- Top of page
Summary: Purpose: A biotechnologic breakthrough for the study of drug permeability across the blood–brain barrier (BBB) would be the use of a reproducible in vitro model that recapitulates the functional, structural, and pathologic properties of the BBB in situ. We developed a humanized dynamic in vitro BBB model (DIV-BBB) based on cocultures of human microvascular endothelial cells (HBMECs) from “normal” and drug-resistant epileptic brain tissue with human brain astrocytes (HAs) from epilepsy patients or controls.
Methods: HBMECs and HAs were cocultured for 28 days in polypropylene capillaries. HBMECs were exposed to physiologic levels of shear stress generated by intraluminal flow. Permeability to [3H]sucrose, [14C]phenytoin, and [14C]diazepam was measured in control and drug-resistant DIV-BBB with and without pretreatment with the MDR1 inhibitor XR9576. BBB integrity was monitored by transendothelial electrical resistance measurements (TEERs). Cell growth and viability were assessed by measurement of glucose consumption and lactate production.
Results: PSucrose and TEER values did not depend on the origin of the endothelium used (epileptic or normal). PPhenytoin was 10-fold less (1.54 × 10−6 cm/s) in drug-resistant BBB models than in controls (1.74 × 10−5 cm/s). MDR1 blockade with XR9576 was effective (3.5-fold increase) only in drug-resistant cultures. PDiazepam in control and drug-resistant DIV-BBB was not affected by XR9576 and did not depend on the epileptic or control origin of endothelia. The overall contribution of epileptic glia to pharmacoresistance was negligible.
Conclusions: These results show that, for the substances used, the humanized DIV-BBB recapitulates the physiologic permeability properties of the BBB in vivo and is also capable of mimicking a drug-resistant BBB phenotype.
Multiple drug resistance is characterized by insensitivity to a broad spectrum of drugs with different chemical nature (Dombrowski et al., 2001; Abbott et al., 2002; Marroni et al., 2003b; Loscher, 2005), presumably acting on different receptors and/or by different mechanisms. Increased expression of drug-efflux transporters such as MDR1 has been found in brain tissue surgically resected from patients with medically intractable epilepsy (Dombrowski et al., 2001). Expression of these drug-extrusion mechanisms could justify the pharmacokinetic feature of pharmacoresistance (Oby and Janigro, 2006). This seems likely because evidence exists to support that overexpression of drug efflux transporters at the blood–brain barrier (BBB) such as P-glycoprotein (P-gp) may reduce levels of antiepileptic drugs (AEDs) in epileptogenic brain tissue (Tishler et al., 1995; Marchi et al., 2004, 2005; Oby et al., 2006).
Despite the numerous animal models of epilepsy developed so far, a significant impediment to the fast and effective development of AEDs has been the paucity of in vivo models that mimic the patterns of multiple drug resistance in humans with epilepsy (Loscher, 2002a). Ideally, a perfect model of AED drug resistance would encompass a therapeutic target comparable to the one to be reached in patients, and mechanisms that either “hide” the target or otherwise prevent the drug from reaching the target itself. Similar considerations apply to an ideal in vitro model of pharmacoresistance. However, in vitro experiments allow the use of resected human tissue, where the efficacy of a given drug treatment can be studied in isolation from other confounding factors. By using cortical brain slices from drug-refractory epilepsy patients, we were able to demonstrate that pharmacokinetic mechanisms such as increased levels of multiple drug resistance transporters at the BBB correlate with reduced levels of AEDs in the epileptic focus of multiple-drug–resistant patients as compared with the surrounding brain tissue. Therefore hampered drug distribution may be an essential contributor to pharmacoresistance (Oby et al., 2006).
We and others developed a strategy to study the passage of drugs from serum into the brain based on a coculture of endothelial and glial cells under dynamic conditions (Stanness et al., 1996; Janigro et al., 1999, 2000; Cucullo et al., 2002). This dynamic in vitro-BBB model has been used in our laboratory to demonstrate (a) the influence of glia on endothelial cell differentiation (Stanness et al., 1997; Cucullo et al., 2002, 2005c); (b) the feasibility of using the DIV-BBB to study the effects of flow on endothelial cell metabolism and gene expression (Cucullo et al., 2002; Desai et al., 2002a; Krizanac-Bengez et al., 2003); and (c) the expression of multiple drug resistance genes in endothelial cells from human epileptic brain (Dombrowski et al., 2001).
The modeling that we used in the past was, however, limited to studies on either “normal” human brain or brain endothelium and glia of animal origin. This has several limitations, chiefly the lack of mechanisms of drug extrusion by MDR1 or other proteins that can mimic those present at the human drug-resistant BBB. Little is known about how pathologic expression of MDR1 in patients affects the passage across the BBB of AEDs and other drugs that are substrates of these proteins.
The exact mechanisms of reluctant distribution of AEDs in multiple-drug-resistant epileptic brain is also confounded by the fact that in epilepsy patients, the BBB appears to be impaired, allowing passage of normally excluded molecules (for a review, see Oby and Janigro, 2006). It is thus possible that this impaired selective permeability may be an additional, albeit paradoxic, mechanism of multiple drug resistance in epileptic brain. Thus modeling of drug permeation across the epileptic BBB may also encompass the use of strategies aimed at “opening” the BBB.
The purpose of our study was thus twofold: on the one hand, we wanted to develop a human model of the BBB in vitro that expresses multiple drug-resistance mechanisms normally present in human epileptic brains. To achieve this, we used endothelial cells and glia isolated from either normal or epileptic brain, the latter having been previously extensively characterized in terms of drug-extrusion molecule expression (Dombrowski et al., 2001). In addition, we studied how the normal and epileptic BBB affect the transport of polar and nonpolar molecules under conditions of BBB failure. These studies were designed to mimic as much as possible the pharmacokinetic properties of the vasculature of the human epileptic, multidrug-resistant brain.
- Top of page
The purpose of this study was to establish whether a humanized DIV-BBB cell-culture system can be used to mimic the in situ pharmacokinetic properties of the human BBB in normal brain or in patients affected by drug-refractory epilepsy. We wished to demonstrate preliminary evidence that a novel in vitro humanized BBB model may be useful to study drug permeation under therapeutically realistic conditions. In this model, we studied electrical resistance, metabolism, permeability to a well-established paracellular marker (sucrose), a highly permeable compound (diazepam; DZP), and an antiepileptic drug (phenytoin; PHT), which is a substrate for MDR transport. Sucrose is commonly used as a marker of the paracellular pathway and has been almost universally acknowledged to be a good marker of BBB integrity. Furthermore, sucrose, as TEER, does not depend on metabolism of the cell or specific transporters into or out of the brain. The other marker chosen for these experiments (PHT) represents an MDR1 substrate, and differences in permeability between the epileptic and control models can be used as indicators of drug resistance. The third marker chosen, DZP, is not transported to our knowledge by any multidrug-resistance molecules but moves across the BBB by passive diffusion.
In the first set of experiments, we tested the feasibility of coculturing epileptic and “normal” HBMECs with normal human astrocytes. In particular, we wished to understand if these cells can establish a viable in vitro BBB. This was achieved by monitoring TEER, metabolism of the cells growing in the DIV-BBB, and by measuring permeability to the tracer of paracellular leakage [3H]-sucrose. Note that under dynamic (flow) conditions, endothelial cells develop a morphology that closely resembles the endothelial phenotype in situ (Ott and Ballermann, 1995), contributing to much higher TEER values and drug-permeability values, closely resembling in vivo conditions. This model has better predictive value than do static cultures (Santaguida et al., 2006).
Permeability values to this marker (Stanness et al., 1997) were respectively 3.92 × 10−7± 0.61 cm/s in control BBB modules and 4.37 × 10−7± 0.53 cm/s (n = 4) in cocultures with epileptic endothelial cells. These values are similar to sucrose permeability in vivo (Davson and Segal, 1995). We found no differences (p < 0.05) between permeability values of control and epileptic DIV-BBB modules (Fig. 1A). This demonstrates that human endothelial cells exposed to dynamic growth conditions form a BBB phenotype in vitro regardless of the “control” or “epileptic” origin of endothelial cells used.
Figure 1. Human brain microvascular endothelial cells (HBMECs) from control and epileptic brain cocultured with human astrocytes (HAs) and exposed to flow to develop blood–brain barrier (BBB) properties. A: Permeability measurements of [3H]-sucrose in control and epileptic in vitro BBB models. Both models develop a very tight barrier to this well-established paracellular marker. No statistically significant differences in sucrose permeability (Psuc) were observed. B: HBMECs develop a very tight barrier characterized by a stable, high (>1,100 ohm/cm2) transendothelial electrical resistance (TEER). No statistical differences were observed between control and epileptic dynamic in vitro BBB (DIV-BBB) modules. All the experiments were repeated in quadruplicate (n = 4), and permeability values are given as ±SEM. C: Glucose consumption and lactate production in both control and epileptic cocultures demonstrate an almost identical, predominantly anaerobic, metabolic pathway. No statistical differences between control and epileptic DIV-BBB were observed.
Download figure to PowerPoint
A fundamental property of the BBB is the formation of an impediment to the passage of polar molecules and selective permeability to ions and other nutrients/substances. An index of this endothelial “tightness” is the TEER (Stanness et al., 1997). Note that the development of high TEER (>1,100 ohm/cm2) was not statistically different (p < 0.05) between control and epileptic cocultures (Fig. 1B). Taken together, the results so far presented demonstrate that endothelial cells from epileptic brain are capable of forming a viable and functional BBB that, in terms of tightness and permeability to polar molecules, does not differ from the “normal” counterparts.
The assessment of glucose consumption/lactate production provides useful information on the metabolic pathway (anaerobic versus aerobic) of endothelial–glial cocultures grown under dynamic conditions. A previous study (Desai et al., 2002a) demonstrated that exposure to shear stress induces changes in the expression pattern of several metabolic enzymes including upregulation of NADH-producing enzymes [Krebs cycle dehydrogenases and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] accompanied by a simultaneous decrease in NADH-depleting pathways [e.g., lactate dehydrogenase (LDH)] and diminished production of lactate (Desai et al., 2002a). Thus the assessment of an aerobic metabolism is a hallmark of the establishment of a functional BBB. The results in Fig. 1C show that both normal and epileptic HBMECs differentiate in a BBB phenotype. In both cases, the ratio between glucose consumption and lactate production was ≈1, indicating increased propensity toward an aerobic metabolic pattern. We found no significant metabolic differences between epileptic and normal endothelium (p < 0.05).
CNS drugs in general and AEDs in particular are intentionally designed to be lipophilic. Lipophilicity improves passage across the BBB and subsequently increases drug distribution into the brain. We first tested the capacity of DZP, a highly lipophilic and commonly used AED, to permeate across a humanized in vitro BBB. Fig. 2A shows the permeability values of DZP in control BBB (4.75 × 10−3± 0.65 cm/s) or in BBB consisting of epileptic endothelium (2.93 × 10−3± 0.71 cm/s). These data indicate that the in vitro BBB established in our model has a permeability to DZP almost identical to that observed in vivo. In addition, although numerous AEDs have been shown to be substrates of MDR1 (Potschka et al., 2004; Schmidt and Loscher, 2005; Volk and Loscher, 2005; van Vliet et al., 2006), DZP moves across the BBB by passive diffusion (Yamazaki et al., 2001). Our permeability data are in agreement with this finding because (a) no significant difference was found in DZP permeability (p > 0.05) between control and epileptic DIV-BBBs (Fig. 2A); and (b) pretreatment with the MDR1 blocker XR9576 did not produce any significant effect on DZP permeability (see Fig. 2B).
Figure 2. Diazepam permeability in control and epileptic dynamic in vitro blood–brain barrier (DIV-BBB): a side-by-side comparison of humanized models. A: No significant difference was observed between permeability of diazepam (PDIAZ) in control and epileptic DIV-BBB. B: Pretreatment with the MDR1 inhibitor XR9576 did not affect the permeability to diazepam regardless the expression of MDR1. C: BBB opening after exposure to hyperosmotic mannitol facilitates the paracellular passage of diazepam across the microvascular brain endothelium. This effect was identical regardless of the epileptic or nonepileptic origin of the vascular endothelium.
Download figure to PowerPoint
BBB opening after exposure to hyperosmotic mannitol is commonly used to enhance chemotherapeutic drug penetration of the BBB to treat patients with metastatic or primary brain tumors (Rapoport, 2000; Brown et al., 2004). Hyperosmotic opening of the BBB is mediated by vasodilatation and shrinkage of cerebrovascular endothelial cells, with widening of the interendothelial tight junctions to an estimated radius of 200 Å, thus facilitating the paracellular passage of substances across the microvascular brain endothelium (Rapoport, 2000; Brown et al., 2004). Opening of the BBB in vitro by hypertonic mannitol solution demonstrated an almost complete loss of selective permeability to DZP. As expected, the effect was identical (p < 0.05) regardless of the epileptic (2.82 × 10−2± 1.2 cm/s) or normal (2.33 × 10−2± 0.80 cm/s) origin of endothelial cells (Fig. 2C).
These results suggested that (a) The DIV-BBB system can effectively reproduce the permeability characteristics on the BBB in vivo; (b) disruption of normal or epileptic BBB can be achieved with hyperosmotic mannitol; (c) the integrity and tightness of the BBB in epileptic brain was similar to that observed in normal brain; and (d) permeability to DZP was not affected by blockade of MDR1 by XR9576. However, the latter finding could be the result of insignificant MDR1 expression in the epileptic model. To rule out this hypothesis, we repeated the same experiments with PHT, which is at least in part an MDR1 substrate.
In contrast to the results previously shown with DZP, permeability values of PHT in control (1.74 × 10−5± 0.44 cm/s) or epileptic endothelium (1.54 × 10−6± 0.76 cm/s) differed (p < 0.05) by an order of magnitude (Fig. 3A). In addition, although pretreatment with XR9576 did not affect PHT permeability in control, epileptic cocultures demonstrated ≈3.5-fold increase in PHT permeability after exposure to the MDR1 blocker (Fig. 3B). Similar to that previously observed with DZP, the permeability to PHT was increased after the osmotic opening of the BBB with mannitol (Fig. 3C). No statistical differences between control and epileptic in vitro BBB were observed.
Figure 3. Phenytoin permeability in control and epileptic dynamic in vitro blood–brain barrier (DIV-BBB). A: Phenytoin is 10-fold less permeable in epileptic than in control BBB (p < 0.05). B: The 3.5-fold increase in phenytoin permeability (PPHEN) was observed in epileptic in vitro BBB pretreated with XR9576 (p < 0.05). C: Similar to diazepam, the osmotic BBB opening with mannitol increased phenytoin permeability regardless of the epileptic or nonepileptic origin of the endothelium.
Download figure to PowerPoint
To assess epileptic astrocytes' contribution to pharmacoresistance, we established parallel DIV-BBB models in which normal HBMECs were cocultured with astrocytes isolated from normal or epileptic brains. TEER was identical in both modules regardless of the origin of astrocytes (data not shown). Once the BBBs were formed, we performed permeability experiments to sucrose, PHT, and DZP (Fig. 4). Note that no differences in permeability were seen regardless of the astrocytes' origin (epileptic or normal).
Figure 4. Epileptic astrocytes do not contribute to endothelial pharmacoresistance. No significant differences were observed between permeability to sucrose (Psuc), phenytoin (PPHEN), and diazepam (PDIAZ) in dynamic in vitro blood–brain barrier (DIV-BBB) established by coculturing brain microvascular endothelial cells (HBMECs) with normal or epileptic astrocytes.
Download figure to PowerPoint
- Top of page
The most important outcome of this research is the development of an in vitro model of pharmacoresistance to AEDs based on coculture of human endothelium with glia. We propose to further the development of this model and extend this research to other AEDs. Our results support the use of this model for drug screening, but several issues remain to be addressed. These are listed in the following paragraphs.
Figure 5 summarizes the results of this study. The use of a humanized DIV-BBB models allowed permeability measurements with a close correlation with that measured in vivo (S = 0.83; Fig. 5A). These data demonstrate that the humanized DIV-BBB represents an excellent system to assess drug permeability with a degree of accuracy comparable to what one expects at the in situ human BBB. The contribution of pathologic expression of multiple drug resistance transporters is clearly maintained in our model. This is demonstrated by the significant difference in PHT permeability between control and epileptic cocultures. This difference was partially abolished by MDR1 blockade with XR9576 (Fig. 5B). Osmotic opening of the BBB by hyperosmolar mannitol as a mean to improve drug permeability was successfully achieved in the DIV-BBB (Fig. 5B), regardless the origin of the brain microvascular endothelium used (from normal or epileptic brain) or the lipophilicity of the compounds tested.
Figure 5. Correlation between permeability in vivo and in vitro. A: Permeability to sucrose, phenytoin, and diazepam in control dynamic in vitro blood–brain barrier modules (DIV-BBB) versus published in vivo data (Davson and Segal, 1996; Patsalos et al., 1996; Walker et al., 1996; Cucullo et al., 2005b). Dashed line, Idealized relation if the data in vivo were identical to that in vitro. Note how permeability obtained in the DIV-BBB accurately reflects the in vivo scenario. B: Summary of permeability of the compounds tested across the in vitro BBB comprising either normal or epileptic brain microvascular endothelial cells under different experimental conditions.
Download figure to PowerPoint
We intentionally used PHT as a prototype of an AED that undergoes active extrusion by multiple drug resistance molecules. Several studies have pointed out that PHT is indeed an MDR1 substrate (Potschka and Loscher, 2001; Rizzi et al., 2002), but other results suggest a minor role (Maines et al., 2005; Awasthi et al., 2005). Our results have shown a small but significant (p < 0.05) effect of the MDR1 blocker XR9576, suggesting that at least a small portion of drug resistance to PHT may be due to MDR1 overexpression. Because the P-gp inhibitor had no effect on the permeability of PHT in normal brain, which still expresses a constitutive low level of MDR1, it appears that only significant MDR1 increases above “normal” levels are required to promote measurable drug extrusion. However, given the broad range of genes encoding for multiple drug resistance proteins that are expressed by epileptic brain endothelial cells isolated from surgical specimens (Dombrowski et al., 2001), the contribution of other transporters may also play a role (e.g,, RLIP76) (Awasthi et al., 2005).
Regardless of the mechanism of drugs extrusion, one remarkable result was that in presumably non–multiple drug-resistant brain, PHT permeability was identical to that predicted by its oil/water partition coefficient. In contrast, tissue from drug-resistant epilepsy patients provided endothelial cells forming a BBB with significantly lower PHT permeability. Based on our tissue-screening experiments (Dombrowski et al., 2001), we expect that this behavior may be consistently seen in similar endothelial cells. Another interesting finding is that functional expression of drug-resistance mechanisms present in freshly isolated brain persists in cultured and passaged cells. The contribution of glial cells and intraluminal flow to this phenomenon is currently being investigated. Preliminary results (data not shown) suggest that flow per se does not cause induction or downregulation of MDR1 in brain endothelial cells. The role of glia remains unknown, but because normal endothelial cells have a very low expression of MDR1 in comparison to their epileptic counterpart, if any, the role of glia is related to maintenance of expression rather than induction. This also is supported by the fact that permeability to PHT in DIV-BBB established with normal HBMECs was not affected by the origin (from normal or epileptic brain) of glial cells (Fig. 4). This suggests that astrocytes have a minor contribution to the establishment of pharmacoresistance and that overexpression of MDR1 in astrocytes might instead play a role in cell survival (Marchi et al., 2004).
Several reports suggest that the epileptic BBB is altered both in terms of permeability and functional expression of glucose transporters (for a review, see Gronlund et al., 1996; Cornford et al., 2000). Our in vitro results suggest that the intrinsic capacity of endothelial cells isolated from epileptic brain is not different from their “normal” counterparts. Both cells promptly and reproducibly responded to flow and glia as expected, based on our previous work (Cucullo et al., 2005a). Thus it appears that factors other than intrinsically altered endothelial cell properties are involved in the described leakiness of the BBB. We did, however, test the capacity of our system to react to factors known to alter BBB permeability. We focused on the clinically relevant use of hyperosmotic agents to demonstrate that (a) BBB permeability can be transiently altered in the DIV-BBB; (b) these changes follow a time course comparable to that observed in vivo (Marchi et al., 2003); and (c) drug permeability is massively increased after disruption of the endothelial tight junctions.
The fact that a leaky BBB, as suspected to be in epilepsy patients, allows greater passage of drugs appears fairly obvious and yet is in sharp contrast with the clinical reality. Increasing evidence suggests that AED penetration in human epileptic brain is decreased (Oby et al., 2006) despite an apparent increased permeability. Thus other factors, including perhaps perivascular edema or different pKa values in epileptic brain, may be involved.
As soon as new AEDs have been introduced into the market, hope that drug resistance will be overcome is rapidly overtaken by lack of significant improvement in multiple drug resistant epilepsy patients. The antiepileptic activity of these drugs was, for the most part, defined by acute seizure models such as subcutaneous pentylenetetrazol seizure tests and rat kindling. The clinical evidence to date would suggest that none of these models, albeit useful, is likely to identify those therapeutics that will effectively manage the refractory seizures. Data from this and other laboratories (reviewed in Loscher, 2002b; Oby and Janigro, 2006) suggested that the BBB plays a crucial role in drug resistance. We envisage that the transendothelial permeation properties of novel compounds obtained by using a “drug resistant” humanized BBB model are more likely representative and predictive of the brain uptake of such compounds by the “epileptic” brain.