Phase I study of the histone deacetylase inhibitor panobinostat in sickle cell disease Free

Introduction: Reactivation of HbF in adult life is a proven effective therapy for β-hemoglobinopathies. Hydroxyurea (HU) is the only FDA approved drug for sickle cell disease (SCD) that ameliorates disease severity by inducing fetal hemoglobin (HbF). Several other classes of drugs, including histone deacetylase inhibitors have been shown to increase HbF production in cell cultures and mouse models of SCD. Since HU is underutilized in SCD, there is an unmet need to develop alternate HbF inducing agents. Therefore, the purpose of our study was to identify a safe and effective oral agent that induces HbF for the treatment of SCD.

Method. We designed a Phase I trial of the pan-histone deacetylase inhibitor Panobinostat (PAN) in subjects with severe SCD (SS and Sb⁰thalassemia) who are intolerant of HU (NCT01245179). Eligibility criteria included age ³18 years, use of protocol-defined contraception, and severe SCD phenotype, defined by 2 hospitalizations or 3 ED/outpatient-treated vaso-occlusive episodes in the previous year, acute chest syndrome within 5 years, recurrent leg ulcers or priapism, or history of stroke. Participating subjects were followed every 1-4 weeks and underwent physical exam and laboratory tests including CBC, reticulocytes (retics), CMP, HbF by HPLC, and F-cells, HbF and F-retics by flow cytometry. Peripheral blood mononuclear cells were isolated from whole blood on D1, D57 and D85 of PAN treatment using density gradient centrifugation. Whole cell protein extraction was performed and STAT5, acetylated p53, NFκB expression levels determined by Western blot. Subsequent studies used CD71⁺ erythroid cells purified from red blood cells using CD71 microbeads. The purified CD71⁺ cells, were crosslinked with 1% formaldehyde and processed for chromatin immunoprecipitation (ChIP) assay using acetylated histone H3 and H4 (AcH3 and AcH4) antibody.

Results. The first 3 subjects (Cohort 1) received PAN 15 mg PO MWF continuously throughout the 12-week treatment period. Since no dose limiting toxicities (DLT) were observed in any of the 3 subjects, 2 additional subjects (Cohort 2) were enrolled at PAN 20 mg MWF, 3 weeks on/1 week off x 12 weeks. All five subjects who have completed the study tolerated treatment well, without any DLTs or drug related adverse events. One subject had a VOC with resultant hospitalization and transfusion, which was determined unrelated to PAN treatment. The CBC, retics, and CMP results did not show significant changes between Day 1 and Day 85. By HPLC analysis, in peripheral blood the average HbF in Cohort 1 were 9.1% on Day 1 and 8.2% on Day 85 (p=0.79), however, in Cohort 2 the HbF increased from 14.9% on Day 1 to 19.6% on Day 85 (p=0.15). To support HbF induction, flow cytometry showed Cohort 1 had an average increase from Day 1 to Day 85 in F-cells from 7.6% to 19.7% (p=0.01), F retics from 1.8% to 5.2% (p=0.04) and a 3.1-fold increase in HbF by mean fluorescence intensity (MFI). Cohort 2 subjects showed increased F-cells from 12.9 to 20.6 and a 2-fold increase in MFI. Western blot assessed changes in cellular proteins known to be affected by PAN. For Cohort 1, subject 2002 had a 2-fold (p=0.03) increase in acetylated NFκB and subject 2003 showed a 60% decrease (p=0.026) in total STAT5; however, there was no significant changes in protein levels noted for subject 2004. Likewise, there were no significant change in plasma cytokine levels during the study period. Lastly, to determine the ability of PAN to alter chromatin structure, we performed chromatin immunoprecipitation (ChIP) assay which showed that PAN significantly increased enrichment of AcH3 and AcH4 at the locus control region hypersensitive site 2 and HBG promoter by D85. By contrast, no significant changes in histone marks were observed for the HBB promoter.

Future Plans/Conclusions: A third subject will be enrolled to complete Cohort 2, and if no DLTs are observed, dose escalation to Cohort 3 (20 mg MWF continuous x 12 weeks) will be initiated, for a total of 12 additional subjects to be enrolled. Our findings in both Cohorts support the ability of PAN to increase HbF in vivo. The observation of an increase in HbF by HPLC in Cohort 2 is encouraging. The findings from ChIP assay suggest that PAN promotes an open chromatin configuration at the HBG promoter. This report supports developing PAN as a treatment option for individuals with SCD.