A Novel Oral Prodrug AN233 Induces Fetal Hemoglobin Expression in Sickle Cell Disease Mice and Baboons

Introduction. Individuals with sickle cell disease (SCD) experience a spectrum of clinical symptoms including anemia, pain, and progressive organ damage. However, fetal hemoglobin (HbF) induction is the best strategy to ameliorate disease severity. Hydroxyurea (HU) is the only FDA-approved HbF inducer for individuals with SCD, but it is not effective for all people. Previous clinical trials demonstrated the ability of parenteral arginine butyrate to induce HbF, but it was not effective by oral administration. To address this barrier, Nudelman and Rephali developed AN-233, comprised of δ-aminolevulinate and butyric acid, which is metabolized by cellular esterases to active drug. Subsequently, we demonstrated HbF induction by AN-233 in sickle erythroid cells and activation of γ-globin expression in β-Yac mice treated by intraperitoneal injections. In this study, we determine the optimal dose of oral AN-233, which activates γ-globin transcription in β-Yac mice, followed by treatment of Townes SCD mice to establish whether AN-233 produces sufficient plasma butyrate levels to mediate HbF induction.

Methods. We treated β-Yac mice (n=12) with oral AN-233 (300 and 400 mg/kg), 5 days/week for 4 weeks. Tail vein blood evaluated complete blood counts with differential (CBC), and flow cytometry quantified F-cells and mean fluorescence intensity (MFI). Next, we treated fragile SCD mice (n=12) with oral 300 mg/kg AN-233 alone or combined with HU (100 mg/kg), 5 days/week for 6 weeks. Body weights and blood collected at weeks 0, 3, and 6 weeks determined the effects of AN-233 on the CBC, γ-globin transcription, and F-cells and HbF levels. Spleens harvested at 6 weeks were used for H & E stain. Using a triple-quadrupole mass spectrometer, coupled with separation of compounds by reverse phase HPLC, plasma AN-233, and butyrate levels were measured. Finally, we treated three anemic baboons with AN-233 (300 mg/kg; 5 doses) subcutaneously and quantified CBC, F-cells, and HbF levels on various days post-treatment.

Results. Initial treatment of β-YAC mice with both doses of oral AN-233 was well tolerated and there were no deaths; the CBC remained stable without signs of toxicity. We observed a concentration-dependent increase in F-cells 2.6-fold (p=0.01) and 3.6-fold (p=2.0 E-07) for the 300 mg/kg and 400 mg/kg doses respectively, compared to a 2.9-fold increase for HU (p=0.0005). Since low dose AN-233 induced HbF without toxicity, we evaluated AN-233 (300 mg/kg) alone or combined with HU in fragile SCD mice. After 6 weeks of treatment, no adverse effects occurred on weights and CBC; one mouse died in each group including water supporting equal survival. Levels of γ-globin increased 2.5-fold (p=0.004), 1.6-fold (p=0.003), and 4.0-fold (p=0.006) respectively for AN-233, HU, and combination treatments; β^S-globin mRNA levels did not change significantly. Parallel enhancement of F-cells 10.1-fold (p=0.001), 12.5-fold (p=0.01), and 13.8-fold (p=0.0005) for AN-233, HU, and combination therapy was produced. Significantly, higher MFI levels verified increased HbF protein per cell for all treatment groups and combination treatment produced a mild additive effect. Spleen analysis revealed a significant decrease in weights for the three treatments compared to control. For the water group, the H & E stain showed increased loss of lymphoid tissue, hemorrhagic infarcts, and ischemia. By contrast, AN-233 and HU restored lymphoid elements with enhanced hemosiderin-laden macrophages and megakaryocytes indicative of regeneration and extra-medullary hematopoiesis. By mass spectrometry, plasma AN-233 levels decreased by 3 h with a steady rise in butyrate from 3-9 h supporting drug activation in vivo. Lastly, three anemic baboons treated with AN-233 subcutaneously confirmed increases in F-cells from 31.2% (baseline) to 47.9% (p<0.025) and HbF protein levels increased 1.7-fold (p<0.015). Of note, redness and swelling occurred at the injection site for AN-233 treatments.

Conclusion. Our findings in SCD mice confirmed absorption and metabolism of oral AN-233, which produced sufficient rises in plasma butyrate to activate γ-globin transcription and HbF expression without adverse toxic effects. Moreover, baboon studies confirmed the ability of AN-233 to increase F-cells and HbF levels. This report supports developing AN-233 as a treatment option for individuals with SCD.