2-Fluorofucose (2FF) blocks the fucosylation and the tethering of sialyl-Lewisx tetrasaccharide and structural variants on leukocytes and red blood cells to P- and E-selectins on activated endothelial cell surfaces. Because P- and E-selectin are required for vaso-occlusion in murine sickle cell disease (SCD), we investigated whether 2FF would inhibit vaso-occlusion in SCD mice. Microvascular stasis was measured in subcutaneous venules in NY1DD and HbSS-Townes SCD mice with dorsal skin-fold chambers after infusion of hemoglobin or exposure to hypoxia/reoxygenation. 2FF in drinking water or administered by gavage inhibited stasis in sickle mice in a dose-responsive manner. Significant inhibitory effects on stasis were seen 1 day post-treatment. 2FF treatment of SCD mice also significantly reduced leukocyte rolling and adhesion along the vessel walls of SCD mice and the static adhesion of neutrophils and sickle red blood cells isolated from 2FF-treated SCD mice to resting and activated endothelial cells. Total white blood cell counts increased in response to 2FF. NF-ĸB activation and VCAM-1 and E-selectin expression were inhibited in the livers of SCD mice consistent with an overall decrease in vascular inflammation and ischemia-reperfusion physiology. Pretreatment with 2FF completely eliminated heme-induced lethality in HbSS-Townes mice, consistent with the observed anti-inflammatory and anti-adhesive properties of 2FF in SCD mice. These data suggest that 2FF may be beneficial for preventing or treating vaso-occlusive crises in SCD patients.

Carbon monoxide (CO) at low, non-toxic concentrations has been previously demonstrated to exert anti-inflammatory protection in murine models of sickle cell disease (SCD). However CO delivery by inhalation, CO-hemoglobin infusion or CO-releasing molecules presents problems for daily CO administration. Oral administration of a CO-saturated liquid avoids many of these issues and potentially provides a platform for self-administration to SCD patients. To test if orally-delivered CO could modulate SCD vaso-occlusion and inflammation, a liquid CO formulation (HBI-002) was administered by gavage (10 ml/kg) once-daily to NY1DD and Townes-SS transgenic mouse models of SCD. Baseline CO-hemoglobin (CO-Hb) levels were 1.6% and 1.8% in NY1DD and Townes-SS sickle mice and 0.6% in Townes-AS control mice. CO-Hb levels reached 5.4%, 4.7% and 3.0% within 5 minutes in NY1DD, SS and AS mice respectively after gavage with HBI-002. After ten treatments, each once-daily, hemoglobin levels rose from 5.3g/dL in vehicle-treated Townes-SS mice to 6.3g/dL in HBI-002-treated. Similarly, red blood cell (RBC) counts rose from 2.36 x 106/μL in vehicle-treated SS mice to 2.89 x 106/μL in HBI-002-treated mice. In concordance with these findings, hematocrits rose from 26.3% in vehicle-treated mice to 30.0% in HBI-002-treated mice. Reticulocyte counts were not significantly different between vehicle and HBI-002-treated SS mice implying less hemolysis and not an increase in RBC production. White blood cell counts decreased from 29.1 x 103/μL in vehicle-treated versus 20.3 x 103/μL in HBI-002-treated SS mice. Townes-SS mice treated with HBI-002 had markedly increased Nrf2 and HO-1 expression and decreased NF-κB activation compared to vehicle-treated mice. These anti-inflammatory effects were examined for the ability of HBI-002 (administered orally once-daily for up to 5 days) to inhibit vaso-occlusion induced by hypoxia-reoxygenation. In NY1DD and Townes-SS sickle mice, HBI-002 decreased microvascular stasis in a duration-dependent manner. Collectively, these findings support HBI-002 as a useful anti-inflammatory agent to treat SCD and warrants further development as a therapeutic.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80-102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and hemopexin supplementation. A single infusion of haptoglobin or hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or hemopexin. Haptoglobin and hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

Recent evidence indicates that hemolysis in sickle cell disease (SCD) promotes inflammation via innate immune signaling through toll-like receptor 4 (TLR4). Free heme released by hemolyzed red blood cells can bind to myeloid differentiation factor-2 (MD-2) and activate TLR4 pro-inflammatory signaling on endothelium to promote vaso-occlusion and acute chest syndrome in murine models of SCD. MD-2 is co-expressed with TLR4 on cell membranes, but in inflammatory conditions, soluble MD-2 (sMD-2) is elevated in plasma. sMD-2 levels were significantly increased in human and murine sickle (SS) plasma as compared to normal (AA) plasma. Human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells incubated with human SS plasma had significant increases in pro-inflammatory IL-8, IL-6, and soluble VCAM-1 secretion compared to endothelial cells incubated with AA plasma. The increase in HUVEC IL-8 secretion was blocked by depletion of sMD-2 from SS plasma and enhanced by the addition of sMD-2 to AA plasma. The TLR4 signaling inhibitor, TAK-242, inhibited HUVEC IL-8 secretion in response to SS plasma by 85%. Heme-agarose pull-down assays and UV/Vis spectroscopy demonstrated that heme binds to sMD-2. Hemopexin, a high affinity heme-binding protein, inhibited HUVEC IL-8 secretion induced by SS plasma or SS and AA plasma supplemented with sMD-2. These data suggest that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD.