Development and application of a quick and sensitive method to measure intracellular NRTI ( Nucleoside analogue Reverse Transcriptase Inhibitors)-triphosphates
05 / 2004 - 04 / 2008
aanlevering Aids Fonds
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) are the backbone of antiretroviral therapy. NRTIs are intracellularly phosphorylated to their active triphosphate form (NRTI-TP) by host cell phosphorylation enzymes. Intracellularly, a NRTI-TP competes with the corresponding deoxynucleotide-triphosphate (dNTP) for incorporation into the growing viral DNA chain by HIV reverse transcriptase. Incorporation of a NRTI-TP results in premature chain termination, thus suppressing HIV activity. The toxic effects exerted by NRTIs include anaemia, myopathy, neuropathy, (fatal) lactic acidosis, and lypodystrophy. Evidence suggests that mitochondrial toxicity is the common pathway involved in these seemingly different toxicities1. NRTI-TPs compete with dNTPs for incorporation in to mitochondrial DNA (mtDNA). Incorporation of a NRTI-TP leads to premature mtDNA chain termination, loss of mtDNA and thus mitochondrial dysfunction. In vitro studies show that the ratio of the intracellular NRTI-TP concentration and intracellular dNTP concentration (NRTI-TP/dNTP ratio) is the crucial factor in determining the antiviral activity of NRTIs2. In HIV-1 infected adults, the intracellular NRTI-TP concentration correlates with the decline in HIV RNA levels and the increase in CD4 cell counts. In contrast, the plasma NRTI concentrations did not3. The absence of a sensitive high throughput method to measure the intracellular NRTI-TP concentrations (and simultaneously intracellular dNTP concentrations) is a serious setback to study intracellular NRTI-TPs. Even the best current established method is labour intensive, can only analyse up to 150 samples per week, and more importantly needs at least 7 ml of blood4. Studies in HIV-1 infected children are currently not possible due to these limitations. We have developed a new method, based on MALDI mass spectrometry, to measure intracellular NRTI-TPs and dNTPs. Our data show that this method exceeds current methods in sensitivity (= 1 ml blood needed), high throughput capacity (~ 1000 samples per day) and labour intensiveness (method can be fully automated)5. We will use this method for assessing the relationships between intracellular NRTI-TP/dNTP ratios, clinical and virological outcome, and selected laboratory parameters in HIV-1 infected children and adults. We have a large, well-defined cohort of 50 HIV-1 infected children. Since 1997, we are collecting clinical and laboratory parameters from these children. In addition, PBMC samples have been obtained and are stored at -135 *C for future research purposes. Currently, over a 1000 PBMC samples are available of this children cohort. The department of internal medicine (Erasmus MC) has a large, well-defined cohort of 1000 HIV-1 infected adults. Current studies show that the NRTI phosphorylation capacity differs between HIV-1 infected patients and that phosphorylation enzyme activity is an important factor that influences NRTI phosphorylation capacity6, 7. We will assess the correlation between phosphorylation enzyme activity and NRTI-TP/dNTP ratios in cultured PBMCs. This could be of importance to understand why some patients fail on NRTI therapy and develop viral resistance. We expect that the activity of the phosphorylation enzymes and the NRTI-TP/dNTP ratios differ between cytoplasm and mitochondria, since 1) Phosphorylation enzymes differ between mitochondria and cytoplasm. 2) NRTI-TPs cannot passively diffuse from cytoplasm to mitochondria. 3) NRTI-TPs can be actively transported in mitochondria. Elucidating these differences between cytoplasm and mitochondria are important for understanding why some patients suffer from NRTI toxicity and others do not. We will measure the NRTI-TP/dNTP ratios in mitochondria of cultured cells and correlate these values to the activity of the phosphorylation enzymes in the mitochondria. For this purpose, cell types will be used that are related to the observed NRTI induced mitochondrial toxicity, for example adipocytes (lypodystrophy) and myocytes (myopathy).
NRTIs zijn de ruggengraat voor HIV therapie. Deze middelen worden in de cel omgezet in de werkzame stof, de NRTI-trifosfaten. Er is nog steeds weinig bekend over de relatie tussen de dosering van NRTIs enerzijds, behandelsucces, ontwikkeling van virale resistentie, bijwerkingen en de intracellulaire concentratie van NRTI-trifosfaten anderzijds. Dit komt mede door het gebrek aan een snelle en gevoelige methode om deze concentraties te bepalen. Wij hebben een dergelijke methode ontwikkeld en gaan deze methode toepassen om meer inzicht te krijgen in de bovengenoemde relaties. Dit is van groot belang voor het optimaliseren van de behandeling van HIV geïnfecteerde patiënten.