This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. AMS will be applied to measuring the platinum-DNA adducts for drug pharmacokinetics. In spite of the importance of platinum-based anticancer drugs (cisplatin, carboplatin and oxaliplatin), their mechanisms of action, repair of damaged DNA and pharmacokinetics are unclear because of the detection limit of conventional methods (conventional methods have failed in quantifying Pt-DNA adducts with cells incubated with a pharmacological dose of the anticancer agent, which is the reason we need the sensitivity of AMS). In order to address these important issues, 14C-labeled carboplatin and oxaliplatin will be administered to E. coli, human cells, and bladder cancer patients, which may overcome the previous detection limits even at sub-pharmacological doses. The goals are to use AMS to elucidate their in vivo mechanism of action, to correlate Pt-DNA adduct level with cell death using a variety of human cancer cells, to determine the pharmacokinetics of the patients dosed with 14C-labeled carboplatin and oxaliplatin, and to ultimately correlate the phamacokinetic results to individual outcome (patient survival). Experimentally, after dosing a number of human cancer cells or cancer patients with radioactive platinum-based anticancer drugs, cell lysis and extracted platinated DNA will be measured by AMS. These 'real-time pharmacokinetics' will allow determination of which cancer patients will benefit from platinum treatment and which will be resistant to the drugs. Because of the high sensitivity, AMS is the very best technology for realizing these challenging goals. Progress Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and inter-strand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [14C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. The strength of this approach is that only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, thus allowing for the calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 hours. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic crosslinks. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analog, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [14C]carboplatin and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 attomole per 10 micrograms of DNA. This sensitivity may allow the method to be used for clinical applications. Future work will be focused on a small clinical study in which patients are dosed with 14C-labeled carboplatin, samples taken of the course of two weeks. The resulting pharmacokinetics profiles will be correlated to patient outcomes such as tumor size and mortality. Work to date has resulted in the following publication: Hah, S.S.; Stivers, K.M.; de Vere White, R.; Henderson, P.T. Kinetics of Carboplatin-DNA Binding in Genomic DNA and Bladder Cancer Cells As Determined by Accelerator Mass Spectrometry, Chemical Research in Toxicology, 2006, 19, 622-626. A second manuscript is in preparation.
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