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

Abstract

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. Disclosures No relevant conflicts of interest to declare.

PF‐04447943, a Phosphodiesterase 9A Inhibitor, in Stable Sickle Cell Disease Patients: A Phase Ib Randomized, Placebo‐Controlled Study

“…In a second model, using Townes SCD mice, which experienced an acute inflammatory response and vaso-occlusion upon cremaster muscle exposure, pretreatment with the combination of PF-04447943 and hydroxyurea also decreased neutrophil adhesion and increased neutrophil rolling. 21 A response was also observed in the number of neutrophil-platelet aggregates; administration of PF-04447943 alone resulted in a decrease in neutrophil-platelet aggregates with a more pronounced effect observed with the coadministration of hydroxyurea. In a prophylactic setting with repeat dosing, Townes SCD mice treated with PF-04447943 in combination with hydroxyurea twice daily for 4 weeks achieved an increase in neutrophil rolling, a reduction in neutrophil adhesion, a 73% reduction in the formation of neutrophil-platelet aggregates, and an 11% decrease in plasma-soluble E-selectin compared with vehicle-treated mice.…”

“…However, our clinical findings are consistent with changes observed preclinically with a combination of PF-04447943 and hydroxyurea in two models of SCD. 21,22 In these studies, the dynamics of cellular interactions were visualized using intravital microscopy. In the first model, C57BL/6J wild-type mice challenged with tumor necrosis factor alpha, which induces an acute inflammatory response in the cremaster microcirculation characterized by endothelial neutrophil adhesion and the formation of multicellular aggregates, coadministration of hydroxyurea and PF-04447943 prior to the challenge reduced endothelial neutrophil adhesion and formation of neutrophil-platelet aggregates and increased the number and velocity of rolling neutrophils compared with results in saline-treated mice.…”

“…Based on studies in SCD mice, we hypothesized that cellular aggregates and soluble plasma adhesion biomarkers may be impacted. 21,22 Microparticles were not examined in the SCD mouse models.…”

“…17,19 In two SCD models, tumor necrosis factor alpha-treated normal wildtype mice and the Townes model of SCD, 20 PF-04447943, in combination with hydroxyurea as prophylaxis, improved cellular and plasma pharmacodynamic (PD) markers, including cellular aggregates and soluble adhesion plasma markers. 21,22 Previous clinical studies assessed the pharmacokinetics (PKs)/PDs of PF-04447943 in healthy human volunteers 23,24 and demonstrated its beneficial effects on cognition in preclinical models 17,19 and in patients with Alzheimer disease, in whom PF-04447943 was shown to be safe and well tolerated. 25 The objectives of this study were to evaluate the safety, tolerability, PKa, and PD of PF-04447943, with or without coadministration of hydroxyurea, in patients with stable SCD and to evaluate possible biomarkers for use in future SCD studies.…”

“…PF‐04447943 was also shown to achieve improvements in synaptic plasticity and cognitive performance in several rodent cognition models, including working, episodic, and spatial memory, auditory gating, and attention . In two SCD models, tumor necrosis factor alpha–treated normal wild‐type mice and the Townes model of SCD, PF‐04447943, in combination with hydroxyurea as prophylaxis, improved cellular and plasma pharmacodynamic (PD) markers, including cellular aggregates and soluble adhesion plasma markers . Previous clinical studies assessed the pharmacokinetics (PKs)/PDs of PF‐04447943 in healthy human volunteers and demonstrated its beneficial effects on cognition in preclinical models and in patients with Alzheimer disease, in whom PF‐04447943 was shown to be safe and well tolerated …”