Quantification of Gemcitabine Incorporation into Human DNA by LC/MS/MS as a Surrogate Measure for Target Engagement
ABSTRACT In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.
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- "In the current study, we observed heterogeneity in delivery between patients and within pancreatic tumors. Notably, the Advion measurement of gemcitabine DNA incorporation has high inter-assay precision and accuracy , making it unlikely that the variability in gemcitabine DNA incorporation was due to the measurement technique. "
ABSTRACT: There is substantial heterogeneity in the clinical behavior of pancreatic cancer and in its response to therapy. Some of this variation may be due to differences in delivery of cytotoxic therapies between patients and within individual tumors. Indeed, in 12 patients with resectable pancreatic cancer, we previously demonstrated wide inter-patient variability in the delivery of gemcitabine as well as in the mass transport properties of tumors as measured by computed tomography (CT) scans. However, the variability of drug delivery and transport properties within pancreatic tumors is currently unknown. Here, we analyzed regional measurements of gemcitabine DNA incorporation in the tumors of the same 12 patients to understand the degree of intra-tumoral heterogeneity of drug delivery. We also developed a volumetric segmentation approach to measure mass transport properties from the CT scans of these patients and tested inter-observer agreement with this new methodology. Our results demonstrate significant heterogeneity of gemcitabine delivery within individual pancreatic tumors and across the patient cohort, with gemcitabine DNA incorporation in the inner portion of the tumors ranging from 38 to 74% of the total. Similarly, the CT-derived mass transport properties of the tumors had a high degree of heterogeneity, ranging from minimal difference to almost 200% difference between inner and outer portions of the tumor. Our quantitative method to derive transport properties from CT scans demonstrated less than 5% difference in gemcitabine prediction at the average CT-derived transport value across observers. These data illustrate significant inter-patient and intra-tumoral heterogeneity in the delivery of gemcitabine, and highlight how this variability can be reproducibly accounted for using principles of mass transport. With further validation as a biophysical marker, transport properties of tumors may be useful in patient selection for therapy and prediction of therapeutic outcome.Physical Biology 12/2014; 11(6):065002. DOI:10.1088/1478-3975/11/6/065002 · 3.14 Impact Factor
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ABSTRACT: LY2334737 is an orally available prodrug of gemcitabine. The objective of this study was to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of daily administration of LY2334737 with or without erlotinib. Patients with advanced or metastatic cancer were treated with escalating doses of LY2334737 monotherapy or in combination with continuous daily administration of 100 mg erlotinib. LY2334737 was given once daily for 14 days of a 21-day cycle. The study was extended with a bioequivalence trial to investigate a novel LY2334737 drug formulation. A total of 65 patients were treated in this study. The MTD was 40 mg LY2334737. Fatigue was the most frequent DLT for LY2334737 monotherapy (4 patients) followed by elevated transaminase levels (2 patients), both observed at the 40- to 50-mg dose levels. Among the 10 patients in the combination arm, 2 had DLTs at the 40-mg dose level. These were fatigue and elevated liver enzyme levels. The most common adverse events were fatigue (n = 38), nausea (n = 27), vomiting (n = 24), diarrhea (n = 23), anorexia (n = 20), pyrexia (n = 18), and elevated transaminase levels (n = 14). The pharmacokinetics showed dose proportional increase in LY2334737 and gemcitabine exposure. The metabolite 2',2'-difluorodeoxyuridine accumulated with an accumulation index of 4.3 (coefficient of variation: 20%). In one patient, complete response in prostate-specific antigen was observed for 4 cycles, and stable disease was achieved in 22 patients overall. Pharmacokinetic analysis showed that the 2 investigated LY2334737 drug formulations were bioequivalent. LY2334737 displays linear pharmacokinetics and the MTD is 40 mg with or without daily administration of 100 mg erlotinib. Signs of antitumor activity warrant further development.Clinical Cancer Research 07/2011; 17(18):6071-82. DOI:10.1158/1078-0432.CCR-11-0353 · 8.19 Impact Factor
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ABSTRACT: In the last decades, the number of patients receiving chemotherapy has considerably increased. Given the toxicity of cytotoxic agents to humans (not only for patients but also for healthcare professionals), the development of reliable analytical methods to analyse these compounds became necessary. From the discovery of new substances to patient administration, all pharmaceutical fields are concerned with the analysis of cytotoxic drugs. In this review, the use of methods to analyse cytotoxic agents in various matrices, such as pharmaceutical formulations and biological and environmental samples, is discussed. Thus, an overview of reported analytical methods for the determination of the most commonly used anticancer drugs is given.Talanta 10/2011; 85(5):2265-89. DOI:10.1016/j.talanta.2011.08.034 · 3.51 Impact Factor