Tetrahydrouridine Co-Administration Improves Oral Bioavailability and Dampens Inter-Individual Variability of Decitabine Pharmacokinetics In Baboons

The cytosine analogue decitabine binds and depletes the chromatin modifying enzyme DNA methyl-transferase 1 (DNMT1), thereby modifying cellular epigenetics and differentiation. An oral formulation of decitabine would potentially increase the time-above-threshold concentration for depleting DNMT1 while avoiding high peak drug levels that cause DNA damage/cytotoxicity, facilitating outpatient epigenetic-differentiation therapy. The enzyme cytidine deaminase (CDA) is the major barrier to oral absorption and furthermore, causes significant inter-individual variability in decitabine pharmacokinetics. Since there is a narrow therapeutic window for depleting DNMT1 without causing cytotoxicity, decreasing inter-individual variability and improving the predictability of dose-pharmacokinetic relationships is important to the objective of non-cytotoxic epigenetic/differentiation therapy. Therefore, we examined if combining the CDA inhibitor tetrahydrouridine (THU) with decitabine could facilitate oral administration and furthermore, decrease inter-individual variability in decitabine pharmacokinetics. Drugs were administered under ketamine/xylazine anesthesia to normal female baboons by intravenous or subcutaneous injection or oral gavage. Blood samples for pharmacokinetic (PK) analysis were drawn at 7 time-points during anesthesia. Decitabine levels were measured using LC-MS/MS. The AUClast (the area under the curve from the time of dosing to the last measurable concentration) was calculated by the linear trapezoidal method. Oral bioavailability was estimated by the ratio of AUC_oral to AUC_IV, following adjustment of the AUC values for the different doses administered. Minimum doses of THU (400 mg/m², oral gavage) and decitabine (100 mg/m², oral gavage), and optimal timing between THU and decitabine administration (60 minutes), that achieved peak levels of decitabine associated with depletion of DNMT1 without cytotoxicity in normal hematopoietic cells (0.2 – 0.5 μM) were determined. Even with AUClast being calculated over a longer period of time for the decitabine alone group (240 versus 180 minutes), the increase in AUClast in the THU group was significant per mg/kg of decitabine administered (p=0.02, Wilcoxon test) (Table 1 ). Oral decitabine administered 60 minutes after THU produced area under the curve (AUClast) similar to that produced by decitabine alone at double the dose, with a decrease in the inter-individual variability in pharmacokinetics observed with decitabine alone (Table 1 ).

This is the first report of oral THU in combination with oral decitabine. The significant increase in decitabine bioavailability produced by preceding THU administration may actually be larger than reported here, since AUClast estimates for THU-decitabine were calculated over 180 minutes while AUClast estimates for decitabine alone were calculated over 240 minutes to accommodate the period of allowable anaesthesia. Furthermore, the concentration-time profiles suggested that decitabine levels may have continued to increase beyond the last sampling time in some animals, moreso in the THU-decitabine group. Preceding administration of THU could facilitate oral administration of decitabine and concurrently address other limitations of decitabine therapy, including the problem of inter-individual variability, to advance towards the objective of accessible non-cytotoxic epigenetic-differentiation therapy.

Off Label Use: FDA approved decitabine for use in MDS, therefore, its use in thalassemia is off-label. Furthermore, the dose, schedule and route of administration used is also not on the label. Decitabine was used in a study to augment production of fetal hemoglobin in an NIH sponsored trial.