Abstract
HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI) - triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 µL plasma and 1×106 PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.
Highlights
HIV protease inhibitors exert their action inside cells that are susceptible to HIV infection
The lower limits of quantification (LLOQ) obtained in plasma were 167 nM for lopinavir, 14.5 nM for ritonavir, 16 nM for nelfinavir, 16 nM for indinavir, and 3.2 nM for saquinavir, and the precisions and accuracies were within the criteria set by the FDA for bioanalysis [27]
The matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometry (MS) assay was used to determine a pharmacokinetic curve of lopinavir and ritonavir in an HIV-1 infected child receiving Kaletra and two nucleoside reverse transcriptase inhibitors (NRTI) twice daily per os
Summary
HIV protease inhibitors exert their action inside cells that are susceptible to HIV infection. Determination of the intracellular levels of antiretroviral drugs is an analytical challenge, because of the small amount of target cells, e.g. peripheral blood mononuclear cells (PBMCs), which can be obtained from a patient’s blood sample. One million PBMCs can be obtained from 1–2 mL blood. This amount of PBMCs has a total intracellular volume of ,0.4 mL, while 500 – 1000 mL plasma can be obtained from that same blood sample. Much is known about the plasma pharmacokinetics of HIV protease inhibitors in HIV infected children, but no information is available on the intracellular levels of HIV protease inhibitors in this group of patients [11,12,13,14]
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