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

Abstract

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.

Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine

“…Although the murine studies enabled more comprehensive analyses of the effects of THU on DAC pharmacokinetics, dose-exposure extrapolation by body surface area scaling from mice to humans is not useful, because dose for dose, there is a more than 100-fold greater DAC exposure in rodents versus humans. 21 Similarly, there is more than 100-fold greater exposure of the cytidine analog AraC in mice versus monkeys dose for dose. 47 The reasons for this log-scale increase in cytidine analog exposure in rodents compared with primates are unknown.…”

“…21 The modification of these methods for determination of levels in baboon plasma is described in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).…”

“…A significant physiologic barrier to DAC oral bioavailability is the enzyme cytidine deaminase (CDA), which is highly expressed in the gut and liver of humans and mice and metabolizes cytidine, deoxycytidine, and analogs thereof into uridine counterparts that cannot deplete DNMT1. [17][18][19][20] CDA drastically decreases the half-life of DAC to Ͻ 20 minutes in vivo 21,22 from 5-16 hours at 37°C in vitro. 21,22 Furthermore, nonsynonymous single nucleotide polymorphisms in CDA produce person-to-person variability in CDA enzyme activity [23][24][25][26][27][28] and, consequently, clinically significant variation in cytidine analog pharmacokinetics, toxicity, and efficacy that could be amplified with oral administration.…”

“…[17][18][19][20] CDA drastically decreases the half-life of DAC to Ͻ 20 minutes in vivo 21,22 from 5-16 hours at 37°C in vitro. 21,22 Furthermore, nonsynonymous single nucleotide polymorphisms in CDA produce person-to-person variability in CDA enzyme activity [23][24][25][26][27][28] and, consequently, clinically significant variation in cytidine analog pharmacokinetics, toxicity, and efficacy that could be amplified with oral administration. 29 The uridine analog tetrahydrouridine (THU) is a competitive inhibitor of CDA that has been used as a CDA inhibitor in combination with cytidine analogs both preclinically and clinically for some decades without documentation of toxic side effects.…”

Nucleoside analogs: molecular mechanisms signaling cell death

“…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 .…”