Functional feature of a novel model of blood brain barrier: studies on permeation of test compounds

Journal of Controlled Release 76 (2001) 139–147 www.elsevier.com / locate / jconrel

Functional feature of a novel model of blood brain barrier: studies on permeation of test compounds Alessandro Cestelli a , Caterina Catania a , Stefania D’Agostino a , Italia Di Liegro a , Luana Licata a , Gabriella Schiera a , Giovanna Laura Pitarresi a , Giovanni Savettieri b , Viviana De Caro c , Giulia Giandalia c , Libero Italo Giannola c , * a

Dipartimento di Biologia Cellulare e dello Sviluppo Alberto Monroy, Universita` degli Studi di Palermo, Palermo, Italy Istituto di Neuropsichiatria, Policlinico Universitario Paolo Giaccone, Universita` degli Studi di Palermo, Palermo, Italy c Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy b

Received 12 March 2001; accepted 5 July 2001

Abstract Drug delivery to the central nervous system (CNS) is subject to the permeability limitations imposed by the blood–brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical behavior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain microvessel endothelial cells (ECs), grown on top of polycarbonate filter inserts with cortical neuronal cells grown on the outside. Neurons induce ECs to synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals released by cortical co-cultured neurons. The observed effects do not require strict physical contact among cells and neurons. To assess the physiological function of the barrier we examined the transendothelial transfer of three test compounds: dopamine, L-tryptophan and L-DOPA. Polycarbonate filter inserts, where ECs were co-cultured with neurons, were assumed as open two compartments vertical dynamic models. Permeation studies demonstrated that the ECs / neurons co-cultures possess permeability characteristics approaching those of a functional BBB: the system behaved as a selective interface that excludes dopamine permeation, yet permits L-tryptophan and L-DOPA to cross. The movement of test compounds from the donor to the acceptor compartment was observed at a distinct time from the start of co-culture. Transfer was determined using standard kinetic equations. Different performance was observed after 5 and 7 days of co-culture. After 5 days dopamine, L-tryptophan and L-DOPA passively permeate through the membrane as indicated by fittings with a first-order kinetic process equation. After 7 days of co-culture, occludin localizes at ECs periphery, dopamine does not cross the barrier to any further extent, while the transfer of L-tryptophan and L-DOPA fits well with a saturable Michaelis–Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces the characteristic permeability limitations of a functional BBB.  2001 Elsevier Science B.V. All rights reserved. Keywords: Blood–brain barrier (BBB); Brain capillaries endothelial cells (ECs); Dopamine; L-Tryptophan; L-DOPA

*Corresponding author. Tel.: 139-091-6236-191; fax: 139091-6236-124. E-mail address: [email protected] (L.I. Giannola).

1. Introduction Delivery of drugs from blood to the central

0168-3659 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00431-X

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nervous system (CNS) is limited by the selectivity of the blood–brain barrier (BBB), which consists of a single continuous layer of endothelial cells (ECs) lining the intraluminal portion of brain capillaries [1]. In contrast to the peripheral organs, the brain ECs are characterized by unique anatomic specialized intercellular contacts, called tight junctions (TJs), which act as a zipper that closes the interendothelial pores, connects and seals ECs, thus restricting the free movement of substances between blood and interstitial fluid. The transmembrane protein occludin, specifically located within tight junction fibrils, appears to be one of the main elements of brain that play a role in TJ barrier functions [2,3]. TJ components may bind other cytoplasmic molecules to interacting each other. These interactions may contribute to TJ assembly [4]. EC-TJs severely limit water-soluble compounds from entering and leaving the CNS, thus maintaining brain homeostasis [5]. Quantification of drug transfer throughout the brain endothelium in vivo is hampered by a great variety of clearance routes. Moreover, determination of the kinetics of transport in vivo has proved to be difficult due to the poor temporal and spatial resolution and the difficulty of access to the brain side of the endothelium [6]. Various laboratories developed dynamic in vitro models of the BBB by culturing either whole brain capillaries or isolated brain ECs [6,7]. Highly enriched preparations of brain microvascular endothelial cells have been available since the mid-1980s [8]. Nevertheless, the use of in vitro models of BBB has been hampered by a number of difficulties: the procedures required are time-consuming and extremely complex, causing a high level of irreproducibility of the final composition of cell populations [6,9]. Better understanding of the functioning of this important structure was gained by developing immortalized cell lines with stable endothelial traits [10,11]. In a previous work, we have developed a new in vitro system in which ECs were co-cultured with neurons. We demonstrated that neurons are able to induce RBE4.B immortalized rat brain microvessel ECs to synthesize and sort occludin to the cell periphery and that occludin localization is modulated by both compositions of the substratum and soluble signals released by cortical co-cultured neurons. The observed effects do not require strict physical contact

among cells and neurons; indeed, in the culturing apparatus the two cell populations lay almost 1 mm apart [12]. As peripheral localization of occludin in ECs suggests formation of TJs [13], we suppose that a coherent and continuous layer of cells would be created and a functional barrier formed. One of the questions that arose was the feature of the obtained membranous barrier interface [14]. The barrier should reproduce important features in vitro, i.e., it should be highly resistant to solute free diffusion, allowing the passage of certain compounds while excluding others. The rate at which molecules cross the BBB is related to their physicochemical characteristics; lipid solubility, hydrogen bonding, charge, molecular size, ionization profile, flexibility or other parameters play an important role in BBB permeation [15,16]. Very different mechanisms are involved in BBB penetration such as simple transmembrane diffusion, carriermediated saturable transport, as well endo- and transcytosis [17]. Studies in vivo have identified carrier systems for the transfer of amino acids throughout the endothelial cells; the neutral amino acids experience the highest transport rate [9,18]. As other neutral amino acids and small peptides, L-tryptophan crosses the BBB by facilitated transport [19]. Catechol groups confer to a molecule polarity and hydrophilicity. For this reason dopamine and related catecholamines do not cross the barrier [20]. However, L-DOPA, a precursor of dopamine, enters the brain from the blood more easily than would be predicted by its lipid solubility because it has affinity with the neutral amino acid transporter system [21]. L-DOPA as well L-tryptophan is transported using the leucine system (L) large neutral amino acid (LNAA) carrier; this transporter is energy independent [17]. The involved transport system operates in direction of influx from the blood to brain. The presence of efflux transporters from brain to blood has been demonstrated, however they seem to be different from LNAA carrier and functional for detoxication and prevention of nonessential molecules from penetrating the brain [22]. It has been reported that both L-DOPA and Ltryptophan permeate the barrier through saturable kinetics dependent by time, temperature and concentration [23–25]. The purpose of this study was to evaluate transfer

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of L-tryptophan, dopamine and L-DOPA across the barrier, using a simple in vitro model of the brain endothelium, in order to estimate the ability of the fully formed monolayer to act as a selective barrier in vitro. The chosen model compounds are well detectable by spectrophotometric analytical methods and avoid the use of radiolabeled materials.

2. Materials and methods

2.1. Materials Dopamine hydrochloride, L-tryptophan and LDOPA were all purchased from Aldrich (St. Louis, MO, USA) and used as received. Chemicals used were of analytical grade. All other reagents for cell culture were obtained from Sigma (St. Louis, MO, USA) and solutions for cell culture were prepared in endotoxin-free water. RBE4.B immortalized rat brain microvessel endothelial cells were kindly donated by Dr. Franc¸oise Roux, with the permission of Neurotech (Orsay, France).

2.2. Animals Sprague–Dawley rats (Stefano Morini, San Polo d’Enza, Italy) were housed in our institutional animal care facility under direction of a licensed veterinary. Procedures involving animals were conducted according to European Community Council Directive 86 / 609, OJL 358 1, 12 December 1987.

2.3. Cell culture Neurons were prepared from fetal rat cortices at the 16th day of gestation, and cultured in Maat medium, on laminin, as previously described [12]. RBE4.B immortalized rat brain microvessel endothelial cells were plated on collagen I (2 mg / cm 2 ), and maintained in DME–Ham’s F-12 (2:1) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 300 mg / ml geneticin and 1 ng / ml basic fibroblast growth factor in humidified 5% CO 2 –95% air at 378C. Half-confluent RBE4.B cells, originally plated on collagen I, were coped to serum-free Maat medium with daily changes by which serum-supplemented medium was progressively substituted with medium

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selective for neurons. At the end of this period, that lasted 1 week, confluent cultures were detached with trypsin-EDTA, and replated at the same concentration on collagen IV-coated (5 mg / cm 2 ) translucent polycarbonate filters (transparent Transwell inserts, 23 mm diameter, 0.4 mm pore size, Falcon). Filters were then placed on top of 2-day-old primary neuronal cultures. As control, RBE4.B cells were also plated on collagen IV dishes, which were placed on companion wells without neurons. Both co-cultures and control cultures were fed for an additional week daily changing the medium in the insert chamber before harvesting cells for further analyses. Cells to be processed for immunofluorescence were instead plated on clear dishes precoated as above.

2.4. Immunofluorescence All procedures were performed at room temperature. RBE4.B endothelial cells, co-cultured which neurons for at least 7 days, were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, and then permeabilized with 0.1% Triton X-100 in PBS for additional 15 min. After rinsing, cells were preincubated for 60 min with saturating solution (50% fetal calf serum, 3% bovine serum albumin in PBS), in a wet chamber, rinsed in PBS, incubated with anti-occludin rabbit polyclonal antibody (Santa Cruz Biotechnology), at a dilution of 1:500. After rinsing, cells were further incubated for 60 min with rhodamine-conjugated anti-rabbit secondary antibodies (Boehringer) and finally rinsed. Transparent membranes were then mounted in 2% 1,4-diazobicyclo(2,2,2)octane (DABCO, Sigma) in 20 mM Tris, pH 8–glycerol (1:9, v / v) on glass slides, sealed with nail varnish, examined by microscopy (Olympus BX-50 microscope equipped with a Vario Cam B / W camera) and elaborated (image-pro / plus Media Cybernetics) (Fig. 1).

2.5. Permeation studies Once neuronal / endothelial co-cultures were accomplished, the used polycarbonate Transwell inserts were considered for permeability studies as an open two-compartments vertical side-by-side dynamic model. The upper chamber, where microvessel endothelial brain cells were cultured on microporous bed,

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2.6. UV analysis and determination of concentrations

Fig. 1. Segregation of occludin to the periphery of RBE4.B brain capillary endothelial cells cultured for 7 days on top of cortical neurons. Cells were immunostained with rabbit polyclonal antioccludin antibody as described in the text. Peripheral localization of occluding implies formation of TJs. Bar520 mm.

was used as the donor compartment. The lower chamber, accessible to sampling, where neurons were compartmentalized, was used as the acceptor chamber. The donor and the receiver chambers contained 2 and 2.5 ml of Maat medium, respectively, to equilibrate the hydrostatic pressure at the interface of the two compartments. The microporous bed, on top of which ECs were layered, had a surface area of 4.15 cm 2 . The whole system was mixed in synchronous fashion with a mounting drive console and kept in humidified 5% CO 2 –95% air at 37.060.28C during experiments. Permeation experiments started with substitution of ECs feeding medium in the upper chamber (donor) with an equivalent volume of Maat medium in which the appropriate test compound was dissolved. The neuronal medium in the lower chamber (acceptor) was substituted with an equal volume of fresh Maat medium. The amount of substance transferred to the acceptor chamber was monitored with time. Every 10 min, up to 2 h, samples of acceptor fluid (0.5 ml) were withdrawn from the receiver chamber. To avoid saturation phenomena, maintaining the sink conditions and the original volume of the acceptor compartment, each sample volume was replaced with equal volume of fresh Maat medium. Each model compound was administered in concentration of 0.05, 0.10 and 0.15 mg / ml. Each experiment was repeated six times for each starting drug concentration.

The amount of compound transferred into the acceptor medium was monitored by UV spectrophotometric analysis with a Shimadzu UV–Vis Model 1601 spectrophotometer. Absorbance measurements were highly reproducible, linearly related to concentrations over the range 0.2–15 mg / 100 ml and suitable to determine the absorbance of compounds in culture medium. Dopamine and L-DOPA were detected at wavelengths of 276 and 278 nm, respectively, using the appropriate calibration curve and blank. L-Tryptophan was determined using the UV second-order derivative spectrophotometric technique [26]. The second-order derivative of the absorption curve showed valleys at 270.5, 280.5 and 288.5 nm. The valley at 288.5 nm was the best one to distinguish L-tryptophan from the others components of the culture medium. As sampling implied removal of drug aliquots from the acceptor phase, the total amount of transferred material was corrected taking into account the removed fraction in preceding samplings. At the end of each experiment, the residual drug amount in the donor chamber was also determined. The sum of drug amount in the donor and acceptor phases, respectively, matched the original drug content within 2–5%. Reproducibility was within 65% of the mean.

2.7. Mathematics In uptake studies, where transfer of molecules was measured as a function of time, the data of quantitative analysis were plotted versus time. Standard kinetic equations were fitted to the experimental results. Uptake kinetics were analyzed by means of a curve fitting software (Curve Expert 1.34 for Windows) and treated according to a first-order model or a saturation Michaelis–Menten pattern. For a first-order transfer, a complete concentration versus time profile was calculated using the following equation:

F

S

ADKt Mt 5 M` 1 2 exp 2 ]] V,

DG

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where Mt is the amount of test compound transferred at the time t, M` is the amount transferred at the time t5`, A is the membrane area, D and K are the coefficients of diffusion and partition, respectively, V is the volume of receptor compartment and , is the thickness of the endothelial layer. As ADK /V, is the same in every experiment, the equation becomes: Mt 5 M`s1 2 e 2btd A validation of the model may be obtained taking the logarithm of both sides of the latter equation to get:

S

D

Mt log 1 2 ] 5 2 bt M` This equation represents a straight line. Therefore, converting the concentration values to their logarithm and plotting the data against time, a straight line indicates the best fit with a first-order transfer. The saturable Michaelis–Menten (MM) type processes was treated according to the equation: dM Vmax 2 Mt ]]t 5 ]]] dt Km 1 Mt where dMt / dt is the differential rate of change in drug concentration with time, Vmax is the limiting velocity as concentrations get very large, Km is the Michaelis–Menten constant and Mt is the concentration of drug that may undergo transfer. Validation of the most appropriate model used for describing drug transfer was accomplished by regression analysis, correlation coefficient values (r), standard error determination (S.E.), analysis of residuals (data not shown) and x 2 values. All experiments were carried out with at least six replications.

3. Results and discussion To assess the transendothelial transfer properties of the fully formed monolayer barrier and establish its selectivity, we examined the kinetic aspects of the transcellular passage of the aforementioned model compounds. Transwell culture dishes are commonly used to culture cells so that the top and bottom of the

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cells can be exposed to different culture media conditions. In our hands, ECs were cultured on one side of the porous filter and neurons on the other, lying the two cell populations almost 1 mm apart. The apparatus was assumed to behave as a twocompartment vertical dynamic system. The movement of test compounds from donor to acceptor chamber was monitored after different time of coculture and the kinetic behavior was observed. Previously, dishes without biological material were used to determine the permeability of polycarbonate membranes to verify the free movement of solutes from donor to the acceptor chamber. Experiments were performed at various times of co-culture and different conditions. First, passage of drug across the polycarbonate membranes precoated with collagen IV without cells was observed; second, passage was measured across a layer of ECs cultured in the absence of neurons and across a layer of ECs cocultured with neurons for 5 days. Finally, the movement of drug was assessed across a layer of ECs co-cultured with neurons for 7 days. In all instances, under conditions differing from the last mentioned, the permeability of membranes to the selected substances was much greater. When dopamine was administered in the donor chamber, at every concentration value, free diffusion was observed across the polycarbonate membranes precoated with collagen IV without cells and across a layer of ECs cultured in the absence of neurons. In the presence of a layer of ECs co-cultured with neurons it was observed that dopamine passage gradually decreases with co-culture time. The passage of dopamine throughout a layer of ECs alone and throughout a layer of ECs cultured in presence of neurons for 5 days is comparatively shown in Fig. 2a. In experiments carried out after 5 days of coculture it was observed that after 1 h from the start of permeation the transferred amount of dopamine throughout the cell layer was almost threefold less than that observed throughout the layer of ECs alone. Data reported in Fig. 2 refer to the administration of 0.1 mg / ml of drug in the donor compartment. An analogous trend was observed when 0.05 or 0.15 mg / ml of dopamine was administered in the donor chamber. As confirmed by linear regression analysis, correlation coefficient, standard error and x 2 values, the

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Fig. 3. Permeability to dopamine with culture time of a monolayer of (쏆) ECs cultured alone and (j) ECs cultured in presence of neurons. Percent amount of dopamine, which crosses the barrier, was determined after 1 h from the start of experiments. Results are the means of six experiments. At the end of each experiment the sum of drug transferred to the acceptor compartment and the residual content in the donor chamber matched the amount of administered dopamine.

Fig. 2. Dopamine transfer: (a) expressed as Mt versus time (m) throughout a layer of ECs alone (r50.9995, S.E.560.0017, x 2 51.77?10 25 ) and (j) throughout a layer of ECs cultured in presence of neurons for 5 days (r50.9985, S.E.560.0016, x 2 5 1.48?10 25 ); (b) expressed as logarithm of the diffused drug fraction against time (m) throughout a layer of ECs alone (r5 0.9986, S.E.560.0028) and (j) throughout a layer of ECs cultured in presence of neurons for 5 days (r50.9984, S.E.5 60.0079). Straight lines validate a first-order kinetic process. The reported data refer to the administration of 0.1 mg / ml of model compound in the donor compartment. Results were reported as mean of six experiments.

movement of dopamine fits well with the first-order kinetic equation in every experiment. Moreover, straight lines were obtained when the transferred drug fraction was converted into its logarithm and plotted against time (Fig. 2b), thus validating a simple diffusion process. The permeability to dopamine with culture time is shown in Fig. 3. Drug transfer markedly decreases when the culture time of ECs in presence of neurons

increases. After 7 days of co-culture the barrier remains impermeable to dopamine. Experiments conducted after 10 and 12 days of co-culture verified that barrier endure impermeable to the drug and remains sealed. On the other hand, in analogous experiments carried out with ECs alone, the amount of dopamine that crosses the barrier is high and remains quite regular. These data are in alignment with the previously reported results [12]. Indeed, when immortalized RBE4.B ECs were immunostained for occludin either for 5 or 7 days following starting of co-culture with neurons, by immunofluorescence experiments (data reported in Ref. [12]) we observed that after 5 days cells are occludin positive, but exhibit diffuse staining throughout their cytoplasm, after 1 week they become able to segregate occludin to their periphery. We attributed the time-dependent decrease in permeability to dopamine to localization of occludin in ECs, although other tight junction molecules such as claudins may contribute to regulate junctional structure, thus suggesting that a coherent and continuous layer of cells was created and TJ barrier functions developed. Mathematical modeling of experimental data can

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be an excellent method of exploring the mechanisms involved in the process under investigation. Analysis of the data of drug transfer allows in distinguishing a simple diffusion process from a carrier-mediated passage. Movements occurring exclusively through passive transmembrane diffusion can be described by the first-order kinetic equation. In this case indeed, the rate of passage is affected by the initial concentration of drug in the donor compartment and decreases linearly with time. Fig. 4 shows the results of administration of increasing amounts of L-tryptophan or L-DOPA in the donor compartment. In experiments performed throughout ECs co-cultured with neurons for 5 days,

Fig. 4. Transfer of L-tryptophan and L-DOPA throughout a layer of ECs cultured in presence of neurons for 5 days. Each point was determined as the mean value of six experiments and solves the equation Mt 5M`s12e 2btd. Model compounds were administered in the donor compartment in increasing amounts: (a) L-tryptophan (j) 0.05 mg / ml, (r50.9992, S.E.560.0015, x 2 51.33?10 25 ); (d) 0.10 mg / ml, (r50.9997, S.E.560.0010, x 2 56.13?10 26 ); (m) 0.15 mg / ml, (r50.9994, S.E.560.0017, x 2 51.45?10 26 ); (b) L-DOPA (j) 0.05 mg / ml, (r50.9956, S.E.560.0012, x 2 5 7.47?10 26 ); (d) 0.10 mg / ml, (r50.9962, S.E.560.0027, x 2 5 3.57?10 25 ); (m) 0.15 mg / ml, (r50.9960, S.E.560.0035, x 2 5 6.20?10 25 ).

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the observed data were in line with a passive transmembrane diffusion process. Each compound was administered in the donor compartment at concentration of 0.05, 0.10 and 0.15 mg / ml. As validated by analysis of data points, the movement of both L-tryptophan and L-DOPA fit well with the first-order kinetic equation in every experiment. The results of fittings are reported in Fig. 4. Straight lines were obtained when available drug concentrations values were converted into their logarithm and reported against time (Fig. 5). After 7 days of co-culture, when occludin localizes at ECs periphery and dopamine does not cross anymore the barrier, accumulation in the acceptor compartment in the initial phase showed analogy

Fig. 5. Permeation profile of L-tryptophan and L-DOPA throughout a layer of ECs cultured in presence of neurons for 5 days expressed as logarithm of diffused drug fraction against time. Each point was determined as the mean value of six experiments and solves the equation log(12Mt /M` )5 2bt. Compounds were administered in the donor compartment in increasing amounts: (a) L-tryptophan (j) 0.05 mg / ml, (r50.9994, S.E.5 60.0021); (d) 0.10 mg / ml, (r50.9988, S.E.560.0058); (m) 0.15 mg / ml, (r5 0.9988, S.E.560.0040); (b) L-DOPA (j) 0.05 mg / ml, (r5 0.9960, S.E.560.0049); (d) 0.10 mg / ml, (r50.9970, S.E.5 60.0054); (m) 0.15 mg / ml, (r50.9987, S.E.560.0047).

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with a first-order process; while for extended periods of time drug concentration increased linearly with time. The first-order equation was not adequate anymore to describe the transfer into the acceptor chamber of L-tryptophan and L-DOPA, indicating a different transmembrane transfer mechanism. The values of r, S.E., x 2 and analysis of residuals gave no significant fittings when ECs were co-cultured with neurons for 7 days. Indeed, plotting the logarithm of diffused amount against time a curved line was observed, especially when higher doses were used. This strongly suggested that a nonlinear transfer process was involved. A major factor that imparts nonlinear kinetics may be the saturation of the transfer system for the drug. The first-order process can be replaced with the saturable Michaelis–Menten (MM) type processes.

Because no analytical expression for Mt as a function of t could be found, we used the software Curve Expert 1.34 for Windows to fit the model to our experimental data. Fittings of data indicated that the MM model is the most appropriate for describing L-tryptophan and L-DOPA permeation profiles (Fig. 6). The Michaelis–Menten parameter Km was determined to be 30.862.4 and 95.065.4 mM for L-tryptophan and L-DOPA, respectively. The calculated values of Km are in good agreement with those previously determined by both in vitro and in vivo studies [27–29,19]. The uptake characteristics of these two compounds is compatible with a carriermediated process, thus confirming that after 7 days of co-culture a continuous layer of cells was created and functional barrier formed.

4. Conclusions

Fig. 6. Permeation profiles of (a) L-tryptophan and (b) L-DOPA throughout a layer of ECs cultured for 7 days in presence of neurons: (d) L-tryptophan, (r50.9993, S.E.560.0213, x 2 5 9.60?10 26 ) and (j) L-DOPA, (r50.9988, S.E.560.0006, x 2 5 1.65?10 26 ). Each point was determined as the mean value of six experiments. The solid lines were estimated using a non linear least-square regression analysis program. The Km values were calculated as 30.862.4 and 95.065.4 mM for L-tryptophan and L-DOPA, respectively.

Our results demonstrate that RBE4.B immortalized rat brain microvessel ECs, when co-cultured with neurons, are able to form organotypic slices, which possess similar characteristics to in situ BBB. Indeed, the system does behave as a selective interface that excludes dopamine, whereas allows L-tryptophan and L-DOPA to cross. By mathematical approach, we demonstrate that both L-tryptophan and L-DOPA move across the cultured ECs layer by saturable process, thus indicating that in our model the specific transporters are always present. Permeation studies confirmed that the described co-culture system possess the characteristic permeability limitations of BBB. As a concluding remark, the described model is characterized by a relative ease with which the coculture can be produced in large amounts and reproducibility. It would facilitate the study of selective passage of a great variety of compounds across the BBB, thus allowing new strategies for designing drugs that could be delivered specifically to the brain.

Acknowledgements This research was supported by a grant from M.U.R.S.T., Rome, Italy.

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