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

2.50
Hdl Handle:
http://hdl.handle.net/10541/70421
Title:
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
Authors:
Danson, Sarah; Ranson, Malcolm R; Denneny, Olive; Cummings, Jeffrey; Ward, Timothy H
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.
Affiliation:
Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Paterson Laboratories, Wilmslow Road, Manchester, M20 4BX, UK.
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
Issue Date:
Nov-2007
URI:
http://hdl.handle.net/10541/70421
DOI:
10.1007/s00280-007-0432-9
PubMed ID:
17333193
Type:
Article
Language:
en
ISSN:
0344-5704
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorDanson, Sarah-
dc.contributor.authorRanson, Malcolm R-
dc.contributor.authorDenneny, Olive-
dc.contributor.authorCummings, Jeffrey-
dc.contributor.authorWard, Timothy H-
dc.date.accessioned2009-06-12T15:44:20Z-
dc.date.available2009-06-12T15:44:20Z-
dc.date.issued2007-11-
dc.identifier.citationValidation 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.en
dc.identifier.issn0344-5704-
dc.identifier.pmid17333193-
dc.identifier.doi10.1007/s00280-007-0432-9-
dc.identifier.urihttp://hdl.handle.net/10541/70421-
dc.description.abstractPURPOSE: 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.en
dc.language.isoenen
dc.subject.meshAntineoplastic Agents-
dc.subject.meshAziridines-
dc.subject.meshBenzoquinones-
dc.subject.meshCalibration-
dc.subject.meshCombined Modality Therapy-
dc.subject.meshComet Assay-
dc.subject.meshCross-Linking Reagents-
dc.subject.meshDNA Damage-
dc.subject.meshDose-Response Relationship, Drug-
dc.subject.meshDose-Response Relationship, Radiation-
dc.subject.meshFreezing-
dc.subject.meshHumans-
dc.subject.meshK562 Cells-
dc.subject.meshLeukocytes, Mononuclear-
dc.subject.meshQuality Control-
dc.subject.meshRadiotherapy-
dc.subject.meshReference Standards-
dc.subject.meshReproducibility of Results-
dc.subject.meshSpecimen Handling-
dc.subject.meshTime Factors-
dc.titleValidation 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 trialen
dc.typeArticleen
dc.contributor.departmentClinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Paterson Laboratories, Wilmslow Road, Manchester, M20 4BX, UK.en
dc.identifier.journalCancer Chemotherapy and Pharmacologyen

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