Novel Conjugates of Butyrate and δ-Aminolevulinate Increase γ-Globin Gene Expression and Fetal Hemoglobin Synthesis in Erythroid Progenitors

Pharmacologic induction of fetal hemoglobin (HbF) expression reduces the clinical severity of sickle cell disease (SCD) and improves long-term survival. Research efforts to develop effective and safe therapies have focused on a wide variety of agents however, Hydroxyurea is the only FDA-approved drug proven to mediate HbF induction in about 50% of adult SCD patients. Our group previously reported potent HbF-induction by sodium butyrate through p38 MAPK activation and CREB1 binding to the Gγ-globin cyclic AMP response element (Sangerman et al., Blood 2006). However, oral administration of butyrate derivatives to SCD patients was ineffective due to rapid metabolic inactivation by the liver. Therefore to address this critical barrier of limited treatment options for SCD, we investigated the prodrugs 1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (AN233) and 1-(butyryloxy)butyl 5-amino-4-oxopentanoate (AN908), esters of δ-aminolevulinate (ALA) and butyric acid as HbF inducers in erythroid cells. Oral administration of AN233 and AN908 to BALB/c mice increased total hemoglobin levels without causing tissue toxicity (Rephaeli et al., Eur J Pharm Sci 2016).

To test the ability of prodrugs to induce HbF, both K562 and KU812 cells and the immortalized human umbilical cord blood-derived erythroid progenitor 1 (HUDEP1) model were used. Treatment of K562 cells with 0.125-0.50 mM concentrations of AN233 produced cell toxicity at the 0.50 mM concentration, however >90% viability was observed by Trypan blue exclusion for 0.25 mM AN233 without evidence of caspace-3 cleavage. Using this concentration of AN233, a 1.5-fold and 6-fold (p<0.05) increase in γ-globin mRNA and HbF levels respectively, were observed supporting robust intracellular activation of AN233. Flow cytometry analysis confirmed a 1.5-fold increase in the percent of HbF positive cells (F-cells). Preliminary studies in HUDEP1 cells showed 0.25 mM AN233 increased γ-globin expression by 1.5 fold and HbF protein synthesis by 2.2 fold. Mechanistically, AN233 mediated a 53% decrease in phosphorylation levels of the kinase, eIF2α (eukaryotic initiation factor 2α) which favors conditions for protein synthesis. As further evidence that both prodrugs induce HbF synthesis in different cell types, KU812 cells expressing the γ-globin and β-globin genes were treated with AN908 (0.125-0.50 mM). In contrast to AN233, cell toxicity was not observed at the 0.50 mM AN908 concentration where 93% cell viability was maintained. Treating KU812 cells with 0.25 mM AN908 for 48 h produced a 1.4-fold increase in γ-globin transcription and HbF synthesis by western blot. In addition, F-cell levels increased from 45% to 70% and HbF levels (by mean fluorescence intensity) 2.2-fold (p<0.05). Interestingly, when 0.25 mM AN908 was combined with hydroxyurea (100 μM) we observed an additive increase in HbF and F-cells levels.

Collectively, these findings support the oral active prodrugs AN233 and AN908 as HbF inducers in erythroid cells. Furthermore, the combination of AN908 and hydroxyurea produced an additive effect without increasing cell toxicity. Studies are underway to determine the EC₅₀ of both prodrugs in normal and sickle primary erythroid progenitors and mechanisms of γ-globin regulation through epigenetic modifications.