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Novel PDE9i therapy for sickle cell disease
Lung homogenate myeloperoxide (MPO) and arginase were also determined (see Online Supplementary Methods).
Hemoglobin S-Townes vaso-occlusive crisis model. HbSS- Townes mice42 (6-17 weeks old, n=3 per group) were treated with vehicle (0.08% w/v methyl cellulose), 100 mg/kg of HU, 10 or 30 mg/kg of IMR-687, or 100 mg/kg HU + 30 mg/kg IMR-687 in their drinking water. On day 7 of treatment, the mice were implanted with dorsal skin-fold chambers (DSFC). Three days later, on day 10 of treatment, mice with DSFC were anes- thetized, placed on a special intravital microscopy stage, and 20- 23 flowing subcutaneous venules in the DSFC window were selected and mapped. Mice were then placed in a hypoxic atmosphere chamber (7% O2/ 93% N2) for 1 hour (h), after which they were returned to room air. All the selected venules were re-examined after 1 and 4 h of re-oxygenation in room air, and the number of static (no flow) venules was counted and expressed as percent stasis. After this, mice were euthanized and plasma hematocrit, bilirubin, Hb and heme were measured and WBC, RBC, sickled RBC and HbF+ RBC quantified (see Online Supplementary Methods).
Results
Phosphodiesterase enzyme selectivity
To determine the selectivity of IMR-687 for the phos- phodiesterase 9A, 33 recombinant human PDE were incu- bated in vitro with increasing concentrations of IMR-687 and their activity determined. The IC50 of IMR-687 for PDE9A1 and PDE9A2 were 8.19 nM and 9.99 nM, respec- tively. IMR-687 inhibited PDE9A with more than 800-fold greater potency than PDE1A3, PDE1B, PDE1C, PDE5A2, with IC50 values of 88.4 mM, 8.48 mM, 12.2 mM, and 81.9 mM, respectively (Table 1). Significant inhibition of the other 27 PDE enzymes tested, including PDE4 and PDE10, was not observed (Table 1).
cGMP and fetal hemoglobin induction in erythroid cells
To determine if IMR-687 would increase cGMP levels in an erythroid cell line, actively growing K562 cells were cultured in media containing increasing concentrations of IMR-687 or HU. cGMP levels were assessed using a non-
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C
Figure 1. IMR-687 recapitulates the cGMP and fetal hemoglobin (HbF) induc- tion mechanism of hydroxyurea (HU) in erythroid cells. (A) Phosphodiesterase 9A inhibitor (IMR-687) (triangles) and HU (squares) treatment for 6 hours (h) in erythroid K562 cells increases cGMP in a dose-dependent manner. (B) IMR-687 (triangles) and HU (squares) treatment in erythroid K562 cells for 72 h also induced HbF in a dose-dependent manner, evaluated by an ELISA assay using an antibody against human HbF. (C) HU (red symbols) demonstrates greater cytotoxicity than IMR-687 (black symbols) as assessed by cell counts at the end of a 72-h culture with each drug. *P<0.05; **P<0.01; ***P<0.001. Data are presented as means±Standard Errors.
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