Oral tetrahydrouridine and decitabine for non-cytotoxic epigenetic gene regulation in sickle cell disease: A randomized phase 1 study

Abstract

Background

Sickle 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 findings

To 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 (C _max) 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.

Conclusion

Administration 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 registration

ClinicalTrials.gov, NCT01685515

Abstract

In a clinical trial, Yogen Saunthararajah and colleagues target DNA methyltransferase with small molecules to reactivate fetal hemoglobin which inhibits polymerisation of mutated sickle cell hemoglobin.

Author summary

Why was this study done?

• Sickle cell disease, one of the most frequent inherited diseases in humans, is driven by aggregation and precipitation of less soluble sickle cell hemoglobin in red blood cells—this destroys red blood cells and blocks blood vessels, causing organ damage, suffering, and early death.

• Fetal hemoglobin, expressed in red blood cells until infancy, mixes with sickle cell hemoglobin and promotes its dissolution. Individuals who naturally express high levels of fetal hemoglobin beyond infancy thus receive some protection from sickle complications.

• To mimic this natural state using drugs, one relevant observation was that fetal hemoglobin is increased during recovery of bone marrow from extreme stress. This led to evaluation and approval of the cytotoxic (cell killing) drug hydroxyurea, the only drug approved to treat sickle cell disease. This approach to fetal hemoglobin induction, however, is limited in potency and sustainability.

• Our goal was to increase fetal hemoglobin by inhibiting an enzyme, DNA methyltransferase 1 (DNMT1), involved in shutting off the fetal hemoglobin gene from infancy onward.

What did the researchers do and find?

• We used the drug decitabine to inhibit DNMT1. However, decitabine is very rapidly inactivated in the body by another enzyme, cytidine deaminase (CDA). We therefore combined it with a CDA inhibitor, tetrahydrouridine (THU). Oral THU and decitabine were given to patients with severe, symptomatic sickle cell disease who had not derived benefit from the standard treatment of hydroxyurea.

• Oral THU-decitabine was safe and well-tolerated by the patients.

• Measurement of decitabine levels in the blood confirmed that THU enabled oral absorption of very small doses of oral decitabine. Measurements of DNMT1 protein levels and DNA methylation confirmed that this decitabine exposure was sufficient to deplete DNMT1 from cells.

• DNMT1 depletion by decitabine produced large increases in fetal hemoglobin and increased numbers of healthy red blood cells.

What do these findings mean?

• Oral THU-decitabine to deplete DNMT1 safely increased fetal hemoglobin and numbers of healthy red blood cells.

• Since this was a first-in-human study, only a small number of patients were treated and for a fairly short time. Further clinical studies are therefore needed.