title: In vivo quantification of active decitabine-triphosphate metabolite: a novel pharmacoanalytical endpoint for optimization of hypomethylating therapy in acute myeloid leukemia.
abstract: Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 μM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Wang
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Jiang
authors.initials: J
authors.lastname: Wang
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Santhanam
authors.forename: Josephine
authors.initials: J
authors.lastname: Aimiuwu
authors.forename: U V Vijaya
authors.initials: UV
authors.lastname: Saradhi
authors.forename: Zhongfa
authors.initials: Z
authors.lastname: Liu
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schwind
authors.forename: Alice
authors.initials: A
authors.lastname: Mims
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
authors.forename: Michael R
authors.initials: MR
authors.lastname: Grever
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Villalona-Calero
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klisovic
authors.forename: Alison
authors.initials: A
authors.lastname: Walker
authors.forename: Ramiro
authors.initials: R
authors.lastname: Garzon
authors.forename: William
authors.initials: W
authors.lastname: Blum
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Chan
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000378:metabolism; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D000077209:Decitabine; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D011122:Polyphosphates / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D011122:Polyphosphates; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine; C005692:triphosphoric acid