Elsevier

Brain Research

Volume 927, Issue 2, 15 February 2002, Pages 153-167
Brain Research

Research report
A co-culture-based model of human blood–brain barrier: application to active transport of indinavir and in vivo–in vitro correlation

https://doi.org/10.1016/S0006-8993(01)03337-6Get rights and content

Abstract

The growing array of in vitro models of the blood–brain barrier (BBB) which have been used makes it difficult to draw firm conclusions concerning the BBB penetration of HIV-1 protease inhibitors. What is needed is a combined in vivo and in vitro study on biological models that mimic as closely as possible the normal human BBB, to establish whether and how indinavir crosses the BBB. We developed a new human BBB model using primary endothelial cells and astrocytes. The biological relevance of this model was checked with respect on the one hand, to the close relationship between the log of drug permeability coefficient normalized to molecular weight and the log of the 1-octanol/water partition coefficient, and on the other hand to the functional P-glycoprotein (P-gp) expression. We employed this model to perform transport studies with indinavir and showed that the rate of in vitro indinavir transport from the basal to apical compartment was higher than the rate of apical to basal transport. Pretreatment of the BBB model with the P-gp inhibitor, quinidine, significantly increased apical to basal transport. Intracellular indinavir accumulation was increased in BBB as a result of inhibition of active transport. These data were correlated with the indinavir-mediated P-gp ATPase modulation showing that indinavir specifically interacted with a binding site on P-gp. Moreover, the activation of P-gp ATPase by indinavir was inhibited by quinidine. In addition, the in vivo brain to plasma concentration ratio of indinavir into mice showed that indinavir concentration was up to five times higher in the brain of mdr1a(−/−) mice than in the brain of mdr1a(+/+) mice. All these results confirm the role of P-gp in preventing the passage of indinavir across BBB and thus its entry into the central nervous system (CNS). Our human BBB model represents a useful tool for the evaluation of drug penetration into the CNS.

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.

References (54)

  • L. Stähle et al.

    Indinavir in cerebrospinal fluid of HIV-1 infected patients

    Lancet

    (1997)
  • M. Teifel et al.

    Establishment of the permanent microvascular endothelial cell line PBMEC/C1-2 from porcine brain

    Exp. Cell Res.

    (1996)
  • Q. Wang et al.

    Effect of the P-glycoprotein inhibitor, cyclosporin A, on the distribution the brain: an in vivo microdialysis study in freely moving rats

    Biochem. Biophys. Res. Commun.

    (1995)
  • N.J. Abbot et al.

    Development and characterization of a rat brain capillary endothelial culture: towards an in vitro blood brain barrier

    J. Cell Sci.

    (1992)
  • K.L. Audus et al.

    Characterization of an in vitro blood–brain barrier model system for studying drug transport and metabolism

    Pharm. Res.

    (1986)
  • O. Benveniste et al.

    Comparative IL-2/IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SIVmac251

    Proc. Natl. Acad. Sci. USA

    (1996)
  • O. Benveniste et al.

    Techniques for quantification of cytokine mRNAs

    Cytokines Cell Mol. Ther.

    (1998)
  • P. Black

    HTLV-III, AIDS and the brain

    New Engl. J. Med.

    (1988)
  • A. Cheret et al.

    Cytokines mRNA expression in mononuclear cells from different tissues during acute SIVmac251 infection of macaques

    AIDS Res. Hum. Retroviruses

    (1996)
  • C. Cordon-Cardo et al.

    Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood brain barrier sites

    Proc. Natl. Acad. Sci. USA

    (1989)
  • C. Cordon-Cardo et al.

    Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

    J. Histochem. Cytochem.

    (1990)
  • M.P. Dehouck et al.

    An easier, reproducible and mass production method to study the blood–brain barrier in vitro

    J. Neurochem.

    (1990)
  • S.E. Devine et al.

    Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance

    Proc. Natl. Acad. Sci. USA

    (1992)
  • L. Fenart et al.

    Inhibition of P-glycoprotein: rapid assessment of its implication in blood brain barrier integrity and drug transport to the brain by an in vitro model of the blood brain barrier

    Pharm. Res.

    (1998)
  • D.H. Gabuzda et al.

    Immunohistochemical identification of HTLV-III antigen in brain of patients with AIDS

    Ann. Neurol.

    (1986)
  • P.J. Gaillard et al.

    Astrocytes increase the functional expression of P-glycoprotein in an in vitro model of the blood–brain barrier

    Pharm. Res.

    (2000)
  • M. Garrigos et al.

    Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase: further experimental evidence for a multisite model

    Eur. J. Biochem.

    (1997)
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