Intracellular disposition of AZT in lymphocytes: New tools for clinical pediatri

DESCRIPTION (provided by applicant): Azidothymidine (3'-azido-3'-deoxythymidine, AZT) is the most widely used drug to reduce mother-to child transmission of HIV in most countries. To be effective, AZT must undergo multiple phosphorylation steps in the lymphocytes to the prodrug triphosphate derivative. It is this derivative that is a substrate for viral reverse transcriptase, resulting in termination of the viral replication and thus mitigating the progression of viral growth. There are hazards associated with genotoxicity (later life cancer development) and mitochondrial function that place new methods for assessment of drug disposition in neonates and children a high priority. There is very little known about the drug disposition of nucleoside/nucleotide analogs in pediatrics populations; given the magnitude and frequency to which the youngest of children globally are exposure to these drugs, better clinical pharmacology tools are a high priority. We now have the tools to study drug disposition in pediatrics using Accelerator Mass Spectrometry (AMS). AMS achieves attomole (10-18) sensitivity for 14 C isotopic labels, the lowest levels of any known analytical instrument. Perinatal exposures and infant incorporation of 14 C-AZT may be quantified by AMS safely using permissible blood samples. For Phase 1 of this SBIR, we propose to develop and validate a method to quantify intracellular AZT triphosphate concentrations and final DNA incorporation within lymphocytes isolated from defensibly small blood volumes ( work will demonstrate capability for quantitation of pharmacodynamic parameters, intracellular concentrations and DNA incorporation, that may provide robust measures of target cell exposures from circulating doses. For Phase 2, we will apply this methodology in pediatric populations exposed to AZT to validate the platform and gain new insights into the pediatric response to AZT therapy. The ultimate aim of this work is to obtain a robust analytical platform to be applied as a service for clinical pharmacology to improve pediatric extrapolation from adult therapies. This work is significant as it could answer critical questions including appropriate oral dosing of genotoxic drugs such as nucleoside analogs, during pregnancy, labor and the immediate postpartum period, as well as investigating infant DNA incorporation of the active drug metabolites following in utero exposure. This proposal describes a new method made possible by a key technological innovation: ultra sensitive detection of 14 C-labeled compounds by Accelerator Mass Spectrometry. AMS enables the study of drug metabolism and toxicity in pediatric patients by allowing direct quantitation of intracellular uptake of a minute quantity of labeled drug metabolites from a very small blood sample. The isotopic label is radioactive, but the amount needed for AMS tracing is less than an infant's natural 14 C content. This work is significant as it could answer critical questions including appropriate oral dosing of genotoxic drugs such as nucleoside analogs, during pregnancy, labor and the immediate postpartum period, as well as investigating infant DNA incorporation of the active drug metabolites following in utero exposure. Phase I will demonstrate lack of matrix effects and the linearity of quantitation as well as quantify cellular drug content in archived blood samples. Phase 2 will demonstrate the relations between plasma and cellular concentrations in two large on-going clinical trials within the Pediatric AIDS Clinical Trials Group (PACTG) and HIV Perinatal Transition Network (HPTN).