Tracking Decitabine Incorporation into Malignant Myeloid Cell DNA in vitro and in vivo by LC-MS/MS with Enzymatic Digestion

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2\u27-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C-18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy

Gender effects on cytidine analogue metabolism and myelodysplastic syndrome treatment outcomes

In vivo, half-lives of cytidine analogues such as 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), are determined largely by cytidine deaminase (CDA), an enzyme that rapidly metabolizes these drugs into inactive uridine counterparts. Genetic factors influence CDA activity, and hence, could impact 5-azacytidine/decitabine levels and efficacy, a possibility requiring evaluation. Using an HPLC assay, plasma CDA activity was confirmed to be decreased in individuals with the CDA SNP A79C. More interestingly, there was an even larger decrease in females. Explaining the decrease in enzyme activity, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In mathematical modeling, the detrimental effect of shortening half-life of S-phase specific therapy was amplified in low S-phase fraction disease (e.g., MDS). Accordingly, in multivariate analysis of MDS patients treated with 5-azacytidine/decitabine, overall survival was significantly worse in males

p53 independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the cytosine analogue decitabine that deplete the chromatin modifying enzyme DNA methyl-transferase 1 (DNMT1) without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine up regulated the key late differentiation factors CEBPε and p27/CDKN1B, induced cellular differentiation, and terminated AML cell-cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xeno-transplanted AML cells was abrogated but normal hematopoietic stem cell (HSC) engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S-phase specific decitabine therapy. In xeno-transplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared to conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy

Betacellulin Induces Increased Retinal Vascular Permeability in Mice

BACKGROUND: Diabetic maculopathy, the leading cause of vision loss in patients with type 2 diabetes, is characterized by hyper-permeability of retinal blood vessels with subsequent formation of macular edema and hard exudates. The degree of hyperglycemia and duration of diabetes have been suggested to be good predictors of retinal complications. Intervention studies have determined that while intensive treatment of diabetes reduced the development of proliferative diabetic retinopathy it was associated with a two to three-fold increased risk of severe hypoglycemia. Thus we hypothesized the need to identify downstream glycemic targets, which induce retinal vascular permeability that could be targeted therapeutically without the additional risks associated with intensive treatment of the hyperglycemia. Betacellulin is a 32 kD member of the epidermal growth factor family with mitogenic properties for the retinal pigment epithelial cells. This led us to hypothesize a role for betacellulin in the retinal vascular complications associated with diabetes. METHODS AND FINDINGS: In this study, using a mouse model of diabetes, we demonstrate that diabetic mice have accentuated retinal vascular permeability with a concomitant increased expression of a cleaved soluble form of betacellulin (s-Btc) in the retina. Intravitreal injection of soluble betacellulin induced retinal vascular permeability in normoglycemic and hyperglycemic mice. Western blot analysis of retinas from patients with diabetic retinopathy showed an increase in the active soluble form of betacellulin. In addition, an increase in the levels of A disintegrin and metalloproteinase (ADAM)-10 which plays a role in the cleavage of betacellulin was seen in the retinas of diabetic mice and humans. CONCLUSIONS: These results suggest that excessive amounts of betacellulin in the retina may contribute to the pathogenesis of diabetic macular edema

Tracking Decitabine Incorporation into Malignant Myeloid Cell DNA in vitro and in vivo by LC-MS/MS with Enzymatic Digestion

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2\u27-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C-18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy

Increased Neovascularization in Mice Lacking Tissue Inhibitor of Metalloproteinases-3

In light of recent reports suggesting that genetic variants near TIMP-3 influence susceptibility to age-related macular degeneration, the results from this study imply that TIMP-3 may regulate the development of the choroidal vasculature and is a likely contributor to increased susceptibility to choroidal neovascularization