Intracellular Concentrations of Non-Nucleoside Reverse Transcriptase Inhibitors and its Potential Role on Apoptosis in Peripheral Blood Mononuclear Cells

Abstract

Background: Efavirenz (EFV) and nevirapine (NVP) are non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are frequently used in combination with other antiretrovirals for the treatment of HIV-infected persons. Little information is available regarding the intracellular concentrations (ICs) of EFV and NVP in peripheral blood mononuclear cells (PBMCs) and its potential role for cellular toxicity. Methods:PBMCs from healthy adult donors were treated with or without the mean peak steady-state levels (Cmax) of EFV (12.4µM) and NVP (17.0µM) in human plasma during antiretroviral therapy multiplied by 0.5, 1.0, 2.0 and 4.0. After 48 hr treatment, ICs of EFV and NVP were measured using liquid chromatoagraphy-ion trap/mass spectrometry. The degree of apoptotic cells and mitochondrial membrane potential in PBMCs were measured by flow cytometry. Results:The mean log ICs of x1.0 Cmax NVP in PBMCs (2.00 ± 0.23 µM) were significantly lower than the one of x1.0 Cmax EFV (2.95 ± 0.22 µM) (P < 0.01). Similar significant differences of mean log ICs were observed when the concentration of NNRTIs were x0.5 Cmax (1.62 ± 0.26 μM vs. 2.87 ± 0.13 μM, P < 0.01) and x2.0 Cmax (1.99 ± 0.39 μM vs. 3.11 ± 0.21 μM, P < 0.01). Furthermore, apoptotic PBMCs were lower than PBMC treated with the concentrations of NVP above the plasma Cmax observed clinically in patients as compared to those treated with comparable concentrations of EFV (P < 0.01). Conclusion:These in vitro data suggest that ICs of NVP in PBMCs are significantly lower than ICs of EFV in PBMC and are also associated with less apoptotic PBMCs. The clinical relevance of this observation remains to be elucidated. [18,19,20]; however, no study compared the differences in ICs of EFV and NVP using an in vitro model. Materials and Methods Peripheral blood mononuclear cells (PBMCs) isolationPBMCs were obtained from healthy adult donors (n = 5, multiple ethnicities and race) using a Ficoll-Paque (GE Healthcare) density gradient according to the manufacturer's instructions. EFV and NVP treatment of PBMCsIsolated PBMCs were counted and incubated in 3 mL of serum free media (AIM-V, Invitrogen, Carlsbad, CA) at a concentration of 1.0 x 10 6 /mL in 6-well plates (Corning, Corning, NY). EFV was purchased from Morevak Biochemicals (Brea, CA) and NVP was provided by Boehringer Ingelheim (New York, NY). Both compounds were reconstituted in 50% ethanol. The cells were treated with or without the mean peak steady-state levels (Cmax) of EFV and NVP in human plasma during antiretroviral therapy multiplied by 0.5, 1.0, 2.0 and 4.0. The cells were also incubated with vehicle (50% ethanol) with the highest concentration of ethanol (0.78%) used to reconstitute EFV for each assay. The Cmax of EFV and NVP were 12.4μM and 17.0μM, respectively. The cells were treated for 48 hr at 37°C at 5% CO 2 . The assays contained cells treated with each treatment condition in duplicates and were repeated five times using different donor samples. Measurement of intracellular concentrations of EFV and NVP ...

10.4172/jaa.1000036

“…Highly active antiretroviral therapy (HAART) has significantly changed the morbidity and mortality associated with HIV-1 infection. A nonnucleoside reverse transcriptase inhibitor (NNRTI) combined with two or three nucleoside reverse transcriptase inhibitors have shown better results [27,28].…”