BackgroundSickle cell disease (SCD), a congenital hemolytic anemia that exacts terrible global morbidity and mortality, is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells (RBCs). Fetal hemoglobin (HbF) interferes with this polymerization, but HbF is epigenetically silenced from infancy onward by DNA methyltransferase 1 (DNMT1).Methods and findingsTo pharmacologically re-induce HbF by DNMT1 inhibition, this first-in-human clinical trial (NCT01685515) combined 2 small molecules—decitabine to deplete DNMT1 and tetrahydrouridine (THU) to inhibit cytidine deaminase (CDA), the enzyme that otherwise rapidly deaminates/inactivates decitabine, severely limiting its half-life, tissue distribution, and oral bioavailability. Oral decitabine doses, administered after oral THU 10 mg/kg, were escalated from a very low starting level (0.01, 0.02, 0.04, 0.08, or 0.16 mg/kg) to identify minimal doses active in depleting DNMT1 without cytotoxicity. Patients were SCD adults at risk of early death despite standard-of-care, randomized 3:2 to THU–decitabine versus placebo in 5 cohorts of 5 patients treated 2X/week for 8 weeks, with 4 weeks of follow-up. The primary endpoint was ≥ grade 3 non-hematologic toxicity. This endpoint was not triggered, and adverse events (AEs) were not significantly different in THU-decitabine—versus placebo-treated patients. At the decitabine 0.16 mg/kg dose, plasma concentrations peaked at approximately 50 nM (Cmax) and remained elevated for several hours. This dose decreased DNMT1 protein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by approximately 10%, and increased HbF by 4%–9% (P < 0.001), doubling fetal hemoglobin-enriched red blood cells (F-cells) up to approximately 80% of total RBCs. Total hemoglobin increased by 1.2–1.9 g/dL (P = 0.01) as reticulocytes simultaneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Also indicating better RBC quality, biomarkers of hemolysis, thrombophilia, and inflammation (LDH, bilirubin, D-dimer, C-reactive protein [CRP]) improved. As expected with non-cytotoxic DNMT1-depletion, platelets increased and neutrophils concurrently decreased, but not to an extent requiring treatment holds. As an early phase study, limitations include small patient numbers at each dose level and narrow capacity to evaluate clinical benefits.ConclusionAdministration of oral THU-decitabine to patients with SCD was safe in this study and, by targeting DNMT1, upregulated HbF in RBCs. Further studies should investigate clinical benefits and potential harms not identified to date.Trial registrationClinicalTrials.gov, NCT01685515

The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2M administered 2؋/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the ␥-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. IntroductionThe deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1), a key chromatin-modifying enzyme, and thereby modify cellular epigenetics, gene expression, and differentiation. 1 Potential clinical applications include increasing erythropoiesis and fetal hemoglobin (HbF) expression to treat hemoglobinopathies, 2 inducing terminal differentiation in malignant cells, [3][4][5][6][7] and increasing self-renewal of normal hematopoietic stem cells. [8][9][10] Certain aspects of DAC's pharmacology and mechanism of action influence its clinical activity; unlike cytidine analogs such as cytarabine (AraC) or gemcitabine, the sugar moiety of DAC is unmodified. Therefore, at low concentrations, DAC does not terminate DNA chain synthesis 11,12 and can deplete DNMT1 without causing significant DNA damage or cytotoxicity both in vitro and in vivo. 2,3,[11][12][13][14][15] However, at high concentrations, similar to other nucleoside analogs, DAC is cytotoxic. Another important aspect of DAC action is that it is S-phase specific, so exposure timing critically influences efficacy. 13,16 Considering these properties of DAC, for the objective of noncytotoxic DNMT1 depletion, the ideal DAC concentration-time profile is low peak drug levels but extended time above minimum concentrations required to deplete DNMT1. Oral administration of DAC could be more likely to produce this concentration-time profile than parenteral administration and would have major logistical advantages. 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...

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Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.