HIV Antiretroviral Drug Metabolism and Transport Mechanisms at the Blood Brain Barrier

Abstract ID 93889 Poster Board 429 Neurologic deficits associated with human immunodeficiency virus (HIV) infection demonstrate the need to eliminate viral reservoirs in the brain with antiretroviral therapy (ART). First-line ART drugs are substrates of transporters at the blood brain barrier (BBB), including MRP4, BCRP, and P-gp as well as nucleoside kinase drug metabolizing enzymes, such as creatine kinase-brain type, adenylate kinase 2, pyruvate kinase, deoxycytidine kinase, phosphoglycerate kinase 1, and cytidine/uridine monophosphate kinase 1. Transporters and metabolizing enzymes at the BBB may influence ART availability in the brain, yet their activity and protein expression are largely uncharacterized in the cells comprising the BBB. To determine whether ART metabolizing enzymes and transporters at the BBB may impact ART distribution in the brain, we performed proteomics by mass spectrometry, western blots, and in-vitro activity assays using liquid chromatography with tandem mass spectrometry (LC-MS/MS) on primary human monocultures of BBB-resident cells, including brain endothelial cells, pericytes, and astrocytes. We identified several ART-relevant transporters and metabolizing enzymes that were differentially expressed among the BBB-resident cell types, indicating capabilities for cell-specific and region-dependent ART metabolism and transport. We next performed efflux transporter activity assays via fluorescence-activated cell sorting and identified diverse ART transport capacities of BBB cells, where pericytes had the highest P-gp activity but the least MRP4 activity. Interestingly, BCRP had comparable functional activity in all BBB cell types. We also evaluated BBB ART metabolism by performing an in-vitro tenofovir activation assay by LC-MS/MS and determined that all BBB cells had the metabolic capacity to convert tenofovir to its pharmacologically active anabolite, tenofovir-diphosphate. Interestingly, astrocytes demonstrated the highest capacity to convert tenofovir to tenofovir-diphosphate, suggesting cell-specific differences in metabolism at the blood brain barrier. Our findings indicate that the BBB contains cell-specific capabilities to metabolize and transport ART, suggesting that ART availability in the brain may be limited by the active and prevalent ART metabolizing enzymes and efflux transporters at the BBB, thus impacting the ability to effectively treat HIV.