Multi-Modal Mechanisms and Anti-Sickling of Novel Sulfated Non-Anticoagulant Low Molecular Weight Heparin in Sickle Cell Disease

The pathogenesis of Sickle Cell Disease (SCD) comprises a complex interplay of factors associated with vascular endothelial activation, intense inflammation, and increased sickle cell adhesion. Microvascular occlusion in SCD is initiated by adhesion of sickle red blood cells (RBCs) to the endothelium, leading to acute painful vasoocclusive crisis (VOC) and clinical morbidity. Current treatment strategies remain sub-optimal and are limited by significant side effects. The inherent complexity of SCD makes it unlikely that a single therapeutic strategy will be universally beneficial. We have previously shown that the low molecular weight heparin (LMWH) tinzaparin significantly shortened both duration of VOC crisis and hospitalizations by ~40%, and resulted in statistically significant and rapid reduction of pain). However, safety concerns associated with the narrow therapeutic index (bleeding risks) of LMWH are a major barrier to dose escalation/optimization of treatments.

We have developed a novel sulfated non-anticoagulant LMWH, named S-NACH, with an extensive range of bioactivities that would constitute a multi-modal approach to management of SCD. We generated and significantly optimized S-NACH for VOC to: 1) exert its beneficial activities without causing hemostatic (bleeding) side effects that are associated with the clinical use of LMWHs; and 2) incorporate an additional, potent direct anti-sickling property besides its anti-selectin and anti-inflammatory activities.

We conducted in vitro and in vivo investigations on the efficacy of S-NACH on the biophysical properties of RBCs. For the in vitro study, 21 subjects comprising 12 SCD patients with hemoglobin (Hb) SS on hydroxurea and 9 normal subjects with Hb AA of both sexes and of different ages were randomly recruited. To assess the effects of S-NACH on the sickling, the SS blood samples were incubated under hypoxia (2% O₂ gas, balance N₂ gas) at 37°C for 1.5 h, in the absence (control) or presence of 1, 5, or 10 ug/mL of S-NACH or LMWH. For the in vivo study, we obtained pre-treatment samples from Townes' SCD mice (n=6 mice/treatment group) and treated the mice subcutaneously with PBS or 30-100 mg/kg S-NACH. Two hours after treatment, blood samples were evaluated for the percentage of sickled cells in pre- and post-administration samples using Leishman's stain and wet smears.

Incubation with S-NACH in vitro under hypoxia showed a dose-dependent, significant inhibition of sickling (up to 80%) in samples from all subjects while LMWH showed no anti-sickling effect. S-NACH had no effect on the osmotic fragility of both AA and SS RBCs. Importantly, we observed a 40-50% decrease in levels of circulating sickled cells in treated SCD mice, an effect that persisted for up to 6 h. Our in vitro studies show that the direct anti-sickling effect is partly due to dose-dependent modification of Hb S to the high-affinity adduct form and the corresponding increase in oxygen affinity, as demonstrated with cation HPLC and oxygen equilibrium analyses. Summarily, our previous findings showed the efficacy of S-NACH as anti-adhesive and anti-inflammatory in SCD, and our current results demonstrate the direct anti-polymerization action of S-NACH on sickle RBCs. Our data document for the first time the supplemental direct anti-sickling effects of a novel S-NACH derivative, suggesting a rational mode of action for these effects and make a compelling case for future studies. Planned detailed structural studies of our S-NACH derivatives complexed with Hb are expected to further illuminate the anti-sickling properties. Our novel Nanoformulated S-NACH for subcutaneous and oral administration would facilitate broader investigation of this promising molecule with multiple modes of action in animal models, with relatively quick translation to successful studies in individuals with SCD.