Loughborough University
Thesis-2012-Zayed.pdf (3.76 MB)

Development of ICP-MS assays for the study and prediction of the efficacy and side effects of Pt-based drugs in cancer chemotherapy

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posted on 2012-03-07, 12:49 authored by Aref Zayed
Pt-based drugs are important cytotoxic agents that are used in the chemotherapeutic regimes of ~50% of all cancer patients. However, the efficacy of these drugs is often limited by drug toxicity and tumour resistance. Determination of the cellular pharmacokinetics and pharmacodynamics of Pt-drugs is important for understanding their molecular mechanisms of action and toxicity, and may be used, therefore, to predict the outcome of the treatment. ICP-MS is the most sensitive technique for the determination of Pt in biological samples and can offer robust, fast and accurate quantitations for studying pharmacokinetics and pharmacodynamics of Pt-drugs in patients. This thesis describes the development of a set of ICP-MS based assays for the determination of Pt-DNA adducts and Pt sub-cellular distribution in leukocytes of cancer patients and human cancer cell lines following treatment with Pt-based chemotherapy. It is ultimately aimed to use these assays in the clinic to predict the effectiveness and toxicity of Pt-based chemotherapy in individual patients, and offer those who would respond to the treatment personalised drug doses. Alternatively, patients who would not benefit from these drugs would be offered other forms of treatment. Pt DNA adduct formation was determined in leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability and excellent reproducibility of the assay. The sensitivity of the technique enabled quantitation of as little as 0.2 Pt adducts per 106 nucleotides using 10 µg of patient DNA. It was shown that Pt/P ratio was robust against DNA matrix effects, and was considered more reliable approach, with Eu as internal standard, for estimating Pt adducts per nucleotide compared to using Pt data in combination with DNA concentration measured by UV. Comparison of in vivo Pt-DNA adduct formation with the patients clinical notes suggested possible correlation between the adduct formation in leukocytes and toxicity. Speciation methods employing HPLC with complementary ICP-MS and ESI-Ion Trap-MS detection were developed and used for characterisation of oxaliplatin bi-functional adducts with mono-nucleotides and di-nucleotides. Further, a fast and sensitive LC-ICP-MS assay was developed and used for the quantification of oxaliplatin GG intra-strand adducts in human cancer cell lines. The assay, which has a detection limit of 0.22 Pt adduct per 106 nucleotides based on a 10 μg DNA sample, is suitable for in vivo assessment of the adducts in patients undergoing oxaliplatin chemotherapy. Combining the ICP-MS quantitation with a cell fractionation procedure allowed, for the first time, the detailed quantitation of entire sub-cellular Pt-drug partitioning in patient leukocytes in vivo, and in human cancer cell lines in vitro, following exposure to variety of Pt-drugs. The studies showed that Pt broadly follows the total protein content of the individual sub-cellular compartments with the majority being scavenged in the cytosol compartment.



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© Aref Zayed

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A Doctoral Thesis. Submitted in partial fulfillment of the requirements for the award of Doctor of Philosophy of Loughborough University.

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