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 (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.

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.

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: DL, JD, and YS have patent applications around decitabine and around the combination of tetrahydrouridine and decitabine. In addition, JD and YS are consultants to EpiDestiny, that has licensed oral THU-decitabine for development as a treatment for sickle cell disease. EpiDestiny did not fund this clinical trial, and had no role in the study design, collection or analysis of the data, preparation of the manuscript, or the decision to publish. None of the authors have received any payments related to conduct of this clinical trial.

Figures

Fig 1. Fetal hemoglobin (HbF) blocks polymerization of deoxy sickle hemoglobin (HbS), the root cause of sickle cell disease (SCD) pathophysiology, and is the most powerful known disease modifier.

(A) Polymerization of deoxy HbS drives all SCD pathophysiology; In contrast to HbF, normal adult hemoglobin (HbA, ẞ-chains) can participate in polymerization. (B) The gene for HbF (HBG) is silenced by DNA methyltransferase 1 (DNMT1). Although DNA-binding factors, e.g., BCL11A, direct this silencing, the biochemical work of epigenetic repression is executed by chromatin-modifying enzymes, amongst which DNMT1 is central. Decitabine depletes DNMT1 and can do so without cytotoxicity because in contrast to other cytidine analogues (e.g., cytarabine) the deoxyribose moiety (green dotted circle) is natural, although higher concentrations do cause anti-metabolite effects and DNA damage, in part by degradation into uridine counterparts that misincorporate into DNA. (C) Several pharmacologic limitations of decitabine hinder safe, effective, practical clinical translation. The limitations have a common cause, the enzyme cytidine deaminase (CDA). Tetrahydrouridine (THU) inhibits CDA. No toxicities have been found for THU in animals or humans.

Fig 2. Design rationale, schema, flow, and patient characteristics.

(A) Decitabine regimens approved by the US Food and Drug Administration (FDA) to treat myeloid malignancy utilize cytotoxic decitabine doses (red zone), requiring pulse-cycled administration to recover from cytotoxic side effects. This clinical trial instead escalated oral decitabine doses from almost zero (0.01 mg/kg) gradually upward to find the minimum doses required to deplete DNA methyltransferase 1 (DNMT1) without cytotoxicity (green zone) when administered after oral tetrahydrouridine (THU) 10 mg/kg. Because such doses are non-cytotoxic, they can be administered frequently in distributed fashion to increase the fraction of target cells subject to S-phase–dependent DNMT1 depletion. (B) The study schema. (C) Flow of patients through the trial. (D) Patient clinical characteristics at baseline. Abbreviations: ACS, acute chest syndrome; AVN, acute vascular necrosis; CVA, cerebral vascular accident; ED/Hosp, emergency department/hospital; PE/DVT, pulmonary embolus/deep vein thrombosis. (E) Patient demographics and baseline laboratory variables. Median and range showed for continuous variables. P values Wilcoxon ranked sums 2-tailed or Fisher exact test.

Fig 3. Adverse events (AEs).

(A) AEs with possible relatedness to the study drug (AEs other than “unrelated”) per judgement of the treating clinical teams. (B) All grade 3 AEs occurring in study drug—and placebo-treated patients. These grade 3 AEs were sickle cell complications judged to be unrelated to the study drug by the treating clinical teams. (C) Statistical comparison of grade 3 AEs between placebo and decitabine dose-level patients. R function poisson.test(), an exact test, was used. Abbreviation: ED/Hosp, emergency department/hospital.

Fig 4. Adverse events (AEs) in placebo and decitabine dose-level patients (cohorts 1–5).

(A) All AEs as per Common Terminology Criteria for Adverse Events (CTCAE) v 4.0. There were no grade 4 AEs. (B) Statistical comparison of all pain AEs in drug-treated versus placebo cohorts. R function poisson.test(), an exact test, was used.

Fig 5. Pharmacokinetics (PK) and pharmacodynamics (PD) of oral tetrahydrouridine (THU)-decitabine.

(A) Decitabine PK. Samples for PK analysis were obtained in 12 of the 15 patients who received the study drug. Times are listed in hours after administration of oral decitabine. Data points are measured values and curves are fits using the R package PKLMfit. The inset shows a close-up of hours 0–4. Decitabine was quantified by a validated liquid chromatography tandem mass spectrometry (LCMS/MS) method. (B) DNMT1 protein levels in peripheral blood mononuclear cells (PBMC) measured by flow cytometry in cohorts 4 and 5 (decitabine 0.08 mg/kb and 0.16 mg/kg, respectively) and in all placebo-treated patients. Analyses were blinded to treatment assignment. Shown are means of 2 independent measurements. (C) Methylation of long interspersed nuclear elements (LINE-1) repetitive element CpGs (3 individual CpGs) in PBMC in cohorts 4 and 5 placebo and drug-treated patients. Analyses were blinded to treatment assignment. Measurements were made by pyrosequencing. Shown are means of 2 independent measurements.

Fig 6. Fetal hemoglobin (HbF) induction in cohort 4 and 5 patients.

(A) HbF percentage (HbF%) over time in cohorts 4 and 5 (decitabine 0.08 mg/kg and 0.16 mg/kg, respectively) patients. (B) Statistical analysis of the rates of change in HbF% with increasing doses of decitabine (0 = placebo); P value determined by linear regression. (C) Change in HbF% from pretreatment to week 8 or 10. (D) Absolute HbF levels. (E) Proportion of red blood cells (RBCs) expressing high levels of HbF (F-cells) in cohort 4 and 5 patients. (F) Raw F-cell flow cytometry data for cohort 5 tetrahydrouridine (THU)-decitabine—and placebo-treated patients. Abbreviation: FS, forward scatter.

Fig 7. Blood counts and other parameters in cohort 4 (decitabine 0.08 mg/kg) and 5 (decitabine 0.16 mg/kg) tetrahydrouridine (THU)-decitabine–treated patients.

(A) Total hemoglobin. (B) Statistical analysis of the rates of change in total hemoglobin (Hbg) with increasing doses of decitabine (0 = placebo); P value determined by linear regression. (C) Absolute reticulocyte counts (ARC). (D, E) LDH and total bilirubin. These are biomarkers of hemolysis. (F) D-dimer. A biomarker of coagulation activation. (G) C-reactive protein (CRP). A biomarker of inflammation. (H) Platelets. (I) Absolute neutrophil counts (ANC).