Chronic Administration of the PDE9 Inhibitor PF-04447943 Reduces Leukocyte-Platelet Aggregates and Markers of Endothelial Activation in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD), caused by a single amino change in the β-globin gene, exhibits a complex pathophysiology. Hypoxia in the microvascular venous bed leads to inflammation of the endothelium, adhesion of neutrophils, and a decrease in neutrophil rolling and flow velocity. The cell aggregates become trapped in the vasculature through interactions with endothelial cells. The adhesive interactions of the sickle erythrocytes, leukocytes, and endothelial cells obstruct the vasculature leading to vaso-occlusion. The vaso-occlusion can result in endothelial cell dysfunction and inflammation. Soluble adhesion molecules, including soluble E-Selectin, are associated with disease severity (Kato et al, Br.J.Hematol, 2005). PF-04447943, a selective inhibitor of the cyclic guanosine monophosphate-specific phosphodiesterase-9A, is currently in clinical development for the prophylactic treatment of SCD. The Townes mouse model of SCD exhibits many of the pathological symptoms observed in patients. Previously, we demonstrated vaso-occlusion in the Townes model by intravital microscopy (IVM), and that a single dose of PF-04447943 reduced leukocyte-platelet aggregates and the adhesion of neutrophils to the endothelium. Here we examined the pharmacodynamic effects of chronic repeat dosing with PF-04447943 in male Townes sickle cell mice.

Mice were dosed orally with PF-04447943 twice daily for 4 weeks in the presence and absence of hydroxyurea (HU). Treatment effects were evaluated in vivo using IVM after 4 weeks. Surgical exposition of the cremaster muscle elicits inflammation-induced vaso-occlusion in the mouse vasculature and neutrophil rolling, adhesion and neutrophil-platelet aggregates were analyzed using IVM. Administration of PF-04447943 (10 mg/kg) in combination with HU (25 mg/kg) showed a reduction in neutrophil adhesion, an increase in neutrophil rolling on the microvasculature, and a reduction in neutrophil-platelet aggregates compared to vehicle alone. A 50% reduction in neutrophil platelet aggregates was observed when 10 mg/kg of PF-04447943 was administered as a single agent (50%), but this effect was more pronounced with co-administration of PF-04447943 and HU (73%). Significant reductions (11.1%) were observed in soluble plasma E-selectin levels in mice treated with a combination of PF-04447943 and HU for 28 days compared to vehicle treated mice. Analysis of hematological parameters showed a 42% reduction in total peripheral white blood cell count in mice that received a co-administration of PF-04447943 and HU. A small but significant increase was seen in the mean corpuscular hemoglobin (MCH) as well as mean corpuscular volume (MCV).

In conclusion, the phosphodiesterase-9A inhibitor PF-04447943 results in reductions in leukocyte-platelet aggregates and soluble E-selectin in a chronic treatment study in SCD mice. Long term treatment with PF-04447943 may be beneficial in improving pharmacodynamic parameters, as well as markers of inflammation that may mediate vaso-occlusion associated with SCD.

All experiments were within guidelines and were reviewed and approved by Pfizer Institutional Animal Care and use Committee.