Simultaneous Quantitation of Intracellular Zidovudine and Lamivudine Triphosphates in Human Immunodeficiency Virus-Infected Individuals

Abstract

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV).The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.Highly active antiretroviral therapy (HAART) has been used successfully for treatment of human immunodeficiency virus (HIV) since the discovery of protease inhibitors (PIs) (3,4,20). HAART treatment includes a broad category of antiretroviral drug combinations with the goals of decreasing plasma HIV-1 RNA levels below the limit of detection, limiting disease progression, and delaying the appearance of resistant mutants (12). The most common HAART regimen consists of the combination of one PI with two nucleoside reverse transcriptase inhibitors (NRTIs). This triple drug combination has shown dramatic improvements in viral suppression over the combination of the two nucleosides zidovudine and lamivudine (ZDV and 3TC, respectively) (8-10).Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate (TP) moiety to suppress HIV replication. ZDV and 3TC are not active against HIV; they need to be metabolized to 5Ј-ZDV-TP (ZDV-TP) and 5Ј-3TC-TP (3TC-TP) to act as competitive inhibitors of HIV reverse transcriptase or be incorporated into the viral genome (2,7,11,23). Studies conducted with HIV-infected populations have not established any relationship between ZDV or 3TC concentrations in plasma and the efficacy of these agents (19). On the other hand, a recent study showed a linear relationship between ZDV-TP intracellular concentrations and an increase in the percent change in CD4 ϩ cells from baseline in HIV-infected adults (5). Furthermore, several stud- Several approaches have been reported for the individual determinatio...

Lack of Evidence for In Vivo Transformation of Zidovudine Triphosphate to Stavudine Triphosphate in Human Immunodeficiency Virus-Infected Patients

“…All the samples were obtained and processed by the same group at the University of Puerto Rico General Clinical Research Center. The methodology for the processing of the samples has been described in the literature (5,22,23). Liquid chromatography-tandem mass spectrometry.…”

“…Alternative approaches to the direct measurement of nucleotide (d4TTP) concentrations by HPLC-MS/MS have been reported (1,19). Although both the direct and the indirect HPLC-MS/MS approaches differ mainly in the sample processing and the chromatographic conditions, their sensitivities and selectivities are comparable (1,3,22).…”

“…Due to the importance of these findings in the clinical setting, where cross-resistance or toxicity could be generated, we decided to corroborate their results. We monitored the d4TTP signal in patients treated with ZDV using our validated HPLC-MS/MS methodology with limits of quantitation (LOQ) of 2.7 fmol/10 6 cells (total injection, 54 fmol) for d4TTP (5,9,22). Furthermore, in vitro experiments in which ZDV (10 M, 50 M, and 100 M) was incubated in CEM SS cells at 24 and 48 h were performed to corroborate the results reported previously for the conversion of ZDVTP to d4TTP in HIV-infected patients treated with ZDV.…”

Desenvolvimento e validação de método analítico para determinação simultânea de lamivudina, zidovudina e nevirapina em comprimidos dose-fixa combinada por cromatografia líquida de alta eficiência

“…Keneey et al 5 publicaram um método cromatográfico para quantificação de AZT + 3TC em plasma humano, tendo como fase estacionária uma coluna C 18 e como eluente uma mistura de água e acetonitrila. Rodriguez et al, 6 também utilizaram uma coluna C 18 e como eluente, acetonitrila e metanol. Doerge et al, 7 por sua vez, variaram apenas a fase móvel para acetonitrila e ácido fosfórico, sendo considerado, portanto que estes dois últimos mé-todos apresentaram maior custo que o descrito por Kenney.…”

Zidovudine and Lamivudine for HIV Infection

“…61,[69][70][71] The half-life of ZDV-TP is about 7 hours and that for 3TC-TP is about 22 hours. 69 These half-lives for the pharmacologically active moieties are several-fold longer than the parent drug in plasma, and form the pharmacokinetic rationale for the dosing frequencies used in patients today (twice daily for ZDV and once-or twice-daily for 3TC).…”