Flavonoid permeability across an in situ model of the blood–brain barrier

https://doi.org/10.1016/j.freeradbiomed.2003.11.023Get rights and content

Abstract

Understanding mechanisms associated with flavonoid neuroprotection is complicated by the lack of information on their ability to enter the CNS. This study examined naringenin and quercetin permeability across the blood–brain barrier (BBB), using in vitro (ECV304/C6 coculture) and in situ (rat) models. We report measurable permeabilities (Papp) for both flavonoids across the in vitro BBB model, consistent with their lipophilicity. Both flavonoids showed measurable in situ BBB permeability. The rates of uptake (Kin) into the right cerebral hemisphere were 0.145 and 0.019 ml min−1 g−1 for naringenin and quercetin, respectively. Quercetin Kin was comparable to that of colchicine (0.006 ml min−1 g−1), a substrate for P-glycoprotein (P-gp). Preadministration of the P-gp inhibitor PSC833 or GF120918 (10 mg/kg body wt) significantly increased colchicine Kin, but only GF120918 (able to inhibit breast cancer resistance protein, BCRP) affected Kin for quercetin. Naringenin Kin was not affected. The influence of efflux transporters on flavonoid permeability at the BBB was further studied using MDCK-MDR1 and immortalized rat brain endothelial cells (RBE4). Colchicine, quercetin, and naringenin all showed measurable accumulation (distribution volume, Vd (μl/mg protein)) in both cell types. The Vd for colchicine increased significantly in both cell lines following coincubation with either PSC833 (25 μM) or GF120918 (25 μM). Both inhibitors also caused an increase in naringenin Vd; by contrast only GF120918 coincubation significantly increased quercetin Vd. In conclusion, the results demonstrate that flavonoids are able to traverse the BBB in vivo. However, the permeability of certain flavonoids in vivo is influenced by their lipophilicity and interactions with efflux transporters.

Introduction

The blood–brain barrier is formed by the endothelium of brain microvessels, under the inductive influence of associated cells especially astrocytes. Features that distinguish the brain endothelium from that of other organs include complex tight junctions, a low density of pinocytotic vesicles, and the expression of a number of specific uptake and efflux transport systems and metabolic enzymes. It is these properties that make the blood–brain barrier (BBB) a regulatory interface that selectively limits drug delivery to the CNS. In addition, the physicochemical characteristics of drugs such as solubility, molecular weight, lipophilicity, and pKa influence drug permeability (whether by paracellular or transcellular passive diffusion, carrier-mediated transport, or endocytosis/transcytosis), and hence their CNS bioavailability. However, the difficulty in identifying favorable physicochemical properties for CNS penetration significantly hinders discovery of CNS drugs to combat neurological disorders [1]. Thus, interim strategies to retard the development of these disorders may prove beneficial. Recent studies have demonstrated important neuroprotective actions of dietary flavonoids found in fruits, vegetables, and plant-derived drinks [2].

There is considerable evidence implicating oxidative stress as a component in the development of neurological disorders [3], but it is unclear whether flavonoids exert their neuroprotective actions solely as hydrogen-donating antioxidants or independently of such properties. There are several reports describing the potential mechanisms underlying the neuroprotective actions of flavonoids [2], [4], [5], [6], [7], [8], [9]. Evidence from these studies suggests that flavonoid neuroprotection may result from their interactions with different signaling cascades. Identifying the mechanisms associated with flavonoid neuroprotection is complicated by the lack of information concerning their ability to enter the CNS, since few studies have addressed this issue [10]. However, using in vitro models mimicking aspects of the BBB we have recently presented evidence that certain flavonoids and their physiologically relevant metabolites can cross the brain endothelium [11]. The present study extends this investigation using further in vitro and in situ models to elucidate the kinetics of flavonoid uptake and examines whether this is limited by efflux transporters at the BBB.

Two commonly consumed flavonoids were tested, the citrus flavanone naringenin and the flavonol quercetin (Fig. 1). As an in vitro permeability assay, we used the established model of ECV304 cells (expressing a robust endothelial phenotype) cocultured with C6 glioma cells to mimic the glial inductive influence present at the BBB in vivo. Under these conditions the ECV304 cells show up-regulation of brain endothelial features including tight junctional complexity, elevated transendothelial electrical resistance (TEER), and the glucose transporter GLUT-1; the cells show low but detectable expression of P-glycoprotein (P-gp) and transferrin receptor [12], [13]. To extend the study using a model preserving additional physiological features of the in vivo BBB, we used the rat in situ brain perfusion model [14]. The aim was to examine flavonoid distribution into different brain regions and assess whether the efflux transporter P-gp could limit their flux; neither property has been previously reported. P-glycoprotein-mediated efflux was further examined using two established cell lines expressing P-gp, the immortalized rat brain endothelial cell line RBE4 [15] and Madin–Darby Canine Kidney cells transfected with full-length cDNA for human multidrug resistance gene 1 (MDCK-MDR1).

Section snippets

Materials

Standard laboratory reagents of analytical or molecular grade were purchased from Sigma–Aldrich Chemical Co. (Dorset, UK). Cell culture reagents were purchased from Invitrogen (Paisley, UK) and Sigma–Aldrich Chemical Co. (Dorset, UK). ECV304 cells and C6 glioma cell lines used in this study were from the European Collection of Animal Cell Cultures (ECACC, Wiltshire, UK). The immortalized rat brain endothelial cells RBE4 were kindly provided by Dr P.O. Couraud and Dr F. Roux (Neurotech, Paris).

In vitro BBB permeability

We have previously reported the permeability of naringenin across the ECV304/C6 coculture BBB model [11] and, for comparison, the value is shown in Table 1.

In situ BBB permeability

The distribution volume (Vd μl/g) for [3H]naringenin into the right hemisphere was linear as a function of perfusion time over 60 s (r2>.9 in each case) (data not shown). [3H]naringenin was found to enter all seven brain regions studied (Fig. 2). Values for distribution volume ranged from 89 μl g−1 in the medulla to 288 μl g−1 in the

Discussion

The current study provides strong evidence that structurally different flavonoids are able to enter different regions of the brain, albeit to differing extents. This observation derived from in vitro and in situ models of the BBB has significant implications for future studies aimed at identifying the mechanism(s) underlying the neuroprotective effects of flavonoids. Although flavonoids have been shown to possess neuroprotective properties, few studies have specifically examined entry into the

Acknowledgements

Financial support from the Wellcome Trust (Grant 067018) is gratefully acknowledged. The authors also thank Dr. Gunter Kuhnle for his analysis and interpretation of the LC-MS/MS data and Dr. Michael Dobbie for his assistance with the in vitro BBB model.

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    K.A.Y. and M.Z.Q. contributed equally to the preparation of the manuscript.

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