RESUMEN
The nucleotide analog adefovir is an important therapy for hepatitis B viral infection. The study of nucleoside/tide pharmacology has been hampered by difficulties encountered when trying to develop LC/MS/MS methods for these polar analytes. In an attempt to identify a more convenient, selective and sensitive alternative to the analysis of the metabolism of radiolabeled parent nucleotide traditionally used for in vitro cell culture studies, an LC/MS/MS method was developed for the quantitative detection of adefovir and its phosphorylated metabolites in cellular samples. Ion-pairing reversed phase LC using tetrabutylammonium (TBA) and ammonium phosphate had the best compromise between chromatographic separation and positive mode MS/MS detection. Using microbore reverse phase columns and a low flow acetonitrile gradient it was possible to quantitate adefovir, its metabolites and 2'-deoxyadenosine triphosphate. A cross-validation showed comparable levels of adefovir and its metabolites were determined using either LC/MS/MS or radioactivity detection. However, initial methods were conducted at high pH and utilized an acetonitrile step gradient causing unacceptable column life and unpredictable equilibration. Further method optimization lowered the concentration of TBA and phosphate, decreased pH and applied a linear gradient of acetonitrile. This work resulted in a method that was found to have sensitivity, accuracy and precision sufficient to be a useful tool in the study of the intracellular pharmacology of adefovir in vitro and may be more broadly applicable.
Asunto(s)
Adenina/análogos & derivados , Cromatografía Liquida/métodos , Nucleótidos de Desoxiadenina/análisis , Organofosfonatos/análisis , Espectrometría de Masas en Tándem/métodos , Adenina/análisis , Adenina/metabolismo , Línea Celular Tumoral , Humanos , Inmunosupresores/análisis , Inmunosupresores/metabolismo , Inmunosupresores/farmacocinética , Organofosfonatos/metabolismo , Fosforilación , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: An anti-HIV regimen composed of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) disoproxil fumarate (TDF), abacavir (ABC) and lamivudine (3TC) has performed poorly in patients. This study evaluated the combination of TFV, ABC and 3TC for metabolic or antiviral antagonism in vitro. DESIGN: Procedures were developed to evaluate the in vitro metabolism and antiviral activity of drug combinations of TFV, ABC and 3TC in cell types relevant for HIV infection. METHODS: Anabolism of combinations of TFV and ABC were studied over a 24 h period in the human T leukaemic CEM lymphoblast cell line and human primary peripheral blood mononuclear cells (PBMCs) stimulated with human interleukin-2 and phytohaemagglutinin. The anti-HIV activity of combinations of TFV and ABC in the presence or absence of 3TC was studied in stimulated PBMCs infected with the HXB2 strain of HIV-1. RESULTS: Levels of the active metabolites produced from TFV and ABC after incubation with CEM or PBMCs showed no significant change upon introduction of the other NRTI. Moreover, the pool sizes for the natural substrates of 2'-deoxyadenosine triphosphate and 2'-deoxyguanosine triphosphate were also unchanged. In anti-HIV assays in PBMCs, the combination of TFV and ABC was found to be additive with respect to inhibition of HIV replication. Addition of 3TC to the combination did not result in synergistic or antagonistic effects. CONCLUSIONS: The poor efficacy of the triple NRTI regimen of TDF, ABC and 3TC is probably not due to a metabolic drug interaction resulting in antagonism of antiviral activity.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacología , Fármacos Anti-VIH/farmacología , Didesoxinucleósidos/farmacología , VIH-1/efectos de los fármacos , Lamivudine/farmacología , Organofosfonatos/farmacología , Adenina/metabolismo , Fármacos Anti-VIH/metabolismo , Línea Celular , Didesoxinucleósidos/metabolismo , Interacciones Farmacológicas , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Organofosfonatos/metabolismo , TenofovirRESUMEN
Previous studies have suggested that task-oriented biofeedback training may be effective for functional motor improvement. The purpose of this project was to design an interactive, multimodal biofeedback system for the task-oriented training of goal-directed reaching. The central controller, based on a user context model, identifies the state of task performance using multisensing data and provides augmented feedback, through interactive 3D graphics and music, to encourage the patients' self-regulation and performance of the task. The design allows stroke patients to train with functional tasks, and receive real-time performance evaluation through successful processing of multimodal sensory feedback. In addition, the environment and training task is customizable. Overall, the system delivers an engaging training experience. Preliminary results of a pilot study involving stroke patients demonstrate the potential of the system to improve patients' reaching performance.
RESUMEN
Adefovir dipivoxil (ADV) is esterolytically cleaved to the 2'-deoxyadenosine monophosphate (dAMP) analog adefovir, subsequent phosphorylation leads to the formation of the anti-Hepatitis B virus (HBV) agent adefovir-DP. To better understand the mechanism of action of ADV, metabolism studies were done in Hep G2, Huh-7 and primary human hepatocytes. Separation of radiolabeled adefovir metabolites after incubation in Hep G2 cells suggested that adefovir in its mono- and di-phosphorylated forms are the only metabolites formed from adefovir. Incubation of 10 microM adefovir with hepatic cell lines and fresh monolayers of primary human hepatocytes from two donors and analysis of intracellular metabolites by liquid chromatography coupled to tandem mass spectrometry resulted in adefovir-DP levels of approximately 10 pmol/million cells. Adefovir was more efficiently phosphorylated in primary hepatocytes than cell lines with adefovir-DP accounting for 44% versus 26% of total intracellular adefovir after 24 h. Egress studies showed adefovir-DP to have a half-life of 33 +/- 3 h, 10 +/- 1 h, 48 +/- 3 h and 33 +/- 2 h in Hep G2, Huh-7, and primary hepatocytes from two separate donors, respectively. The markedly shorter half-life in Huh-7 cells was inferred to be transport dependent based on its sensitivity to the transport inhibitor MK-571. Effective phosphorylation coupled with a long intracellular half-life and small competing dATP pool sizes in primary hepatocytes forms the cellular metabolic basis for the efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacocinética , Antivirales/farmacocinética , Hepatocitos/metabolismo , Organofosfonatos/farmacocinética , Animales , Transporte Biológico , Células Cultivadas , Nucleótidos de Desoxiadenina/metabolismo , Semivida , Virus de la Hepatitis B/efectos de los fármacos , Humanos , FosforilaciónRESUMEN
The level of systemic exposure to 2',3'-dideoxyinosine (ddI) is increased 40 to 300% when it is coadministered with allopurinol (Allo), ganciclovir (GCV), or tenofovir. However, the mechanism for these drug interactions remains undefined. A metabolic route for ddI clearance is its breakdown by purine nucleoside phosphorylase (PNP). Consistent with previous reports, enzymatic inhibition assays showed that acyclic nucleotide analogs can inhibit the phosphorolysis of inosine. It was further established that the mono- and diphosphate forms of tenofovir were inhibitors of PNP-dependent degradation of ddI (K(i)s, 38 nM and 1.3 microM, respectively). Allo and its metabolites were found to be relatively weak inhibitors of PNP (K(i)s, >100 microM). Coadministration of tenofovir, GCV, or Allo decreased the amounts of intracellular ddI breakdown products in CEM cells, while they increased the ddI concentrations (twofold increase with each drug at approximately 20 microM). While inhibition of the physiological function of PNP is unlikely due to the ubiquitous presence of high levels of enzymatic activity, phosphorylated metabolites of GCV and tenofovir may cause the increased level of exposure to ddI by direct inhibition of its phosphorolysis by PNP. The discrepancy between the cellular activity of Allo and the weak enzyme inhibition by Allo and its metabolites may be explained by an indirect mechanism of PNP inhibition. This mechanism may be facilitated by the unfavorable equilibrium of PNP and the buildup of one of its products (hypoxanthine) through the inhibition of xanthine oxidase by Allo. These findings support the inhibition of PNP-dependent ddI degradation as the molecular mechanism of these drug interactions.