Oral Administration of Dacogen Mesylate Increases HbF and Decreases DNA Methylation of the ε- and γ-Globin Genes in Anemic Baboons (Papio anubis).
Authors: Lavelle Donald , Vaitkus Kestis , Chin Janet , Redkar Sanjeev , Phiasivongsa Pasit , Hankewych Maria , DeSimone Joseph 
Published: April 2020

Increased levels of fetal hemoglobin (HbF) lessen the severity of sickle cell disease. Subcutaneous and intravenous adminstration of a drug that inhibits DNA methyltransferase, 5-aza-2′-deoxycytidine (Dacogen; DAC), increased HbF levels in hydroxyurea-refractory sickle cell patients and experimentally-induced anemic baboons. An orally administered, effective DNA methyltransferase inhibitor would be desirable for increasing compliance of patients in future clinical trials. Previous experiments showed that oral administration of the DNA methyltransferase inhibitor 5-azacytidine was only effective when combined with tetrahydrouridine, a cytidine deaminase inhibitor. Zebularine inhibits DNA methyltransferase when administered orally, however relatively high doses are required and the ability of this drug to increase HbF levels has not been tested in either experimental primate studies or in patients with sickle cell disease. Based upon previous studies in mice demonstrating bioavailability of orally administered DAC, we tested whether a new salt form, DAC mesylate, could increase HbF and cause DNA demethylation when administered orally at doses 8–36 fold higher than effective doses given subcutaneously. Three baboons were rendered anemic by acute phlebotomy for ten days and maintained at a Hct of 20 during the course of drug treatment. HbF levels following the initial bleeding and prior to DAC mesylate administration were 6.3–13.9%. Each baboon received a different orally administered dose of DAC mesylate (18.7mg/kg/day; 9.35mg/kg/day; 4.1mg/kg/day) for ten days. Peak HbF levels achieved in animals receiving these three different doses were 67.8, 61.9, and 17.4, respectively. Peak HbF in the two animals receiving higher doses were comparable to levels observed in these animals following subcutaneous injection of a lower dose of DAC (0.52mg/kg/day). Bisulfite sequence analysis showed that methylation of the ε- and γ-globin genes was decreased &gt;50% in animals treated with 18.7mg/kg and 9.35mg/kg doses, while minimal changes were observed in the animal treated with the lowest dose (4.1mg/kg). Chromatin immunoprecipitation (ChIP) studies showed that the levels of acetylated histones H3 and H4 associated with the β-globin promoter were 5–6 fold higher than with the γ-globin promoter in bled animals. Following DAC mesylate, equivalent levels of acetylated histones H3 and H4 were associated with the γ- and β-globin promoters in the two animals treated with the higher doses of drug. These studies thus demonstrate that orally administered DAC mesylate increased HbF, reduced DNA methylation of the ε- and γ-globin genes, and increased acetylation of histones H3 and H4 associated with the γ-globin promoter in anemic baboons. Future experiments to directly compare the efficacy of DAC mesylate and DAC in baboons and potential Phase I studies to assess the feasibility of oral administration of DAC mesylate in the treatment of patients with sickle cell disease may be warranted.

Effect of Oral DAC Mesylate on HbF and DNA Methylation Animal Treatment DAC Dose (mg/kg/day) ε-globin (% dmC) γ-globin (% dmC) HbF (%) PA 6974 Bled 0 96.6 79.3 6.3 Oral DAC mesylate 18.7 43.3 35.0 67.8 PA 7002 Bled 0 86.6 71.7 13.9 Oral DAC mesylate 9.35 46.6 34 61.9 PA 7001 Bled 0 90.0 78.7 6.3 Oral DAC mesylate 4.1 86.6 74.1 17.4