We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.

The blood-brain barrier (BBB) is composed of microvessel endothelial cells sealed by tight junctions and surrounded by pericytes, neuron endings and astrocyte foot processes. These form a dynamic neurovascular unit which is the first line of defence for the brain against unwanted compounds. The entry of many compounds into the brain, including numerous commercial drugs, is also restricted by ATPbinding cassette (ABC) efflux transporters, including Pglycoprotein [P-gp, ABCB1/multidrug resistance (MDR1)], several multidrug resistance-associated proteins (MRPs) (ABCCs) and breast cancer resistance protein (BCRP) (ABCG2), at the plasma membrane of brain microvessel Address correspondence and reprint requests to Xavier Declèves, PhD, INSERM U705 CNRS UMR 7157, Faculté de Pharmacie, 4 avenue de l'observatoire, Paris 75006, France. E-mail: xavier.decleves@univ-paris5.frAbbreviations used: ABC, ATP-binding cassette; AhR, aryl hydrocarbon receptor; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; CAR, constitutive androstane receptor; C t , crossing-threshold; CYP, cytochromes P450; GFAP, glial fibrillary acidic protein; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; NG2, neuronglial antigen 2; PECAM-1, platelet endothelial cell adhesion molecule 1; Pgp, P-glycoprotein; PXR, pregnane xenobiotic receptor; qPCR, quantitative PCR; SYP, synaptophysin; TBP, TATA box-binding protein. AbstractWe have established the expression patterns of the genes encoding ATP-binding cassette (ABC) transporters and cytochromes P450 (CYPs) at the adult human blood-brain barrier (BBB) using isolated brain microvessels and cortex biopsies from patients with epilepsia or glioma. Microvessel purity was checked by measuring the expression of genes encoding BBB markers: platelet endothelial cell adhesion molecule 1 (endothelial cells), glial fibrillary acidic protein (astrocytes), synaptophysin (neurons) and neuron-glial antigen 2 (NG2) (pericytes). ABCG2 [breast cancer resistance protein (BCRP)] and ABCB1 (MDR1) were the main ABC transporter genes expressed in microvessels, with 20 times more ABCG2 and 25 times more ABCB1 in microvessels than in the cortex. The CYP1B1 isoform represented over 80% of all the CYPs genes detected in microvessels. There were 14 times more CYP1B1 in microvessels than in the cortex, showing that CYP1B1 is mainly expressed at the BBB. p-glycoprotein (ABCB1), BCRP (ABCG2) and CYP1B1 proteins were found in microvessels by western blotting. The expression of genes encoding three transcription factors [pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR)] was also investigated. The AhR gene, involved in the regulation of CYP1B1 expression, was highly expressed in brain microvessels, whereas PXR and CAR genes were almost undetected. This detailed pattern of ABC and CYPs gene expression at the human BBB provides useful information for understanding how their substrates enter the brain.