Anti-Inflammatory Activities of Sulfated Non-Anticoagulant Heparin Derivative Beyond Its Anti-Sickling and Anti-Selectin for the Effective Management of Sickle Cell Disease

Abstract

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by the inheritance of a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases the adhesiveness of RBCs to alter the rheological properties of the blood and trigger inflammatory responses, leading to hemolysis, vasoocclusive crisis, pulmonary complications, plethora, and other pathological sequelae. Glycosaminoglycans, such as low molecular weight heparin (LMWH), have been suggested as treatments to relieve coagulation complications in SCD because of their ability to decrease thrombin generation and sickle cell adhesion. However, they are associated with bleeding complications after repeated dosing. An alternative, sulfated non-anticoagulant LMWH derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectin-dependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice (1,2). S-NACH has been further engineered to possess an aldehyde moiety, which confers anti-sickling properties primarily due to specific interactions with HbS to increase its affinity for oxygen. Our in vitro sickling assay under hypoxic conditions using S-NACH at 0.5 - 2 mM demonstrated that S-NACH significantly reduced the sickling of SS cells and in a concentration-dependent manner, with comparable to that of 1 mM GBT440 (Figure 1 A-C). A similar concentration dependent effect on increasing HbS affinity for oxygen using oxygen equilibrium study was documented. In an vivo animal model using Townes' SCD animals plasma levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, MCP-1, TNF-α, M-CSF, and VEGF were increased in SCD untreated samples in contrast to a significant decrease (*P< 0.001) in S-NACH-treated animals, at both 2 and 6 h. In addition, S-NACH was able to increase the decreased levels of the endogenous anti-inflammatory IL-10 (Figure 1 D). The above set of novel findings about S-NACH established it to be effective for the management of SCD via the modulation of thromboinflammatory pathways, involved in thromboembolism and end organ damage, beside anti-sickling, anti-selectin and without causing any bleeding risk. Reference 1. Alshaiban A, Muralidharan-Chari V, Nepo A, Mousa SA. Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Non-anticoagulant Heparin Derivative. Clin Appl Thromb Hemost. 2016 ;22(3):230-8. 2. Mousa SA: Compositions and method for anti-sickling of red blood cells in sickle cell disease. US Patent 9,822,190, November 2017. Disclosures Mousa: Vascular Vision Pharma Co.: Patents & Royalties.