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    Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial

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    Authors
    Danson, Sarah
    Ranson, Malcolm R
    Denneny, Olive
    Cummings, Jeffrey
    Ward, Timothy H
    Affiliation
    Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Paterson Laboratories, Wilmslow Road, Manchester, M20 4BX, UK.
    Issue Date
    2007-11
    
    Metadata
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    Abstract
    PURPOSE: RH1 is a novel anticancer agent with potent DNA-cross linking activity. RH1 has the potential to be activated within tumors over expressing NQO1, giving maximal antitumour activity with reduced toxicity in normal tissues. RH1 has recently completed a Cancer Research UK sponsored phase I clinical trial at two different centers in the United Kingdom. The comet-X assay was a secondary endpoint in this trial and assay validation was necessary. We describe here this validation process. Whilst it is impossible to cover all variations/conditions of a pharmacodynamic assay, we have strived to evaluate and demonstrate that this assay conforms to the three R's of validation, that is robustness, reliability and reproducibility. METHODS: K562 and peripheral blood mononuclear cells were treated with either radiation alone, or with a combination of radiation and drug. These samples were then embedded in low melting point agarose and subjected to a modified version of the alkaline single cell gel electrophoresis (Comet) assay, described here as the comet-X assay. Variations in the preparation, electrophoresis, storage and scoring of these samples was investigated. In addition radiation and drug dose response curves were constructed. Finally stability of QC standards was investigated over a 30-month period. RESULTS: We have demonstrated a linear radiation-dose response in cells up to 20 Gy and drug induced DNA cross-linking up to 50 nM. From the radiation dose response curves we were able to show that the relative inaccuracy measured against a global mean value was less than 25% and the relative (within day) imprecision was less than 30% over all doses. Between day runs produced an intra assay imprecision of 21.2%. Variables involved in the electrophoresis process showed the voltage across all slides in the tank ranged from 3.1 to -2.0 (mV) whilst the current ranged from 0.8-5.5 mA. QC standards were prepared from PBMCs of healthy donors and frozen at -80 degrees C. The stability of these frozen QC standards was measured over a 30-month period. No significant deterioration in any of the control, irradiated or drug treated samples was observed. CONCLUSIONS: The comet-X assay has been shown to be a robust, reliable and reproducible assay. It is ideally suited for the evaluation of the pharmacodynamic effects of DNA cross-linking agents undergoing early clinical trials. Furthermore, this assay may provide valuable data, in conjunction with pharmacokinetics, when measuring toxicity and efficacy as part of the RH1 phase I clinical trial.
    Citation
    Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial. 2007, 60 (6):851-61 Cancer Chemother. Pharmacol.
    Journal
    Cancer Chemotherapy and Pharmacology
    URI
    http://hdl.handle.net/10541/70421
    DOI
    10.1007/s00280-007-0432-9
    PubMed ID
    17333193
    Type
    Article
    Language
    en
    ISSN
    0344-5704
    ae974a485f413a2113503eed53cd6c53
    10.1007/s00280-007-0432-9
    Scopus Count
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