PDE-9 Inhibition Combined with Hydroxyurea Is Beneficial in Vaso-Occlusive Crisis in Mouse Model of Sickle Cell Disease

Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease (SCD). It is a complex multistep process initiated by the adhesion of fragile red cells and leucocytes, primarily neutrophils, to the hypoxic and inflamed endothelium. Attachment of large and rigid neutrophils to the endothelium, particularly in the microcirculation induces vaso-occlusive crisis by activating neutrophils and forming multicellular aggregates with erythrocytes and platelets. Dysregulated nitric oxide (NO) homeostasis contributes to vascular dysfunction in SCD. Hydroxyurea is the standard of care and the only approved therapy for SCD. Hydroxyurea has been shown to exhibit NO donor properties and may act to increase g-globin expression via the second messenger cGMP. PF-04447943 (PDE9i) is a selective inhibitor of the cGMP specific phosphodiesterase-9A (PDE-9A) enzyme (IC₅₀ 12nM) being developed for the treatment of SCD. Here, we study the combined effects of this PDE-9A inhibitor and hydroxyurea in a mouse model of acute vaso-occlusion.

The effect of PF-04447943 was assessed in the presence and absence of hydroxyurea in vivo using two models, TNF-α treated normal wild-type mice and the Townes model of SCD. C57BL/6J or Townes SCD mice were randomized to treatment with saline or PDE-9 inhibitor alone or in combination with hydroxurea. In wild-type mice, treatment was administered in a prophylactic setting prior to the TNF-α inflammatory challenge or in an acute setting post-TNF-α (0.5 ug/mouse). TNF-α induces a well described acute inflammatory response in the microcirculation associated with neutrophil adhesion to the endothelium and formation of multicellular aggregates. Alexa-488 labeled Ly-6G neutrophil antibody and Dylight-649 labeled CD42c platelet antibody was injected to quantify neutrophils adhered to endothelium and neutrophil-platelet aggregates. Mouse cremaster microvasculature was observed by intravital microscopy.

TNF–α treatment of C57BL/6J mice increased the number of adherent neutrophils and neutrophil-platelet aggregates, and decreased the number of rolling neutrophils compared to vehicle treated mice. Treatment with PDE9i or hydroxyurea alone in TNF-α challenged mice had no significant effect on neutrophil adhesion. Co-administration of 100 mg/kg HU in combination with 10 mg/kg PDE9i prior to TNF-α challenge led to a 69% reduction in neutrophils adhered to the endothelium and 89% reduction in neutrophil-platelet aggregates compared to TNF–α treated mice alone. Neutrophil adhesion was also reduced 59% in mice receiving 50 mg/kg hydroxurea and 1 mg/kg PDE9i. There was a significant increase in the neutrophil rolling number and velocity after co-administration of PDE9i and HU in TNF-α challenged mice. However, mice receiving PDE9i and hydroxurea after theTNF-α challenge did not show significant changes in neutrophil adhesion or aggregates. Plasma levels of sP-Selectin, sE-Selectin, sVCAM-1 and sICAM-1 decreased when animals were given a prophylactic combination treatment with PDE9i and hydroxyurea.

The Townes sickle cell disease mice exhibit an acute inflammatory response upon surgical exposition of the cremaster muscle and show increased number of adherent neutrophils and large neutrophil-platelet aggregates. Prophylactic treatment of these sickle mice with combination of PDE9i and hydroxyurea also showed a 63% reduction in neutrophil adhesion and a 75% reduction in cell aggregates, leading to reduced vaso-occlusion.

In summary, inhibition of PDE9 in mouse models of SCD had beneficial effects in the prophylactic reduction of crisis. Co-administration of PDE9 inhibitor with hydroxyurea, led to a significant reduction in vaso-occlusion associated with sickle cell disease in two distinct animal models of SCD.

All experiments were within guidelines and were reviewed and approved by the site institutional animal care and use committee.