didanosine and 2--3--dideoxyadenosine-5--phosphate

Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.

Topics: Anti-HIV Agents; Antibodies; Biotransformation; Chromatography, Ion Exchange; Deoxyadenine Nucleotides; Didanosine; Dideoxynucleotides; HIV Infections; Humans; Hydrolysis; In Vitro Techniques; Leukocyte Count; Models, Molecular; Neutrophils; Phosphorylation; Radioimmunoassay

2',3'-Dideoxyadenosine-5'-monophosphate (ddAMP) is a key intermediate in the metabolic pathway involved in the activation of the anti-retroviral agent 2',3'-dideoxyinosine (ddI) to 2',3'-dideoxyadenosine-5'-triphosphate (ddATP). The potential phosphorylation of ddAMP by adenylate kinase (myokinase) and pyrophosphorylation by the reverse reaction of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase were investigated. Using ATP as phosphate donor, ddAMP was phosphorylated by adenylate kinase with an efficiency of 8.8% of that for AMP, as estimated from the Vmax/Km ratios. In the presence of PRPP, Escherichia coli and rat PRPP synthetases catalysed the pyrophosphorylation of ddAMP with efficiencies of 52 and 35% of that determined for AMP, respectively. Two carbocyclic phosphonate analogues of ddAMP were not substrates of adenylate kinase. Yet, they were pyrophosphorylated by both PRPP synthetases, albeit less efficiently than ddAMP. In vivo, the usual function of PRPP synthetase is to synthesize PRPP from ribose-5-phosphate and ATP. In the forward reaction ddATP proved to be a substrate as efficient as ATP for rat PRPP synthetase. ddATP was also studied as a potential phosphate donor in the reaction catalysed by adenylate kinase with AMP as phosphate acceptor and found to be as efficient as ATP. The relevance of these in vitro results to the in vivo situation is discussed.

Topics: Adenosine Triphosphate; Adenylate Kinase; Animals; Biotransformation; Deoxyadenine Nucleotides; Didanosine; Dideoxynucleotides; Escherichia coli; HIV; Kinetics; Phosphorylation; Rats; Ribose-Phosphate Pyrophosphokinase; Substrate Specificity

Some 2',3'-dideoxynucleotides, of importance in the enzymology of the anti-HIV compounds, ddA and ddI, have been synthesized and purified by ion-exchange chromatography. 2',3'-Dideoxyadenylosuccinate, an intermediate in the pathway of ddI to ddATP, is converted to ddAMP by AMPS lyase at 1.85% of the efficiency of the natural substrate, adenylosuccinate. Interestingly, ddAMP and other 2',3'-dideoxygenated nucleotides are not substrates for AMP deaminase, another relevant enzyme in the conversion of ddA to ddATP via ddI.

Topics: Adenosine; Adenylosuccinate Lyase; AMP Deaminase; Deoxyadenine Nucleotides; Didanosine; Dideoxyadenosine; Dideoxynucleotides; HIV; Succinates