The goal of this research is to develop new brain drug delivery strategies for azidothymidine (AZT) and other nucleoside reverse transcriptase inhibitors (NRTI) so that the clinical potential of these drugs is enhanced for the treatment of the cerebral component of acquired immune deficiency syndrome (AIDS). Nearly all current drugs in clinical practice for the treatment of AIDS (NRTI, non-NRTI, and HIV protease inhibitors) are not transported from blood in brain in pharmacologically significant amounts owing to the active efflux of these drugs across the blood-brain barrier (BBB). The clinical potential of these drugs could be enhanced for the treatment of cerebral AIDS by the administration of co-drugs, wherein the co-drugs are inhibitors of the BBB active efflux systems. The development of co-drugs is straightforward for HIV protease inhibitors, because these drugs are substrates for a known efflux transporter, p-glycoprotein. However, the development of co-drugs for the NRTI's cannot be achieved presently because the putative active efflux systems for AZT and the other NRTI's has not been defined at the molecular level. The purpose of the proposed studies is to clone, sequence, and express a full length cDNA encoding the AZT active efflux transporter protein localized at the BBB. The availability of the cDNA will allow for the expression of the AZT active efflux transporter in a defined format and this will allow for the subsequent identification of drugs that are inhibitors of the BBB AZT efflux transporter. The following specific aims are proposed: (1) expression cloning of the AZT efflux cDNA using the frog oocyte expression cloning system and RNA derived from a bovine brain capillary cDNA library; (2) DNA sequencing of the cloned cDNA and DNA sequence analysis; (3) expression of the cDNA in COS cells followed by measurements of the Michaelis-Menten Kinetics of radiolabeled AZT transport into these cells; (4) functional analysis of the BBB AZT efflux transporter mRNA and protein using Northern blotting, immunocytochemistry, and electron microscopy of brain and isolated brain capillaries.
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| Pardridge, William M (2005) Molecular biology of the blood-brain barrier. Mol Biotechnol 30:57-70 |
| Schlachetzki, Felix; Pardridge, William M (2003) P-glycoprotein and caveolin-1alpha in endothelium and astrocytes of primate brain. Neuroreport 14:2041-6 |
| Boado, Ruben J (2003) Blood-brain barrier genomics. Methods Mol Med 89:401-18 |
| Pardridge, William M (2003) Molecular biology of the blood-brain barrier. Methods Mol Med 89:385-99 |
| Pardridge, William M (2003) Blood-brain barrier genomics and the use of endogenous transporters to cause drug penetration into the brain. Curr Opin Drug Discov Devel 6:683-91 |
| Shusta, Eric V; Zhu, Chunni; Boado, Ruben J et al. (2002) Subtractive expression cloning reveals high expression of CD46 at the blood-brain barrier. J Neuropathol Exp Neurol 61:597-604 |
| Li, Jian Yi; Boado, Ruben J; Pardridge, William M (2002) Rat blood-brain barrier genomics. II. J Cereb Blood Flow Metab 22:1319-26 |
| Shusta, Eric V; Boado, Ruben J; Pardridge, William M (2002) Vascular proteomics and subtractive antibody expression cloning. Mol Cell Proteomics 1:75-82 |
| Li, J Y; Boado, R J; Pardridge, W M (2001) Cloned blood-brain barrier adenosine transporter is identical to the rat concentrative Na+ nucleoside cotransporter CNT2. J Cereb Blood Flow Metab 21:929-36 |
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