Cedazuridine is a novel cytidine deaminase inhibitor that inhibits the in vivo metabolic degradation of decitabine when administered orally in combination with decitabine (known as ASTX727) in clinical trials. Cedazuridine inhibits degradation of decitabine by inhibiting cytidine deaminase in the gut and liver thereby increasing oral bioavailability of decitabine. To support clinical trial pharmacokinetic studies for ASTX727, a sensitive LC-MS/MS method for the simultaneous quantitation of decitabine and cedazuridine, as well as the epimer of cedazuridine, in human plasma was developed and validated. Decitabine is known for its instability in human plasma over time as well as a previously observed chromatographic interference in certain subjects that was inseparable using reverse-phase chromatography. To stabilize decitabine, tetrahydrouridine (THU) was mixed with the human plasma samples. Chromatographic interference was resolved using normal phase chromatography. In-house synthesized stable-label internal standards for all three analytes were employed to ensure assay robustness. Stability of cedazuridine and cedazuridine-epimer were carefully evaluated and extensive experiments were conducted to ensure no inter-conversion occurs. As a result, a 3-in-1 method (single sample extraction) for the quantitation of cedazuridine, cedazuridine epimer and decitabine has been developed and fully validated. Protein precipitation (PPT) was used to extract all analytes from THU-stabilized human plasma samples. The analytes were separated on two different HPLC columns (reverse phase for cedazuridine and cedazuridine epimer and normal phase for decitabine). The method has been applied for clinical studies to evaluate the pharmacokinetics of cedazuridine, cedazuridine-epimer and decitabine in human.
View further details below