Abstract. Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity-like the cationization strategy-as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics.

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.