten unhealthy ciliated cells were observed in the LCC method. After 21 days, higher level of MUC5AC and 8 mRNA were expressed in ALI culture which confirmed the secretory differentiation of HNE monolayers in vitro. No significant difference in the permeability coefficients of a model hydrophilic marker (14C-mannitol) and a lipophilic drug (budesonide) was observed between the two conditions on day 7. The passage 2–3 of the HNE monolayer using ALI condition retained the morphology and differentiated features of normal epithelium. Thus it would be a suitable model for in vitro nasal drug delivery studies.

In vitro Transport of Fexofenadine.HCl in Deformable Liposomes Across the Human Nasal Epithelial Cell Monolayers

Fexofenadine HCl is non-sedating histamine H1 receptor antagonist that can be used for the treatment of seasonal allergic rhinitis. The objective of this study was to investigate whether the carriers of deformable liposomes can enhance the transepithelial permeability of fexofenadine HCl across the in vitro ALI human nasal monolayer model. Characterization of this model was achieved by bioelectric measurements and morphological studies. The passage 2 and 3 of cell monolayers exhibited the TEER value of on 11 days of seeding and maintained high TEER value for 5 days. The deformable liposome of fexofenadine HCl was prepared with phosphatidylcholine (PC) and cholic acid using extruder method. The mean particle size was about 200 nm and the maximum entrapment efficiency of 33.0% was obtained in the formulation of 1% PC and fexofenadine HCl. The toxicity of the deformable liposome to human nasal monolayers was evaluated by MTT assay and TEER value change. MTT assay showed that it has no toxic effect on the nasal epithelial cells in 2-hour incubation when the PC concentration was below 1%. However, deformable liposome could not enhance the transepithelial permeability and cellular uptake of fexofenadine HCl. In conclusion, the in vitro model could be used in nasal drug transport studies and evaluation of transepithelial permeability of formulations.

Bodipy-FL-verapamil: a fluorescent probe for the study of multidrug resistance proteins.

Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV), have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR) or MRP (multidrug resistance‐related protein) (PANC‐1) and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine) or MRP (MK571 and probenecid) has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood–brain barrier model for drug uptake and transport studies

Well-characterised cell lines represent important tools for the study of endogenous solute or xenobiotic transport. A brain microvascular cell line, b.End3, isolated from mice transformed with the Polyoma virus middle T-antigen is available commercially. Here we report the characterisation of some features of b.End3 of relevance to its use in blood–brain barrier transport investigations. The b.End3 cells displayed a distinctive spindle-like squamous morphology in culture. Clathrin coated pits and numerous uncoated intracellular vesicles were evident within the cells, as was the expression of the vesicle-associated proteins, clathrin, caveolin-1, flotillin and dynamin II. In the presence of C6 astroglial co-culture b.End3 monolayers achieved a maximal transendothelial electrical resistance of 130 Ω cm 2, but lacked real discrimination with respect to the permeation of transcellular and paracellular probes, e.g. permeability coefficients (×10 −6 cm s −1) for propranolol of approximately 23 vs. 16 for sucrose. RT-PCR analysis confirmed the presence within the b.End3 cells of mRNA transcripts for the following transporters: GLUT-1; MCT 1 and 2; OAT1; Oatp1; mdr 1a and 1b; MRP 1 and 5; β-alanine, system L and system y +L amino acid carriers; the nucleoside transporters cNT1 and 2, eNT1 and 2, and the tight junctional elements, ZO-1, JAM, occludin, claudin-1 and -5. The b.End3 cells actively accumulated d-glucose in a sodium-independent manner with characteristics consistant with that of GLUT-1. Functionality for P-glycoprotein efflux was evident as assessed by a rhodamine-123 accumulation and retention assay. The system L LAT1/4F2hc amino acid transporter was examined through uptake of l-leucine and l-phenylalanine and provided K m and V max values of approximately 16 μM and 350–480 pmol/mg protein/10 min, respectively; the affinity of transport for these substrates being weaker, approximately threefold, when the b.End3 cells were grown in the presence of C6 astroglial factors. Although the b.End3 cells appear unsuitable for transendothelial permeability assessments they display characteristics that would allow their worthwhile use in studies addressing blood–brain barrier transport mechanisms.