Reconstitution of the blood-brain barrier in cell culture for studies of drug transport and metabolism

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Advanced

Reconstitution

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22 (1996)

251-264

of the blood-brain barrier in cell culture for studies of drug transport and metabolism A.G. de Boer*, D.D. Breimer

Division of Phurmacology,

LeidenlAmsterdam

Received

Center for Drug Research, University of Leiden, P.O. Box 9.50_?,2.?OURA Leiden, The Netherlands 22 September

3995; accepted

28 July 1996

Abstract In this paper the anatomy of the blood-brain barrier and the properties of peripheral versus brain-microvesselendothelial cells (BMEC) are outlined and the isolation and application of microvessels are discussed. In addition, the isolation and culture of BMEC are described and discussed and the application of this system in drug transportand metabolism-studies, visualization of drug transport routes. and in the study of changes in BBB functionality during inflammation, is illustrated. Keywords:

Anatomy;

In vitro; Isolation; Visualization:

Inflammation

Contents 251 252 252 254 254 254 257 258 Application in drug metabolism studies.. .............................................................................................................................. 259 Application in the visualization of drug transport routes.. .................................................................................................... ......................................................................................... 259 during inflammation.. Study of the changes in BBB functionality 261 ......................................................................................................................................................................... Conclusions.. 261

I. Introduction .......................................................................................................................................................................... barrier: anatomy.. ................................................................................................................................ 1 .l. The blood-brain and cerebrovascular endothelial cells ........................................................................................................... 1.2. Peripheral 2. In vitro BBB models.. ........................................................................................................................................................... ........................................................................................................................................... 2.1. Isolated brain microvessels cells (BMEC) ........................................................................... 2.2. Isolation and culture of brain-microvessel-endothelial of BMEC in drug transport studies ................................................................................................................... 3. Application 4. 5. 6. 7.

1. Introduction The blood-brain-barrier (BBB) comprises the interface between blood and the endothelial cells of the microvessels of the brain. It has many important functions, such as limiting the mainte*Corresponding

author.

0 0169-409X/96/$32.00 PII SOl69-409X(96)00421-8

1996 Elsevier

Science

B.V. All rights

nance of homeostasis in the brain, transport of macromolecular compounds and nutrients into the brain, etc. It also plays an important role in disease states of the brain [l]. Research on BBB transport of drugs has been considerably facilitated and enhanced by the development of BMEC isolation techniques [251. These allow the culture of confluent monoreserved

layers of BMEC and therefore the estimation of the rate and quantity of drug transported across the BBB, and the assessment of BBB metabolism. However. the results obtained by the various in vitro techniques can differ considerably, which hinders the comparison of experimental results and the extrapolation to the in vivo situation. The major causes of these discrepancies are due to the different characteristics of the various in vitro models used. In this section such models will be reviewed and discussed and some examples will be given of their use in studying drug transport and metabolism. The application of confocal-laser-scanning-microscopy (CLSM) will be illustrated which allows the localization of fluorescent compounds during their transport through a confluent monolayer. These monolayers can be used to study also the changed properties and permeability of the BBB during disease states, e.g. inflammation. 1.1. The blood-brain

barrier: anatomy

The concept of the blood brain barrier (BBB) has been developed from the end of the nineteenth century onwards, starting with the German pharmacologist and physiologist Paul Ehrlich [7] and several others [S-lo]. Walter [l l] made a distinction between the BBB and the choroid plexus and a crucial experiment was performed by Reese and Karnovsky [12] and Brightman and Reese [13] who demonstrated that intravenously administered horseradish peroxidase, was indeed confined to the blood compartment by the cerebrovascular endothelial cells. implying that these cells constitute the principal anatomical basis of the BBB. Spatz [lo] and Walter [ll] suggested that a blood-cerebrospinal-fluid (CSF) barrier existed comprising the endothelial and epithelial linage of the choroid plexus, and a brain-CSF barrier comprising the ependyma, which lines the brain tissue and bears a close morphological relationship to renal tubular epithelium. An overview of these barriers is given in Fig. 1 [ 151. The surface area available for exchange at the blood-CSF barrier is limited: approximately a factor 5000 less than that of the blood-brain barrier [16]. Therefore, its large surface area

Fig. 1.The various barriers in the central nervtx~s system: the blood-brain barrier, the blood-cerebrospinal fluid barrier and the brain-cerebra-spinal fluid barrier. Redrawn from [ 151.

renders the BBB the most important barrier for drug delivery to the brain. Besides its main function of maintaining homeostasis in the CNS, the BBB, but also the CSF, are important factors in the kinetics and time course of substances moving into and out of the CNS. 1.2, Peripheral and cerehrovascular W1l.S

endothelial

The endothelium of the microvessels in the brain exhibits various structural differences in comparison to that of other organs (Fig. 2) [17]. Peripheral endothelial microvessels have fenestrations of - 50 nm in diameter between the endothelial cells, enabling almost free exchange of water and solute with the extracellular fluid. These fenestrations are not found in brain endothelial cells [18] while also pinocytotic vesicles are almost absent [19]. Between the cells are very tight junctions that effectively block the transport of large compounds. These tight junctions are considered to be highly dynamic and heterogenous. which regulate paracellular permeability 1201. In vitro experiments with bovine brain microvessel endothelial cells, using a series of thtorescein labelled dextrans with increasing molecular size, revealed the existence of 80 A rectangular pores with a fractional pore area of 0.01% [21]. In vivo such pores may be smaller since pore measurements in these experiments were made in relatively leaky monolayers with-

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Drug Delivery Reviews 22 (1996) 251-264

GLIAL INTERCELLULAR

NON-

NEURAL

NEURAL

CAPILLARY

of brain

ENDFOOT

CLEFT

CAPILLARY

(b)

(a) Fig. 2. The properties

253

microvessel

endothelial

cells versus

out the induced high resistance by astrocytes, astrocyte conditioned medium or CAMP precursors. The endothelial cells representing the BBB contain mitochondria, indicating that in addition to its physical barrier properties, the BBB may also function as a metabolic barrier. The upregulation of the P-glycoprotein efflux pump at the BBB in multi-drug-resistance is other evidence for a barrier function [22,23]. Different cell types present in and surrounding the BBB may influence its permeability and functionality. It has, for example, been shown that astrocytes [24-261 influence in particular the tightness of the tight junctions when co-cultured with cerebrovascular endothelial cells. Similarly, pericytes are attracted by capillary structures of endotheli-

peripheral

endothelial

cells. Redrawn

Blood flow pliglmin

Inferior colliculus (grey matter) Sensorimotor cortex (grey matter) Genu of corpus callosum (white matter) Subfornical organ) organ (circumventricular

1.977 2 257(3) 1.550? llO(3) 394?40(3) 1.008

[17].

al cells [27] while neurons also seem to have an effect on BBB functionality [28]. It should again be stressed that the BBB is not made up of a homogeneous cell of tissue system throughout the brain. Due to the presence of fenestrations, the endothelium of the circumventricular-organs (CVO) such as the area postrema or the median eminence [29] is leakier than other parts of the brain and is therefore not representative for the whole brain. Similarly, the blood flow to the various brain regions may differ considerably, which may also cause quantitative differences in drug transport across the BBB at different sites [29]. In Table 1 information is provided on the blood-flow, the albumin transit time, the Lu-amino-isobutyric (AIB) acid influx and the permeability-surface

Table 1 Physiological measurements and calculations for microvessels in grey and white matter and the subfornical lar organ) of rat brain. Values are mean?zS.E. for number of rats in parentheses. Data from [29] Structures

from

Albumin time, s 0.27 0.23 0.45 3.21

transit

organ

AIB influx tJlg/min 4?0.5(3) 1+0.5(3) 1?0.1(3) 401

(circumventricu-

PS product pligimin 5 1

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A.G. tie Row,

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area product for various regions of the brain. In particular the permeability of the subfornical organ is considerably larger than that for other parts of the brain. Current insights into the anatomical basis of the BBB, as an interface between blood and brain, clearly indicate that, in contrast to what has long been generally accepted, it is not a static homogeneous impermeable barrier. Its permeability is regulated dynamically with special features relating to the absence of fenestrations, the relative lack of pinocytotic vesicles, the presence of tight junctions and mitochondria, the presence of (active) transport systems. the influences of other cell types. like astrocytes, pericytes and neurons. Also certain blood components like hormones and histamine may influence BBB permeability. Since drugs that have to be active in the CNS are administered in case of CNS-diseases, it is also important to know if the BBB is changed in disease state. However, little is known about the changes in BBB functionality in disease state [30,31 J. Several processes may be altered under such circumstances. e.g. various physiological factors such as protein binding capacity and blood flow [31,32]. In addition, it is well known that the BBB may be ‘opened’ during infammation or by tumours. Less is known about the upor down-regulation of transport systems at the BBB in disease state. Information about this may enhance the possibility to target drugs to the brain in specific disease states.

2. In vitro BBB models 2.1. Isolated

brain microvessels

Brain microvessels have been used in the very beginning of BBB research [3,4]. They arc well suited for studying the uptake of nutrients and in localizing or identifying specific enzymes and substrate binding-sites. In addition. isolated brain microvessels have successfully been used in drug disposition studies (uptake into endothelial cells) and in metabolism studies. The major advantage of the microvessel isolation technique is that it provides a system that is quite similar to the in vivo situation with respect

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to the expression of surface molecules and it may therefore be a good screening model to identify selective or specific interactions with the microvessel endothelium (enzymes, adhesion molecules, binding sites), and for uptake studies. However, this model also suffers from a number of drawbacks: it is quite a heterogenous system [33] in the sense that various cell types may be present (endothelial cells, pericytes, fibroblasts, astrocytes). Furthermore. brain microvessels are not suitable for studying transcytosis processes. and in uptake studies the substrate approaches the endothelial cells mainly from the abluminal side [4.34,35]. On the other hand these vessels are useful in the so-called capillary depletion technique [36] where one can differentiate between in vitro transcytosis, adsorption, uptake and sequestration of compounds by the BBB.

J.2. Isolation and culture rndotheliul cells (BMEC)

of

hrain-microvessel-

The isolation and culture of BMEC [2-6,21,28] has tremendously enhanced research in the area of drug transport across the BBB in particular at the (sub)cellular level [37,38]. The availability of cultures on porous membranes or filters has contributed much to the applicability of this system in providing confluent monolayers of BMEC. In addition, co-culture of BMEC with astrocytes has enhanced insight in the functional properties of the in vitro BBB. Increased expression of various endothelial markers like r-glutamyl-transpeptidase ( y-GTP), angiotensin convcrting enzyme (ACE), monoamine oxidase (MAO). etc. [39], has been observed in these co-cultures. Cultured BMEC are useful to study a whole variety of important phenomena. e.g. drug transport across the BBB, drug metabolism, the effect of specific agents and conditions on the functionality of the BBB, etc. Various techniques have been applied for the isolation and culture of BMEC. They comprise, following dispersion of the collected grey matter from the brain. the application of enzymatic [S], combined mechanical and enzymatic [40,41] and mechanical [42] procedures. This results in the isolation and culture of BMEC that may be

A.G. de Boer, D.D. Breimer

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characterized by but also differ in various properties, such as the expression of Von Willebrand Factor (VWF), monoamino oxidase (MAO), yglutamyl transpeptidase (r-GTP), uptake of acetylated low density lipid particles (DiIAcLDL), differences in trans endothelial electrical resistance (TEER), differences in (para- and transcellular) permeability, etc. The main causes that are responsible for this variability will be discussed below. BMEC are isolated from human [43], rat [40], mouse [4], bovine [2,42,44], calf [2], and porcine [45] brain.Various parts of the brain are used for the isolation of BMEC: forebrain, cerebral hemispheres, white and grey matter of the cortex, choroidal microcapillaries, etc. In addition, there may be considerable differences, in particular with respect to the number of viable cells isolated [46]. About 200-250 million cells are isolated from one bovine brain, while for 10 rat brains this number varies from 20-50 million. In addition, it is our experience that BMEC isolated from calf brain grow better than from bovine brain, while seasonal factors seem to have an influence too which may be related to changes in food. hormones, etc. In addition, it has furthermore been reported that the isolation of peripheral endothelial cells from non-smoking humans gave significantly better harvests than from smokers [47]. This clearly illustrates that the source of isolation is an important determinant for the ultimate BMEC obtained. The isolation techniques to obtain brain microvessel endothelial cells vary considerably. These comprise mechanical dispersion of the grey matter of the brain followed by enzymatic treatments [2,5] with various enzymes [4,6], combined mechanical/enzymatic procedures [40,41] or only mechanical procedures with various mesh sizes [40,42]. The enzymatic procedures seem to completely remove the surface molecules from the cells which make the cell culture medium and the extracellular matrix, where the cells are grown on, very determinant in the re-expression of surface molecules. The mechanical procedures seem to be less ‘rough’ in this respect, thereby better preserving the in vivo BBB properties. BMEC obtained by this procedure are therefore very suitable to study various BBB characteris-

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tics, which holds in particular for primary cultures. The isolation procedures differ in selectivity with respect to the co-isolation of contaminating cell types [48] e.g. pericytes, fibroblasts and the presence of venous endothelial cells. Contaminating cells may influence the Trans-Endothelial-Electrical-Resistance (TEER), and drug transport through the monolayer. Furthermore, contamination may lead to erroneous conclusions with respect to the expression of transcellular transport systems and/or surface molecules on BMEC. The procedures which result in the ‘purest’ BMEC monolayers are those which first isolate the brain microvessels [40,42]. Subsequently, these are seeded on an extracellular matrix and the endothelial cells will migrate from these microvessels. After subsequent trypsinization and seeding very pure BMEC monolayers can be obtained. Depending on the purpose of the studies to be undertaken with the BMEC monolayers the use of primary cell cultures [2,5] versus passaged cells [49] is preferred. Primary cells usually have preserved their properties to a certain extent, while passaged cells may exhibit considerably increased or decreased expression of surface molecules [50], y-GTP as a characteristic of brain capillaries and to a lesser extent general endothelial markers like ACE and MAO [39,49]. Since y-GTP has shown not to be a specific characteristic of brain capillaries [51], additional markers, or even better a panel of markers, including Von Willebrand Factor (VWF) and or uptake of DiI-labelled acetylated LDL [52], have to be used to characterize the isolated cells. It has been shown that the extracellular matrix (ECM) and the filter material where the cells are grown on may influence the expression of surface molecules. These may change depending on the type of extracellular matrix applied [53]. In addition to the ECM, hyaluronic acid and also some growth factors seem to be important with respect to the occurrence of angiogenesis [54-561 and this may compromise the development of high resistance monolayers [21]. In addition, components of the ECM, e.g. fibronectin and laminin, have been shown to convert quiescent astrocytes into proliferating ones [57] which may

2%

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induce local differences in the BMEC monolayers. The support layers where BMEC are grown on comprise: collagen ? fibronectin coated regenerated cellulose and collagen ? fibronectin coated polycarbonate aluminumoxide, poly-o-lysine coated materials, polyethylenimide coated materials, positively charged polystyrene, polyester. etc. [.5,58-601. The application of a liquid-liquid interface (fluorocarbon/water) seems to be an interesting one in particular to study cellular migration [61]. Support materials may influence the growth of BMEC. Coated supports may prevent the invasion of endothelial cells into the filter [62], while supports in general may cause dedifferentiation, influence receptor localization [53], change the expression of integrin subunits (in peripheral ECs) [50], etc. In addition the properties of the BMEC may also be changed by the isolation procedure. Trypsin may reduce ACE activity, while trypsinization reduces the number of bradykinin receptors and inhibits endothelial-derived-relaxing-factor (EDRF) release of ECs in response to bradykinin [64]. Thrombin induces alterations in morphology and differences in growth stimulation between organderived ECs (non-brain) were observed in response to thrombin and endothelial-cell-growthfactor (ECGF) [6.5]. Various growth media are used to obtain BMEC and the application of serum [4,6] versus serum free medium is still a matter of debate. Serum free medium is well-defined, although it may lack certain (essential?) and unknown compounds which are present in serum-containing medium. On the other hand serum-containing medium may have a less constant composition. Some suggestions have been to perform experiments while culturing cells in serum-containing medium [66]: 1. Replace the culture medium before the experiment with serum-free medium 2. Subculture in serum-free medium plus medium conditioned by heterologous cells 3. Subculture in serum-free medium plus the extra-cellular matrix from heterologous cells grown in the presence of serum. It is still a matter of experience which medium

Drug Delivery Reviews 22 (lYY6) 2_71-264

is the best choice for good growth of BMEC monolayers that resemble as much as possible the in vivo BBB. The application of astrocyte-conditioned medium (ACM) or the co-culture of astrocytes with BMEC may induce the expression of important enzymes, marker molecules. The expression of surface molecules and enzymes (r-GTP, alkaline phosphatase) in vitro is very much dependent on the application of the astrocyteconditioned medium [40,67]. Astrocytes can be isolated from rat pups according to the procedure of Tio et al. [68] or Cole and de Vellis [69]. Oligodendrocytes are removed after 8 days of culture [70] and subsequently the astrocyteconditioned medium (ACM) is collected during further culture. The astrocytes are characterized by their glial fibrillary acidic protein (GFAP) expression, which may lead to a purity of at least 95%. Growth of BMEC and astrocytes also provide good conditions to obtain BMEC with good functionality [39,49,71]. In addition human fetal astrocytes have shown to induce the expression of factor VIII, the GLUT-l glucose transporter and y-GTP on autologous endothelial cells [72]. Several markers can be induced by ACM [40] or astrocytes [39,49] but mostly not to the full 100%. while the susceptibility to astroglial induction seems to be dependent on the proliferative state of the BMEC [39]. The application of growth factors, like glutamine [74], thrombin [75], vascular permeability factor [76], platelet-derived-endothelial-cell-growth-factor (PD-ECGF) and transforming growth factor-/? [77] enhance the formation of confluent monolayers, but may cause angiogenesis at a later stage. Stimulants can be used to induce certain specific features in confuent monolayers of brain microvessel endothelial cells. It has for example been shown that the application of phosphodiesterase inhibitors and compounds that increase intracellular CAMP levels increase the tightness of tight-junctions (401. This is a very interesting method to create high resistance BMEC monolayers for the study of (hydrophillic) paracellular transport. It is still not clear, however, to what extent these stimulated BMEC monolayers resemble the in vivo BBB. Various antibiotics are used in culturing

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BMEC: gentamycin, penicillin-G, streptomycin, amphotericin-B, fungizone, etc. In particular the use of amphotericin-B leads to monolayers which are very leaky since this compound causes pore formation in the membranes [2,21]. Most transport studies are performed in the absence of stirring. It is known that shear stress [78] and periodic stretching and relaxing (aortic endothelial cells from mini-pigs) [79] may modify the BMEC monolayers. This may have consequences for drug transport studies. In addition, other factors like capillary geometry, and in vivo factors like blood pressure or blood cells may influence drug transport [80]. The application of immortalized cell lines may have some advantages since it avoids the repetitive isolation of BMEC from human and animal brain. An immortalized rat brain endothelial cell line (RBE4) has recently been established [81]. It exhibits many characteristics of the BBB but cell lines usually show a reduced expression of surface molecules, enzymes or transport systems upon passage. These can be induced with ACM or in co-culture with astrocytes, but often these cultures result in relatively leaky confluent monolayers.

3. Application of BMEC in drug transport studies The in vitro reconstituted BBB system is very well suited to study various characteristics of drug transport across the BBB in detail. Once the cell cultures have been characterized (morphologically, biochemically and functionally [5,21,28]) they can be used. The techniques used to study drug transport are the Ussing chamber and the filter plate system [59,80]. The Ussing chamber allows stirring but only a limited number of experiments can be carried out concurrently. The filter plate approach allows the performance of a larger number of experiments concurrently. The permeability of the monolayer can be estimated in terms of a permeability constant [80] or the clearance [21] of a substance. Permeability is clearance divided by the surface area of the monolayer [82]. This surface can only sufficiently be estimated when culturing endothelial cells. In case of epithelial cells the surface

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area cannot easily be estimated due to the occurrence of microvilli. Both permeability and clearance can be considered as functional parameters characterizing drug transport through confluent endothelial monolayers. Passive hydrophillic (series of FITC labelled dextranes with increasing molecular sizes) and lipophilic (series of P-blocking drugs) transport [44] has been studied in BMEC as well as carrier mediated transport [83]. Fig. 3 shows an example of the transport of zidovudine (AZT) through confluent monolayers of BMEC [67,84]. By dividing the amount of drug transported from the apical to the basolateral side by the mean apical concentration during the transport interval, one obtains the cleared volume [21]. Plotting the cumulative cleared volume versus time gives a straight line with clearance as slope. The permeability can be calculated from the control and monolayer experiments according to (21,841: P BBB

CL,, . CLwol = A (%ntrol - CL,>

where A =surface area of the endothelial monolayer (- surface area filter), PBb, is the BBB-permeability (ml/min.cm’), CI,,,, and CL”tWl are the clearances (volume/time, often expressed in kl/min) of the test (filter with cells) and control (filter without cells) experiments. Deviations from linearity may indicate satura-

time fminl Fig. 3. The cumulative cleared volumes of AZT following luminal (apical, open symbols) and abluminal (basolateral, solid symbols) application with (triangles) and without (circles) confluent monolayers of bovine BMEC. Redrawn from [84]. ctr. lum. =control luminal; ctr. ablum. =control abluminal; ml. lum. = monolayer luminal; ml. ablum. = monolayer abiuminal.

tion. inhibition. associated with the existence of time- or dose-dependent transport systems. In addition, following comparison of apical and basolateral exposure of drug to the monolayers. it is possible to identify polarized transport. The in vitro BBB models have been proven quite successful in predicting drug transport across the BBB. Initially, the in vitro results gave overestimation of the in vivo BBB permeability [8S]. This was mainly caused by the absence of astrocytes which can induce many properties of the BBB including high trans-endothelial-electrical resistance due to narrow tight junctions [40]. Co-culture of BMEC with astrocytes resulted in a good correlation between in vitro permeability of various compounds varying from very low to high brain extraction rates and in vivo permeability [86]. This gives, therefore. good perspectives for in vitro screening of drugs with respect to their in vivo BBB-permeability. A very important transport system is the Pglycoprotein (Pgp) efflux pump which is present in the apical (luminal) membrane of the BBB [22,23]. This pump extrudes various compounds. like many cytostatics but also peptides [87] out of the cell. It becomes considerably up-regulated following continuous drug exposure leading to multi-drug-resistance [88]. This may effectively reverse the transport of Pgp substrates from into to out of the brain [89,90]. In addition. it has been shown in mdrla (- / -) mice that the transport of the Pgp-substrate ivermectine was increased considerably 123). In addition, LDL (low density lipoprotein), AcLDL (acetylated low density lipoprotein) and HDL (high density lipoprotein) particles bind to confluent monolayers of BMEC by the LDL receptor [91], the scavenger receptor and the HDL receptor. respectively. It is known that AcLDL binds to peripheral endothelial cells and is taken up via the lysosomal route by the scavenger receptor which is present on several types of endothelial cells 1921. Similar data were found for binding to BMEC 1521. It was shown that there is a specific binding site for AcLDL with a mean apparent affinity of 3.1 nM and a maximal binding ranging from 284 to 626 ng/mg protein. Binding studies of HDL with confluent monolayers of BMEC showed a high affinity

binding site for HDL [93]. Similar data were found by Martin-Nizard et al. following binding of HDL, to bovine BMEC [94]. Binding was absent following trypsin treatment and was inhibited by lipoprotein A-I. In addition, surface bound “‘I-HDL, was not endocytosed but rapidly released into the medium. The LDLreceptor present on bovine BMEC was found to have a molecular weight of 132.000 [91].

4. Application

in drug metabolism

studies

The BBB contains many enzymes which may degrade drugs during their passage through the endothelial cells. These comprise phase I enzymes like cytochrome P450, NADPH-cytochrome P450 reductase, epoxy hydrolases, aldehyde dehydrogenase, xanthine oxidase, xanthine dehydrogenase, ketone and alcohol oxidoreductases. monoamino-oxidase A and B, transaminases. alkaline phosphatase. angiotensin converting enzyme, aminopeptidases, carboxypeptidases, catechol-O-methyl transferase, while the phase II enzymes that may be present at the BBB comprise IJDP-glucuronosyltransferases, phenol sulphotransferase, glutathione S-transferases, etc. (X0.95,96]. Intracellular processing of drugs leading to transport into the lysosomes may also result in degradation. However, it was shown in BMEC cultures that AcLDL particles [52] could escape. to a relatively large extent. metabolic degradation. Fig. 4 shows that degradation of AcLDL was about lo-20-fold lower as compared to AcLDL in peripheral endothelial cells in culture and could not be influenced by chloroyuine and ammonium chloride treatment (not shown) [52]. These compounds, which are known to raise the pH in the lysosomes thereby reducing lysosomal enzyme activity, inhibiting the receptor recycling and therefore ligand uptake (97,981, did not influence the extent of degradation. Similar data were obtained with HDL (931. Presently, there is no satisfactory explanation for the results obtained with 12’I-HDL3 (not endocytosed. see [94]) and the degradation of HDL. Peptidase activity was demonstrated by incubating arginine-vasopressin and some of its

A.G. de Boer,

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t/me /mm1 Fig. 4. Time course of the cell association and degradation of “‘I-AcLDL. Cells were incubated for indicated times with 5 and cell association was determined in kg/ml of “‘1-AcLDL the absence (0) or presence (0) of chloroquine (100 FM). Degradation was also determined in the absence (A) or presence (A) of 100 FM chloroquine. Data from one typical experiment out of live. Redrawn from (521.

fragments with relatively large surfaces (60 cm’) of BMEC [99].

5. Application in the visualization transport routes

of drug

Confocal laser scanning microscopy (CLSM) is a very powerful tool to visualize the transport routes of drugs and the influence of drugs and other (toxic) compounds on changes in intracellular Ca’+-concentration. CLSM has been used to visualize the transport of the octapeptide and somatostatin analogue, octreotide (SMS 201-995, SandostatinTM), across confluent monolayers of BMEC [82]. A device was constructed to be able to perform CLSM on confluent monolayers cultured on filters [lOO,lOl]. Two different fluorescent conjugates of octreotide (FITC- and NBDoctreotide) were used to obtain CLSM images. The peptides did not undergo significant degradation in the presence of brain endothelial cell

Table 2 Clearance,

log-P values

Compound Octreotide NBD-octreotide FITC-octreotide

and molecular

weights

Cf,_, (Fl/min) 2.2~0.4 1.4to.3 1.2t0.1

of octreotide

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monolayers. The transport rates of octreotide expressed as clearance (Cl,,,,) are given in Table 2. Cl,,,, of octreotide and that of the paracellular permeability marker fluorescein correlated well [82]. The conjugates exhibited lower CL,,,, values than octreotide itself, which can be explained by their different log-P values and molecular weights (sizes). On the CLSM images, FITCoctreotide was present only in the intercellular space. while the inside of the cells did not exhibit detectable fluorescence. Based on transport studies and CLSM images it was suggested that octreotide passes the endothelial monolayer primarily via the paracellular route without significant contribution of carrier-mediated transport. This conclusion was based on one hand on the clearance of octreotide and on the other hand on the CLSM images obtained with FITC- and NBD-labelled octreotide. Since the log-P values and molecular weights (sizes) between parent compound and conjugates differ considerably, it cannot be excluded that CLSM imaging with the octreotide conjugates is not representative for octreotide itself. This is one of the limitations with CLSM that requires fluorescent compounds.

6. Study of the changes in BBB functionality during inflammation

The functioning of the BBB as an interface between brain and blood is probably best illustrated by its interaction with cells of the immune system (see Fig. 5) [102,103]. Due to luminal or abluminal stimuli the BBB endothelium may present itself as an antigen-presenting-cell subsequently followed by binding to lymphocytes, monocytes, etc. However, it has been shown [104] that in vitro cultured rat BMEC have an incomplete presentation of (auto)antigens which

and its fluorescent

NBD-

log-P (octanollwater) -0.160 1.130 0.372

and FITC-conjugates.Data pH 7.4

from

[82]

Molecular 1019 1183 1408

weight

260

A.G.

dr Boer.

D.D.

Brertnrr

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Dnrg

D&wry

Rmiews

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Fig. 5. Potential ports of entry for the immune system into the CNS. (a) The BBB, which consists of endothehal cells (EC) with tight junctions between them. greatly impedes the entry of lymphocytes (Ly) and antibodies (Ab) into the CNS. (b) EC may act as antigen-presenting cells and thus attract Ly. (c) Astrocytes may act as antigen-presenting cells and through interaction with EC attract Ly. (d) Ly involved in immune surveillance may encounter CNS antigen and secrete lymphokines and thereby attract other Ly. (e) Ab may enter, e.g. through some of the leaky arcas in the BBB: on interaction with their antigen counterpart in the CNS, they may cause damage to EC. making the BBB permeable. Ab and Ly can enter through the leaks (redrawn from [102]).

could be induced following interferon-y (IFN-7) treatment. Permeability of the BBB may be changed during central nervous system diseases. like multiple sclerosis 130,311, bacterial meningitis [lOS] or tumours [106]. There are indications that the expression of ICAMand ICAMis downregulated in a mammary adenocarcinoma in rats these molecules [107]. while in inflammation seem to be up-regulated [14]. Following stimulation with lipopolysaccharide and interleukin 1 and 6. lymphocyte binding was increased 3-fold and could be blocked by monoclonal anti-VLA4 (Fig. 6) and anti-CDllalCD18 (LFA-1) antibodies [63]. In addition. it has been shown that the trans-endothelial-electrical-resistance (TEER) dropped and transport of fluorescein labelled dextran (molecular weight 4000) and “‘I-bovine serum albumin increased, following a 3-h incubation period with confluent monolayers of BMEC [73]. Similar effects on TEER were seen following incubation of confluent monolayers of rat-brain-microvessel-endothelial cells (RMEC) with tumour necrosis factor-a (TNFcy). interleukin-l/? (IL-I p) and interleukin-6 (IL6). These effects could be largely blocked following preincubation with indomethacin or an IL-l receptor antagonist (de Vries et al. unpublished results). In addition. incubation of confIuent monolayers of RMEC with TNF-cu. IL-lp and

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rats) BBB transport seems to be increased and elimination from the brain reduced [73]. These data indicate that disease state may give rise to opportunities to achieve selectivity in BBB-transport by identifying up-regulated transport systems at the BBB. These regulatory systems may provide the physiological basis for selective drug uptake and enhanced drug delivery (targeting) to the CNS.

7. Conclusions The in vitro BBB is a very useful model to study drug transport to the brain and drug metabolism by the BBB. Much progress has been made in optimizing this system, but several pitfalls still exist which may give rise to biased results. The optimal in vitro BBB systems are those that apply astrocyte-conditioned medium or coculture with astrocytes. Application of phosphodiesterase inhibitors or precursors of CAMP are useful when tight monolayers are needed but may lead to an in vitro BBB system that has less resemblance to the in vivo BBB. The functionality of the in vitro BBB has been shown in experiments with inflammatory stimuli and this may offer interesting possibilities to study the influence of inflammatory related diseases like meningitis, multiple sclerosis, Alzheimer, etc. Obviously, in vitro experiments need to be followed up by suitable in vivo experiments to verify the relevance of in vitro observations.

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