Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

Zhao, Ming; Rudek, Michelle A.; He, Ping; Hartke, Carol; Gore, Steve; Carducci, Michael A.; Baker, Sharyn D.

doi:10.1016/j.jchromb.2004.09.012

Cited by 37 publications

(37 citation statements)

References 8 publications

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“…Following observation of samples in which aza-CR could not be detected, presumably due to presumed breakdown by cytidine deaminase, tetrahydrouridine was added to the plasma supernatant at a final concentration of 100 Amol/L to increase the stability of drug in plasma during storage in the freezer. Tetrahydrouridine increased freezer stability from 7 to f21 days when frozen at À80jC (35,36). Aza-CR was quantified in plasma using a previously described validated liquid chromatography/tandem mass spectrometry method over the range of 5 to 500 ng/mL (20.4 to 2,041.6 nmol/L; ref.…”

Section: Correlative Studiesmentioning

confidence: 99%

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Combined DNA Methyltransferase and Histone Deacetylase Inhibition in the Treatment of Myeloid Neoplasms

et al. 2006

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Optimal reexpression of most genes silenced through promoter methylation requires the sequential application of DNA methyltransferase inhibitors followed by histone deacetylase inhibitors in tumor cell cultures. Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Bisulfite sequencing of the p15 promoter in marrow DNA during the first cycle of treatment showed heterogeneous allelic demethylation in three responding patients, suggesting ongoing demethylation within the tumor clone, but no demethylation in two nonresponders. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Gene demethylation correlated with the area under the aza-CR plasma concentration-time curve. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials. (Cancer Res 2006; 66(12): 6361-9)

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Section: Correlative Studiesmentioning

confidence: 99%

“…Aza-CR was quantified in plasma using a previously described validated liquid chromatography/tandem mass spectrometry method over the range of 5 to 500 ng/mL (20.4 to 2,041.6 nmol/L; ref. 36).…”

Section: Correlative Studiesmentioning

confidence: 99%

Combined DNA Methyltransferase and Histone Deacetylase Inhibition in the Treatment of Myeloid Neoplasms

et al. 2006

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“…Owing to the chemical decomposition that results in short plasma half-lives for azacytidine (1.6 h; Zhao et al, 2004) and decitabine (2.5 h; Liu et al, 2006), efforts have been focused on the development of chemically stable cytosine analogs for epigenetic therapy. The cytosine analog, zebularine [ Figure 5; 1-(b-Dribofuranosyl)-1,2-dihydropyrimidin-2-one], has been shown to mediate epigenetic reactivation of the p16 tumor suppressor gene efficiently in human cancer cell lines and bladder carcinoma xenografts .…”

Section: Cell Death Signalingmentioning

confidence: 99%

Nucleoside analogs: molecular mechanisms signaling cell death

2008

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Nucleoside analogs are structurally similar antimetabolites that have a broad range of action and are clinically active in both solid tumors and hematological malignancies. Many of these agents are incorporated into DNA by polymerases during normal DNA synthesis, an action that blocks further extension of the nascent strand and causes stalling of replication forks. The molecular mechanisms that sense stalled replication forks activate cell cycle checkpoints and DNA repair processes, which may contribute to drug resistance. When replication forks are not stabilized by these molecules or when subsequent DNA repair processes are overwhelmed, apoptosis is initiated either by these same DNA damage sensors or by alternative mechanisms. Recently, strategies aimed at targeting DNA damage checkpoints or DNA repair processes have demonstrated effectiveness in sensitizing cells to nucleoside analogs, thus offering a means to elude drug resistance. In addition to their DNA synthesisdirected actions many nucleoside analogs trigger apoptosis by unique mechanisms, such as causing epigenetic modifications or by direct activation of the apoptosome. A review of the cellular and molecular responses to clinically relevant agents provides an understanding of the mechanisms that cause apoptosis and may provide rationale for the development of novel therapeutic strategies.

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“…45 Recently, an Oasis MCX SPE column was used to extract 5-azacytidine from human plasma and showed acceptable recovery and accuracy for its quantification. 46 We have successfully used this extraction column for decitabine. Due to the high polarity of decitabine and the IS, a common reversed-phase column, such as Vydac C 18 peptidemass, could not retain these compounds efficiently, and their separation could not be achieved.…”

Section: Sample Extraction and Optimization Of Hplc Conditionsmentioning

confidence: 99%

Characterization of decomposition products and preclinical and low dose clinical pharmacokinetics of decitabine (5‐aza‐2′‐deoxycytidine) by a new liquid chromatography/tandem mass spectrometry quantification method

Liu

Marcucci

Byrd

et al. 2006

Rapid Comm Mass Spectrometry

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Aberrant DNA methylation patterns resulting in gene transcriptional repression are observed in numerous cancers. Decitabine, a DNA methyltransferase inhibitor, is being clinically evaluated in patients with hematologic malignancies and solid tumors. Decitabine is rather unstable and decomposes to 1-beta-D-2'-deoxyribofuranosyl-3-guanylurea under basic conditions and several additional unknown products under neutral conditions. This has greatly limited application of pharmacokinetic assays to clinical development of decitabine. In this paper, a high-performance liquid chromatography/ultraviolet multi-stage mass spectrometry (HPLC-UV-MSn) study of the decomposition of decitabine in water and human plasma revealed that these previously unknown products are isomers of the intermediates formyl-1-beta-D-2'-deoxyribofuranosyl-3-guanylurea and 1-beta-D-2'-deoxyribofuranosyl-3-guanylurea. A HPLC tandem mass spectrometry (MS/MS) method for the determination of decitabine concentrations in human and rat plasma has been developed. This method was based on a specific fragmentation pathway of the molecular ion of decitabine at m/z 229 to generate a unique fragment ion at m/z 113 under collision-induced dissociation. Separation of decitabine and the stable internal standard dihydro-5-aza-cytidine from the endogenous interfering substance in plasma extract was carried out on a C18 Aquasil column under an isocratic elution with a mobile phase consisting of 5% water/acetonitrile and 10 mM ammonium formate. The detection of decitabine was via selected reaction monitoring (SRM, 229 > 113), and its ionization was enhanced by post-column addition of acetonitrile. Effects of sample preparation and handling parameters on the stability of decitabine were also evaluated in human plasma at various temperatures. The accuracy and precision of this assay showed a coefficient of variation of <15% over the range of 0.5-25 ng for rat plasma and 0.1-25 ng for human plasma injected on-column. Pharmacokinetics of decitabine in rats following intravenous doses of 1.0 and 5.0 mg/kg were characterized. In the rat, plasma concentration-time profiles were found to follow a biexponential decline and the pharmacokinetics was dose-independent. Application of this decitabine pharmacokinetic assay to human studies is therefore justified and ongoing.

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Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

Cited by 37 publications

References 8 publications

Combined DNA Methyltransferase and Histone Deacetylase Inhibition in the Treatment of Myeloid Neoplasms

Combined DNA Methyltransferase and Histone Deacetylase Inhibition in the Treatment of Myeloid Neoplasms

Nucleoside analogs: molecular mechanisms signaling cell death

Characterization of decomposition products and preclinical and low dose clinical pharmacokinetics of decitabine (5‐aza‐2′‐deoxycytidine) by a new liquid chromatography/tandem mass spectrometry quantification method

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