Pathobiology of pain in sickle cell disease

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Abstract

The overall goal of this program is to improve pain management, behavior deficits, and overall quality of life in patients with sickle cell disease (SCD) patients. While the molecular etiology of SCD has been known for over 70 years, the mechanisms and triggers underlying SCD acute pain episodes and the complex multi-system organ damage in SCD remain incompletely understood. We do know that polymerization of sickle hemoglobin (HbS) and sickling of red blood cells (RBCs) are the root causes of SCD pathobiology. With HbS polymerization, a cascade of events including hemolysis, activation of neutrophils, mast cells, macrophages, and platelets, release of inflammatory cytokines and cell adhesion molecules, and an increase in platelet aggregation ensue. Collectively, this cascade of events contributes to recurrent micro-vaso-occlusion, acute pain episodes, chronic vascular endothelial injury and inflammation, and multisystem organ damage. Using humanized sickle cell mouse models, we conduct mechanistic studies and evaluate the effects of novel therapeutic agents on hematologic parameters, organ dysfunction and SCD pain. Over the last cycle, we showed that pharmacologic inhibition of the NLRP3 inflammasome and of Bruton tyrosine kinase (BTK) decreases thrombus formation and platelet aggregation in vitro and hepatic platelet aggregation in SCD mice. We treated SCD mice with the inhibitors of NLRP3 (MCC950) or BTK (ibrutinib), or vehicle control for 4 weeks. We showed that this long-term pharmacological inhibition of NLRP3 and BTK in SCD mice decreased in vitro platelet aggregation and thrombus formation. Long-term treatment with MCC950 or ibrutinib also decreased platelet aggregates in the liver of SCD mice. These data in turn demonstrate that targeting the NLRP3 inflammasome might be a novel approach for antiplatelet therapy to possibly decrease episodes of vaso-occlusion and organ damage in SCD. We also evaluated the effects of mitapivat, an activator of erythrocyte pyruvate kinase (PKR) in the SCD mouse model. Mitapivat enhances the activity of the erythrocyte glycolytic pathway and as such it has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, which are factors that decrease HbS polymerization and improve erythrocyte membrane integrity. We treated control (HbAA) and sickle (HbSS) mice with mitapivat or vehicle for 4 weeks. Surprisingly, we showed that HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. Therefore, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels. Overall, our findings strongly support for using SCD model systems for preclinical evaluation of novel therapies that might ameliorate the complications of SCD in patients.