Cell-type specific differences in antiretroviral penetration and the effects of HIV-1 Tat and morphine among primary human brain endothelial cells, astrocytes, pericytes, and microglia.

The inability to achieve adequate intracellular antiretroviral concentrations may contribute to HIV persistence within the brain and to neurocognitive deficits in opioid abusers. To investigate, intracellular antiretroviral concentrations were measured in primary human astrocytes, microglia, pericytes, and brain microvascular endothelial cells (BMECs), and in an immortalized brain endothelial cell line (hCMEC/D3). HIV-1 Tat and morphine effects on intracellular antiretroviral concentrations also were evaluated. After pretreatment for 24 h with vehicle, HIV-1 Tat, morphine, or combined Tat and morphine, cells were incubated for 1 h with equal concentrations of a mixture of tenofovir, emtricitabine, and dolutegravir at one of two concentrations (5 µM or 10 µM). Intracellular drug accumulation was measured using LC-MS/MS. Drug penetration differed depending on the drug, the extracellular concentration used for dosing, and cell type. Significant findings included: 1) Dolutegravir (at 5 µM or 10 µM) accumulated more in HBMECs than other cell types. 2) At 5 µM, intracellular emtricitabine levels were higher in microglia than other cell types; while at 10 µM, emtricitabine accumulation was greatest in HBMECs. 3) Tenofovir (5 or 10 µM extracellular dosing) displayed greater accumulation inside HBMECs than in other cell types. 4) After Tat and/or morphine pretreatment, the relative accumulation of antiretroviral drugs was greater in morphine-exposed HBMECs compared to other treatments. The opposite effect was observed in astrocytes in which morphine exposure decreased drug accumulation. In summary, the intracellular accumulation of antiretroviral drugs differed depending on the particular drug involved, the concentration of the applied antiretroviral drug, and the cell type targeted. Moreover, morphine, and to a lesser extent Tat, exposure also had differential effects on antiretroviral accumulation. These data highlight the complexity of optimizing brain-targeted HIV therapeutics, especially in the setting of chronic opioid use or misuse.

Simultaneous determination of intracellular concentrations of tenofovir, emtricitabine, and dolutegravir in human brain microvascular endothelial cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Combination antiretroviral therapy (cART) regimens are recommended for HIV patients to better achieve and maintain plasma viral suppression. Despite adequate plasma viral suppression, HIV persists inside the brain, which is, in part thought to result from poor brain penetration of antiretroviral drugs. In this study, a simple and ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell lysates of an immortalized human brain microvascular endothelial cell line (hCMEC/D3) was developed and validated. Analytes were separated on a reverse phase C18 column using water and 0.1% formic acid in acetonitrile as mobile phases. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.1-100 ng mL-1 for all analytes. Intra- and inter-assay precision and accuracy were within ±13.33% and ±10.53%, respectively. This approach described herein was used to determine the intracellular accumulation of tenofovir, emtricitabine, dolutegravir simultaneously in hCMEC/D3 cells samples.