Research reportA co-culture-based model of human blood–brain barrier: application to active transport of indinavir and in vivo–in vitro correlation
Introduction
Clinical and biological findings have indicated that, in many cases, HIV-1 infection leads to neurological impairment [15], [22], [27], [41]. The central nervous system (CNS) has been described as an immunological sanctuary site, with the potential to act as a viral reservoir [6]. Therefore, drugs for the clinical management of HIV-1 infection must reach the CNS to be effective. However, the passage of drugs from the blood into the CNS is restricted by the presence of the blood–brain barrier (BBB), which separates and isolates the microenvironment of the CNS. Generally three major parameters, drug lipophilicity, molecular size of drugs and protein complex formation, might be involved in the entry of drugs into the brain. In addition, multiple carrier systems on brain endothelial cells regulate the passage of selected drugs and macromolecules across the BBB. Efflux transport systems, e.g., P-glycoprotein (P-gp) and multidrug resistance protein [10], [11], [23], [50], which actively transport agents from the cerebral endothelial cells back into the bloodstream, have a major influence on BBB permeability to hydrophobic and amphiphilic drugs [23], [43], [53]. In order to test these parameters, several in vitro BBB models have been developed, based on: (i) isolated rat microvessels [5], (ii) the monoculture of primary endothelial cells [1], [2], [31], [42], (iii) the co-culture of primary endothelial cells with rat astrocytes [12], [14], [32], (iv) the monoculture of immortalised and transfected porcine brain endothelial cells [34], [49], (v) the co-culture of immortalised human vascular endothelial cells and rat C6 glioma cells [24], [39], and (vi) the co-culture of immortalised embryonic neurones with rat neocortical astrocytes [47].
These in vitro models which never use human primary astrocytes and human primary brain microvascular endothelial cells, make it difficult to draw firm conclusions concerning the drug transport across the human BBB. As a matter of fact, conflicting reports have been published concerning the entry of protease inhibitors into the CNS and the efficacy of these drugs against the virus in the CNS. Indinavir has been detected in the cerebrospinal fluid (CSF) at therapeutic concentration with, in some cases, the stabilisation or reversal of HIV-1-induced encephalopathy [44]. By contrast, Pialoux et al. [38] found no correlation between the use of protease inhibitors, in particular indinavir, and the regression of lesions in HIV-1-infected individuals. To our knowledge, all studies have investigated indinavir levels in the CSF of HIV-1-infected individuals [30], [44], [46]. Little is currently known about the ability of indinavir to cross the BBB, because the concentration of indinavir in the CSF does not necessarily reflect the extent to which this molecule is able to cross the BBB. A recent study showed that the entry of indinavir into the brain is limited by the presence of the drug transporter P-gp [25]. Kim et al. [25] used transgenic mice disrupted for the gene mdr1a as an in vivo P-gp model. However, no pharmacokinetic data were reported to demonstrate the direct involvement of P-gp in limiting the transport of indinavir into the CNS. The authors demonstrated the P-gp-mediated vectorial transport of HIV-1 protease inhibitors, including indinavir, in multidrug resistant (MDR)-L cells and Caco-2 cell lines. In vitro interactions have also been studied in other cell models [29], [45], [52], [54]. By contrast, Glynn and Yazdanian [21] tested a number of anti-HIV-1 drugs using human microvascular endothelial cells cultured alone and reported that only saquinavir showed affinity for the P-gp efflux pump, restricting its access to the CNS. Since indinavir transport by the human BBB is not yet demonstrated, what is needed is a combined in vivo and in vitro study on preparations that mimic as closely as possible the normal BBB to determine whether indinavir crosses the BBB and to determine the mechanisms involved. We report here the development of in vitro human BBB model based on the co-culture of human brain microvascular endothelial cells and primary human astrocytes. We employed this model to perform in vitro indinavir transport studies, correlated this transport with indinavir-mediated P-gp ATPase modulation, and the indinavir uptake into the brain in knockout mdr1a mice.
Section snippets
Chemicals and antibodies
[U-14C]Sucrose (350 mCi mmol−1), dl-[4-3H]propranolol (29 Ci mmol−1) [G-3H]vincristine sulfate (7.5 Ci mmol−1), [N-methyl-3H]verapamil hydrochloride (85 Ci mmol−1) and [G-3H]vinblastine sulfate (9 Ci mmol−1) were obtained from Amersham (Buckinghamshire, UK). [1-Methyl-14C]caffeine (51.20 mCi mmol−1) was purchased from NEN Life Sciences (Boston, MA, USA). [N-Methyl-3H]antipyrine (9.3 mCi mmol−1) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate acetylated low-density
Cell characterisation and validation of the in vitro co-cultured human BBB model
Immunocytochemical labeling showed that astrocytes expressed GFAP (Fig. 1A), and that all the BMVEC were labelled for DiI-acyl-LDL (Fig. 1B). In the co-culture model, BMVEC formed a confluent monolayer within 15 days. The trans-endothelial resistance of the human BBB model increased to 260±130 Ω cm2 and was significantly higher (P<0.05) than the electrical resistance of BMVEC culture alone (61±2 Ω cm2) and astrocytes (37±5 Ω cm2). The electrical resistance of the blank filter was 35±9 Ω cm2. We
Discussion
When designing novel strategies for drug delivery into the brain, we need to know whether therapeutic agents cross the BBB and if so, how they do this. To obtain such information, we require both an in vitro BBB models mimicking the in vivo system and animal models. Our study differs from earlier published works in three respects:
- (i)
The permeability study was conducted with an in vitro model of the BBB based on the co-culture of primary human astrocytes and primary brain microvascular endothelial
Acknowledgements
We would like to thank ‘Sidaction Ensemble contre le SIDA’ for funding, Alex Edelman and associates (Malakoff, France) for English correction.
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