Page 113 - Haematologica March 2020

Novel PDE9i therapy for sickle cell disease
times more potent at 10 mM than a dose of HU that demonstrated cytotoxicity (Figure 1B). Induction of HbF by IMR-687 was observed with the GM-CSF dependent erythroid line UT-7 (data not shown). While HU produced more HbF at higher concentrations, the induction was accompanied by cytotoxicity which was not observed with IMR-687 (Figure 1C).
Improved sickle cell disease phenotypes in vivo in murine model of sickle cell disease
We next tested the impact of IMR-687 and HU on F- cells, RBC sickling and markers of hemolysis in HbSS- Townes mice. After 30 days of treatment at 30 mg/kg/day of IMR-687, we observed a greater than 3-fold increase in the percent of HbF+ F-cells (8.4% in vehicle treated and 27.3% in IMR-687 treated; P<0.001) (Figure 2A) and a cor- responding 2-fold decrease in sickled RBC (56.3% in vehi- cle treated and 24.4% in IMR-687 treated; P<0.0001) (Figure 2B). We saw a similar induction of HbF and reduc- tion in sickled RBC with mice treated with HU doses of 100 mg/kg/day (29.3% F cells and 28.8% sickled RBC). This dose, which resulted in mortality in mice, was higher than the dose employed in patients. At HU doses that were tolerated in mice, the induction of HbF was modest and not significant compared to vehicle control (25 and 50 mg/mL/day increased F-cells to 13% and 18% compared to 8.4% for vehicle). There was a minimal decrease in the percent of sickled RBC with 25-50 mg/kg/day of HU com- pared to vehicle control (percentage of sickled RBC was decreased to 52% and 49%, respectively, compared to 56% for vehicle) (Figure 2A and B).
The significant reduction in the RBC sickling by IMR- 687 produced a corresponding decrease in markers of hemolysis. This was seen in a reduction of free plasma Hb (Figure 2C ) where IMR-687 reduced plasma free Hb levels over 55%. HU treatment also reduced free Hb levels in a dose-dependent fashion with the highest dose, 100 mg/kg, reducing levels by approximately 55%.
Consistent with the reduction in hemolysis and reduc- tion in free Hb, plasma bilirubin levels and LDH activity, markers of hemolysis46 were significantly increased in vehicle treated SS mice compared to AA mice and reduced over 2-fold in IMR-687 treated mice (4.7 mg/dL, P<0.01 and 102 AU, P<0.05) (Figure 2D and E). The impact of the 100 mg/kg HU treatment was less pronounced, reducing bilirubin levels to 5 mg/dL ( P<0.01) and LDH levels to 121 AU (not significant). HU dosed at 25 and 50 mg/kg did not produce a significant reduction in either marker of hemol- ysis.
Red cell lysis results in the release of Hb which con- sumes the plasma pool of NO and increases the vascu- lopathy associated with SCD7 nitrite generated in the plasma from an excess of NO produced by endothelial NO synthase (eNOS), can be converted back to NO as lev- els drop, acting as a biochemical reserve for NO.47 In HbSS-Townes mice, plasma nitrate levels are 41% lower than those in control AA mice (0.56 mg/mL vs. 0.95 mg/mL) (Figure 2F). Hemolysis results in the release of Hb and heme, which acts as a scavenger of NO. Treatment of SS mice with 30 mg/kg of IMR-687 increased plasma nitrite levels almost 2.5-fold to 1.39 mg/mL ( P<0.05). HU in a dose-dependent manner increased nitrite levels as well, with a peak of 1.23 mg/mL in the 100 mg/kg dose group; however, these changes were not significant and were modest at therapeutic doses of HU. The difference in
plasma nitrite levels in IMR-687 and 100 mg/kg HU treat- ed mice were not significantly different.
Hemolysis also results in the release of arginase which reduces NO bioavailability and is correlated with SCD mortality.10 IMR-687 reduced lung arginase 25% (P<0.0001) (Figure 2G) compared to vehicle controls. This effect was less pronounced in the mice treated with 100 mg/kg of HU.
Reticulocytosis reflects the bone marrow’s response to anemia due to hemolysis. IMR-687 treated mice demon-
mice for 30 days results in reduced immune cell activity. White blood cell (WBC) counts are elevated in Townes-HbSS mice above normal controls (A). Townes- HbSS mice were dosed orally for 30 days with IMR-687 at 30 mg/kg or hydrox- yurea (HU) at 100 mg/kg. Treatment with IMR-687 or HU reduced circulating WBC counts (A) (n=3). NS: not significant; *P>0.05. Data are presented as means±Standard Errors. This decrease in WBC counts is not seen in normal mice, rats or dogs dosed with IMR-687, including long-term 9-month toxicology studies in dogs dosed orally daily with 10, 25, or 50 mg/kg of IMR-687 (B). Along with the reduction in circulating WBC levels in IMR-687-treated Townes mice, there was a significant reduction in lung myeloperoxidase activity (C) (n=7). **P<0.01; ***P<0.001; ns: not significant (P>0.05). Data are presented as means±Standard Errors. MPO: myeloperoxide.
A
B
C
Figure 3. Treatment of phosphodiesterase 9A inhibitor (IMR-687) in sickle
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