Fatty Acid-Binding Protein 5 Mediates the Uptake of Fatty Acids, but not Drugs, Into Human Brain Endothelial Cells.

The purpose of this study was to examine the involvement of fatty acid-binding protein 5 (FABP5), a lipid-binding protein expressed at the blood-brain barrier (BBB), in fatty acid and drug uptake into human brain endothelial cells. Following transfection with siRNA against hFABP5, human brain endothelial cell (hCMEC/D3) uptake of lipophilic ligands with varying affinity to FABP5 was assessed with intracellular concentrations quantified by liquid scintillation counting, HPLC, or LCMS/MS. The in situ BBB transport of [3H]-diazepam was also assessed in wild type and FABP5-deficient mice. hFABP5 siRNA reduced FABP5 expression in hCMEC/D3 cells by 39.9 ± 3.8% (mRNA) and 38.8 ± 6.6% (protein; mean ± SEM), leading to a reduction in uptake of [14C]-lauric acid, [3H]-oleic acid, and [14C]-stearic acid by 37.5 ± 8.8%, 41.7 ± 11.6%, and 50.7 ± 13.6%, respectively, over 1 min. No significant changes in [14C]-diazepam, pioglitazone, and troglitazone uptake were detected following FABP5 knockdown in hCMEC/D3 cells. Similarly, no difference in BBB transport of [3H]-diazepam was observed between wild type and FABP5-deficient mice. Therefore, although FABP5 facilitates brain endothelial cell uptake of fatty acids, it has limited effects on brain endothelial cell uptake and BBB transport of drugs with lower affinity for FABP5.

Fatty Acid Binding Proteins Expressed at the Human Blood-Brain Barrier Bind Drugs in an Isoform-Specific Manner.

PURPOSE: To examine the expression of fatty acid binding proteins (FABPs) at the human blood-brain barrier (BBB) and to assess their ability to bind lipophilic drugs. METHODS: mRNA and protein expression of FABP subtypes in immortalized human brain endothelial (hCMEC/D3) cells were examined by RT-qPCR and Western blot, respectively. FABPs that were found in hCMEC/D3 cells (hFABPs) were recombinantly expressed and purified from Escherichia coli C41(DE3) cells. Drug binding to these hFABPs was assessed using a fluorescence assay, which measured the ability of a panel of lipophilic drugs to displace the fluorescent probe compound 1-anilinonaphthalene-8-sulfonic acid (ANS). RESULTS: hFABP3, 4 and 5 were expressed in hCMEC/D3 cells at the mRNA and protein level. The competitive ANS displacement assay demonstrated that, in general, glitazones preferentially bound to hFABP5 (Ki: 1.0-28 µM) and fibrates and fenamates preferentially bound to hFABP4 (Ki: 0.100-17 µM). In general, lipophilic drugs appeared to show weaker affinities for hFABP3 relative to hFABP4 and hFABP5. No clear correlation was observed between the molecular structure or physicochemical properties of the drugs and their ability to displace ANS from hFABP3, 4 and 5. CONCLUSIONS: hFABP3, 4 and 5 are expressed at the human BBB and bind differentially to a diverse range of lipophilic drugs. The unique expression and binding patterns of hFABPs at the BBB may therefore influence drug disposition into the brain.