The impact of highly active anti-retroviral therapy (HAART) on AIDS related dementia is less than the effect of drug therapy on AIDS-related illness not involving the brain, and this is attributed to the poor HAART drug penetration into the central nervous system (CNS). Whereas HAART drugs may enter cerebrospinal fluid (CSF) owing to transport across the choroid plexus or blood-CSF barrier, the actual penetration of the drugs into brain parenchyma is restricted owing to limited transport across the brain capillary endothelial wall, which forms the blood-brain barrier (BBB) in vivo. Many of the HAART drugs should cross the BBB owing to lipid-mediated transport of small molecules. However, these drugs are actively effluxed from brain back to blood by either p-glycoprotein (in the case of the protease inhibitors) or non-p-glycoprotein BBB active efflux transporters (AET) [in the case of the nucleoside reverse transcriptase inhibitors (NRTIs)]. While drug companies are working on the development of """"""""co-drugs"""""""", which are p-glycoprotein inhibitors, no work is being done on the non-p-glycoprotein AETs at the BBB, because the molecular identity of these transporters is not known. The purpose of the present grant is to continue work on the molecular cloning of BBB AETs that transport the NRTIs using the frog oocyte expression cloning system and cloned RNA obtained by in vitro transcription of a rat or rabbit cDNA library in the pSPORT vector. With this methodology, we cloned the rat BBB adenosine transporter, CNT2, and showed that dideoxyinosine (DDI) is transported by BBBCNT2. As an alternative to the amphibian expression system, we will also use a mammalian COS cell expression cloning system, which has been reduced to practice in the past funding period. BBB cDNAlibraries will be prepared from both rat and rabbit brain capillary-derived polyA+RNA, because there are species differences in BBB active efflux transport of drugs. Once the full-length cDNAs are cloned and sequenced, the amino acid sequence of the AET system is deduced. From this sequence, we will prepare anti-peptide antisera, which will be used in confocal microscopy, and immuno-gold electron microscopy. These immunochemical studies will (a) demonstrate the specific expression in brain of the AET at the BBB, and (b) localize the site of transporter expression to either the luminal or abluminal brain endothelial membrane. This work provides the basis for future drug discovery of AET blockers, which can be used as co-drugs to increase CNS penetration of HAART drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH061138-06
Application #
6883955
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Joseph, Jeymohan
Project Start
1999-12-01
Project End
2008-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
6
Fiscal Year
2005
Total Cost
$270,229
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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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
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
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, 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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