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A.G. Gilmartin et al.
thrice weekly for a total of six doses and terminated after the final dose on day 12. In previous studies, lower doses of decitabine administered daily resulted in no significant HbF induction in a 2-week timeframe, while higher doses administered daily or every other day were not tolerated (Online Supplemental Table S1). In the current decitabine study, mean HbF levels were increased by up to 2-fold at the 0.4 mg/kg dose, and by up to 4.4-fold at the 0.8 mg/kg (Figure 5A). However, 0.8 mg/kg was not tolerated based on the death of two of six animals. Bone marrow samples from surviving decitabine-treated animals exhibited decreased -53 bp DNA methylation by 4%, 9%, and 13% respectively in the 0.2, 0.4, and 0.8 mg/kg dose groups (Figure 5A).
As further characterization of the effects of dosing with GSK3482364 in the SCD mouse model, complete blood counts were taken at all doses. Compared to vehicle-treat- ed animals, GSK3482364-treated SCD mice had no remark-
able changes in peripheral blood cell counts or red cell indices (Table 1). Consistent with peripheral blood cell counts, histopathology of sternal bone marrow from the high dose group of animals treated with GSK3482364 (100 mg/kg) demonstrated no overt abnormalities in marrow cellularity or hematopoietic cell composition (six of six mice; Figure 6). Notably, in the comparable 12 day study, dose levels of decitabine that caused HbF induction (≥0.2 mg/kg ) also caused dose-dependent decreases in multiple blood cell counts (Online Supplemental Table S2), indicating that these dose regimens were all cytotoxic to some extent in the SCD mice. While lower doses of decitabine (0.1-0.2 mg/kg) administered either daily or every other day were poorly effective at inducing HbF in our previous 12 day studies (Online Supplemental Table S1), we acknowledge that alternative non-cytotoxic dose regimens or longer study durations might better optimize the efficacy and tol- erability of decitabine in these mice.
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Figure 4. Effects of GSK3482364 and decitabine in vivo. (A) Percentage fetal hemoglobin (%HbF) by high- performance liquid chromatography (HPLC) in whole blood of transgenic sickle cell mice treated with GSK3482364 for 12 days. Bars represent mean %HbF +/- standard deviation from 4-6 mice. (B) Representative histograms from HPLC assay; HbF peaks are indicated with arrowheads. (C) %F-cells for blood samples from treated mice were determined by flow cytometry. (D) Representative flow cytometry staining of HbF and nucleic acid content for blood cells. Asterisks indicate significance by 1-way ANOVA. (*P<0.01; **P<0.001). b.i.d.: twice daily; p.o.: orally.
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haematologica | 2021; 106(7)