Murine Model Of Sickle Cell Disease Research Articles

Nitric Oxide Resistance in Priapism Associated with Sickle Cell Disease: Mechanisms, Therapeutic Challenges, and Future Directions.

Patients with sickle cell disease (SCD) display priapism, a prolonged penile erection in the absence of sexual arousal. The current pharmacological treatments for SCD-associated priapism are limited and focused on acute interventions rather than prevention. Thus, there is an urgent need for new drug targets and preventive pharmacological therapies for this condition. This review focuses on the molecular mechanisms linked to the dysfunction of the NO-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) pathway implicated in SCD-associated priapism. In murine models of SCD, reduced NO-cGMP bioavailability in the corpus cavernosum is associated with elevated plasma hemoglobin levels, increased ROS levels that inactive NO, and testosterone deficiency that leads to eNOS downregulation. We discuss the consequences of the reduced cGMP-dependent PDE5 activity in response to these molecular changes, highlighting it as the primary pathophysiological mechanism leading to excessive corpus cavernosum relaxation, culminating in priapism. We also further discuss the impact of intravascular hemolysis on therapeutic approaches, present current pharmacological strategies targeting the NO-cGMP-PDE5 pathway in the penis, and identify potential pharmacological targets for future priapism therapies. In men with SCD and priapism, PDE5 inhibitor therapy and testosterone replacement have shown promising results. Recent preclinical research reported the beneficial effect of treatment with haptoglobin and NO donors. Significant strides have been made in understanding the pathophysiology of SCD-associated priapism. Significance Statement This review discusses the molecular changes that reduce NO-cGMP bioavailability in the penis in SCD and highlights pharmacological targets and therapeutic strategies for the treatment of priapism, including PDE5 inhibitors, hormonal modulators, NO donors, soluble guanylate cyclase stimulators, haptoglobin, hemopexin, and antioxidants.

Apohemoglobin-Haptoglobin Complex Improves Cardiac and Pulmonary Function in Homozygous Townes (HbSS Townes) Mice

The constant ischemia-reperfusion injury and the release of hemoglobin and heme in patients with sickle cell disease (SCD) can affect the heart, lungs, and vasculature. While treatments have improved survival, cardiovascular complications are becoming more prevalent, including pulmonary hypertension and left ventricular diastolic dysfunction. Pulmonary hypertension is associated with increased mortality risk and linked to intravascular hemolysis, leg ulcers, renal insufficiency, iron overload, and liver dysfunction. Chronic anemia leads to heart chamber dilation and left ventricular mass increase, often accompanied by left ventricular diastolic dysfunction, both impacting exercise capacity. Additionally, it can cause Acute Chest Syndrome (ACS), which is characterized by inflammation and blockage of small blood vessels in the lungs. In some cases, the heart may enlarge (cardiomegaly) due to the increased workload it faces in pumping blood through narrowed or obstructed blood vessels. Sudden death is also emerging as a concern, necessitating further cardiac investigations and risk assessment for effective management. These issues are speculated to result from the high amount of hemoglobin and heme released into circulation. In this study, a synthetic protein scavenger for heme and Hb, the apohemoglobin (apoHb) was stabilized with haptoglobin (Hp) to create the apoHb-Hp complex, was used to study the cardiac and pulmonary changes in SCD mice resulting from heme and Hb. Homozygous Townes (HbSS Townes) mice (B6; 129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB)Tow/Hbbtm3(HBG1,HBB)Tow/J) were used as the murine model of SCD, and HbAA Townes mice (homozygous for Hbatm1(HBA)Tow and Hbbtm3(HBG1,HBB)Tow) were used as the phenotypically controls. The results conclusively illustrate the benefits of using apohb-Hb, as it improves cardiac output, decreases pulmonary hypertension, and alleviates the immune response that is expected. In conclusion, our study provides strong evidence supporting the significant cardiovascular implication of heme and hemoglobin. We have demonstrated that apoHb-Hp complex effectively neutralizes these harmful activities, rendering these heme and free Hb nonreactive, leading to a marked reduction in hemolysis induced endothelial activation. These findings have important implications, suggesting that apoHb-Hp complex plays a protective role for cardiac and pulmonary cells exposed to elevated levels of heme and free Hb, a condition commonly seen in SCD. Furthermore, our study's consistency with prior research reinforces the role of apoHb-Hp complex in preventing P-selectin expression. Our SCD mouse model experiments reveal that apoHb-Hp complex effectively ameliorates vaso-occlusion, regardless of whether the vaso-occlusion was triggered by Hb or heme-independent factors like hypoxia-reoxygenation. Notably, the apoHb-Hp complex also demonstrated some efficacy, indicating that the protective effect extends beyond simple heme clearance and TLR4 signaling inhibition. While our results are promising, further experiments are required to gain a deeper understanding of the apoHb-Hp complex activity in both clearance-dependent and independent mechanisms of resolving vaso-occlusion. Overall, our research highlights the potential therapeutic value of the apoHb-Hp complex in combating the detrimental effects of Hb and heme, and offers valuable insights into its protective role in managing vaso-occlusive events associated with conditions like SCD.

Moderate hypoxia induces metabolic divergence in circulating monocytes and tissue resident macrophages from Berkeley sickle cell anemia mice.

Human and murine sickle cell disease (SCD) associated pulmonary hypertension (PH) is defined by hemolysis, nitric oxide depletion, inflammation, and thrombosis. Further, hemoglobin (Hb), heme, and iron accumulation are consistently observed in pulmonary adventitial macrophages at autopsy and in hypoxia driven rodent models of SCD, which show distribution of ferric and ferrous Hb as well as HO-1 and ferritin heavy chain. The anatomic localization of these macrophages is consistent with areas of significant vascular remodeling. However, their contributions toward progressive disease may include unique, but also common mechanisms, that overlap with idiopathic and other forms of pulmonary hypertension. These processes likely extend to the vasculature of other organs that are consistently impaired in advanced SCD. To date, limited information is available on the metabolism of macrophages or monocytes isolated from lung, spleen, and peripheral blood in humans or murine models of SCD. Here we hypothesize that metabolism of macrophages and monocytes isolated from this triad of tissue differs between Berkley SCD mice exposed for ten weeks to moderate hypobaric hypoxia (simulated 8,000 ft, 15.4% O2) or normoxia (Denver altitude, 5000 ft) with normoxia exposed wild type mice evaluated as controls. This study represents an initial set of data that describes the metabolism in monocytes and macrophages isolated from moderately hypoxic SCD mice peripheral lung, spleen, and blood mononuclear cells.

Molecular and cellular effects of in vivo chronic intravascular hemolysis and anti-inflammatory therapeutic approaches

Intravascular hemolysis (IVH) occurs in numerous inherited and acquired disorders, including sickle cell disease (SCD), malaria and sepsis. These diseases display unique symptoms, but often share complications, such as vasomotor dysfunction and pulmonary hypertension. Consequently, in vivo models are needed to study the effects of continuous intravascular hemolytic processes, independently of the molecular alteration or extrinsic factor that leads to erythrocyte destruction. We gave twice-weekly low-dose phenylhydrazine (LDPHZ) to C57BL/6 J mice for 4 weeks, and measured parameters indicative of anemia, hemoglobin-clearance pathways, inflammation and iron turnover, comparing these to those of a murine model of SCD, which displays associated IVH. LDPHZ administration provoked discreet anemia in mice and significant reticulocytosis, in association with hemoglobin/heme-clearance pathway protein depletion. Mice subjected to chronic hemolysis displayed elevated leukocyte counts and plasma levels of interleukin (IL)-1β, TNF-α, IL-6, soluble ICAM-1, endothelin-1 and anti-inflammatory IL-10, closely emulating alterations indicative of systemic inflammatory and endothelial activation in SCD, and confirming chronic IVH in itself as a serious complication. Discreet accelerations in hepatic and splenic iron turnover also occurred in LDPHZ mice, without alterations in liver damage markers. Examining the effects of two therapies on hemolysis-induced inflammation, the administration of hydroxyurea (and to a lesser extent, l-glutamine) significantly abrogated hemolytic inflammation in mice, without apparent inhibition of hemolysis. In conclusion, the isolation of chronic IVH, a common disease mechanism, using this model, may allow the study of hemolysis-specific sequelae at the cellular and systemic level, and the investigation of candidate agents that could potentially counter hemolytic inflammation.

GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model.

The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.

GBT021601 Prevents Microvascular Dysfunction and Improves Hypoxia-Reoxygenation-Induced Vaso-Occlusion in a Murine Model of Sickle Cell Disease

Background: In sickle cell disease (SCD), a single nucleotide change in the β-globin gene coding sequence results in the production of hemoglobin S (HbS), which polymerizes when deoxygenated and leads to red blood cell (RBC) sickling, hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Pharmacological increase of the oxygenated HbS concentration in RBCs has been shown to be a well-tolerated and effective approach to inhibiting HbS polymerization, reducing RBC sickling, and improving oxygen (O2) delivery by reducing anemia and improving blood rheology. GBT021601, a potent second-generation small-molecule HbS polymerization inhibitor, is currently under investigation and has previously been shown to increase hemoglobin (Hb), improve hemolysis, and improve RBC health in a murine model. However, little is known regarding its impact on vaso-occlusion and microvascular dysfunction. Objective: To examine the effects of GBT021601 on SCD-induced microvascular dysfunction, capillary perfusion and density, Hb-O2 oxygen affinity, and Hb and hematocrit levels in a murine model of SCD. Methods: Capillary perfusion was measured by functional capillary density (FCD, a marker of capillary perfusion defined as the number of capillaries with RBCs in transit) in a murine model of SCD with an implanted dorsal window chamber (Figure). HbSS Townes mice were treated with GBT021601 (75 mg/kg) by oral gavage daily for 14 days and were compared with untreated HbSS and HbAA Townes mice. Results: Chronic treatment of HbSS mice with GBT021601 led to increased Hb-O2 affinity, evidenced by a reduction in blood p50 (partial O2 pressure at which Hb is 50% saturated with O2) from 32 mm Hg to 14 mm Hg. GBT021601 significantly reduced anemia by increasing total Hb and hematocrit from 8.3 g/dL and 26% in untreated HbSS mice to 11.2 g/dL and 37% in GBT021601-treated HbSS mice, respectively (P<0.01). Moreover, GBT021601 treatment prevented microvascular dysfunction (reduced RBC velocities and blood flow) in the arterioles and venules of HbSS mice. Increased capillary density is indicative of greater oxygen transport. Challenging HbSS mice with hypoxia (8% O2 for 1 hour) followed by reoxygenation (21% O2) induced vaso-occlusion, causing reduced microvascular blood flow and FCD in untreated mice. However, in GBT021601-treated HbSS mice, microvascular blood flow and FCD were significantly higher than those in untreated HbSS mice. Unlike in untreated HbSS mice, microvascular blood flow and FCD returned to baseline values after 72 hours of reoxygenation in treated HbSS mice. These results suggest that by increasing HbS-O2 affinity and thereby inhibiting HbS polymerization, GBT021601 may attenuate hypoxia-induced vaso-occlusion and impaired capillary perfusion. Furthermore, GBT021601 treatment in HbSS mice reduced both circulating adhesion markers and leukocyte-endothelium interactions in venules after hypoxia and reoxygenation. Conclusions: GBT021601 increased microvascular perfusion, reduced vaso-occlusion, and improved the hypoxia-induced reduction in capillary perfusion in HbSS mice, suggesting that chronic GBT021601 treatment prevents SCD-induced microvascular dysfunction and mitigates the rebound vaso-occlusion induced by hypoxia and reoxygenation in a murine model of SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased Hemoglobin Affinity for Oxygen with GBT021601 Reduces Pulmonary Hypertension and Lung Vascular Inflammation in a Murine Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin gene coding sequence that results in the production of hemoglobin S (HbS). Under conditions of red blood cell (RBC) deoxygenation, HbS polymerizes and leads to RBC sickling, resulting in hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Despite improvements in treatment options, SCD remains a debilitating condition, and most patients experience progressive end-organ damage with associated complications and repeated hospitalizations. Pulmonary hypertension (PH), which is associated with increased mortality, is a common complication in patients with SCD, with nearly 10% of the SCD population reporting PH. PH in patients with SCD is multifactorial and may be caused by hemolysis, nitric oxide scavenging, and/or tissue hypoxia. Pharmacological increase of the concentration of oxygenated HbS (oxyHbS) in RBCs by voxelotor, a first-generation HbS polymerization inhibitor, reduces RBC sickling and preserves oxygen (O2) delivery by reducing anemia and improving blood rheology. Limited data are currently available on whether inhibiting HbS polymerization by increasing oxyHbS concentration may improve end-organ damage and associated complications, including PH. Objective: To examine the effects of GBT021601, a small-molecule, investigational, second-generation HbS polymerization inhibitor, on hemoglobin (Hb)-O2 affinity, PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Methods: Townes HbSS mice treated with GBT021601 (75 mg/kg orally once daily for 14 days) were exposed to acute hypoxia (8% O2) for 1 hour and reoxygenated for 72 hours. Untreated HbAA Townes mice were used as controls. Lung inflammation was assessed in terms of accumulation of inflammatory cells, activation of nuclear factor kappa B subunit 1, and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. To evaluate the impact of GBT021601 treatment on Hb-O2 affinity and anemia, p50 (partial O2 pressure at which Hb is 50% saturated with O2) and hematocrit levels were measured in SCD mice. In vivo pulmonary hemodynamic measurements included pulmonary artery pressure and right atrial pressure, whereas right ventricular hypertrophy was assessed using the Fulton index, which is the ratio of right ventricular weight to the weight of the left ventricle plus septum. Results: The total number of inflammatory cells and the specific numbers of lymphocytes, macrophages, and neutrophils were lower in untreated HbAA Townes mice and in HbSS Townes mice treated with GBT021601 compared with vehicle-dosed HbSS mice (Figure, panel A). Chronic treatment with GBT021601 increased Hb-O2 affinity, reducing the p50 in SCD mice from 32 mm Hg to 14 mm Hg. The treatment significantly reduced anemia, increasing hematocrit from 27.2% in the vehicle-dosed HbSS mice to 38.5% in the mice treated with GBT021601. Chronic GBT021601 treatment in HbSS mice also reduced pulmonary artery and right atrial pressure compared with those in untreated HbSS mice. Additionally, GBT021601 reduced right ventricular hypertrophy in HbSS mice, as evidenced by a reduction in the Fulton ratio (Figure, panel B). Conclusions: GBT021601 reduced PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Our findings suggest that reducing RBC sickling, anemia, and hemolysis by increasing HbS affinity for O2 plays a critical role in modulating the immune system in PH and lung vasculature. These findings highlight the potential therapeutic efficacy of GBT021601 based on its ability to increase HbS affinity for O2 and prevent HbS polymerization and RBC sickling to reduce PH in SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Thrombophilia in Sickle Cell Disease with Isoquercetin: Results of a Fixed Dose, Single Center, Phase II Randomized Clinical Trial

Introduction: Sickle cell disease (SCD) is characterized by elevated markers of hemostatic activation and thrombophilia manifesting as arterial stroke and venous thromboembolism (VTE). Anticoagulation for VTE in SCD is associated with an increased risk for bleeding. In cancer patients at high risk for VTE, the flavonoid Isoquercetin (IQ) lowered D-dimer and soluble P-selectin possibly by inhibiting plasma protein disulfide isomerase. In a murine model of SCD, IQ with Hydroxyurea (HU) reduced platelet accumulation and fibrin deposition following laser-induced injury. Given these results, we tested the hypothesis that IQ would be relatively safe and reduce thrombo-inflammatory biomarkers in steady state SCD patients. Methods: We conducted a phase II randomized, double blind, placebo-controlled study at the NIH Clinical Center. Eligible patients had SCD, were 60 days remote from an acute VOC or blood transfusion and were on a stable dose of HU. Study subjects (n=46) were randomly allocated to receive either Isoquercetin 1000 mg daily (n= 23) or placebo (n= 23) for 28 days and were monitored at the beginning and at completion of study. The primary endpoint of the study was the ELISA measured change in plasma sP-selectin levels after 28 days of placebo or study drug exposure. Analysis was performed on an intention to treat principle. D-dimer levels and Thrombin anti-thrombin (TAT) complex were measured by Immunoturbidimetric assays and ELISA. Statistical analyses was performed using analysis of covariance (ANCOVA) and Wilcoxon rank-sum test. Results: The mean (± SD) age of the study population was 40 years (± 11.57), and 56% were female. The majority had HbSS disease and were receiving HU therapy. A subset (28%) reported a prior history of venous thrombosis (VTE) and four of these were on extended duration oral anticoagulation (Table 1). Baseline characteristics between the two study groups were relatively similar. For the primary endpoint, at the end of study the effect of IQ on the change in plasma sP-selectin levels after adjusting for the baseline was not significant (Figure 1) (IQ mean change= 0.10 ± 6.53 vs. placebo mean change= 0.74 ± 4.54; p= 0.66). Since treatment with HU could confound a treatment effect of IQ in a small sample, we compared the change of sP-selectin among those receiving and not receiving HU but did not find any significant difference (p= 0.67). In a sensitivity analysis we did not find an interaction between HU and IQ treatment (p= 0.48). A similar examination for the potential role of VTE history (n= 5 in IQ and n= 8 in placebo arm) as a confounder did not show a VTE effect on change in sP-selectin (p= 0.68) nor an interaction between VTE history and treatment (p= 0.65). In addition to the primary outcome, we explored the effect of IQ treatment on traditional biomarkers of thrombosis. In contrast to studies in patients with cancer, IQ exposure for 28 days did not have a noticeable effect on change from baseline in either D-dimer levels (IQ mean change= 0.15 mcg/ml ± 0.92 vs. placebo mean change= -0.10 mcg/ml ± 2.11; p= 0.82) or thrombin-antithrombin (TAT) complexes (IQ mean change= 0.09 ng/ml ± 1.60 vs. placebo mean change= -0.05 ng/ml ±1.29; p= 0.57). Conclusion: Short term Isoquercetin exposure in steady state SCD did not appreciably lower plasma sP-selectin but was well tolerated, safe and did not have bleeding side effects. Clinical studies of higher dose IQ with greater exposure duration may yield more measurable effects on antithrombotic activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GBT021601 Increases Hemoglobin and Improves Red Blood Cell Deformability without Rebound Hyperviscosity upon Drug Clearance in a Sickle Mouse Model

Background: Whole-blood viscosity and red cell deformability are markedly abnormal in sickle cell disease (SCD) due to sickling of red blood cells (RBCs). Increased whole-blood viscosity is a risk factor for vaso-occlusive crisis (VOC), avascular necrosis, and proliferative retinopathy. Blood viscosity is strongly impacted by hemoglobin (Hb) levels and RBC deformability. GBT021601 is a potent next-generation sickle hemoglobin (HbS) polymerization inhibitor, with anti-sickling properties, that increases the oxygen affinity of Hb. In preclinical studies, treatment with GBT021601 achieved higher levels of Hb than the US Food and Drug Administration-approved first-generation compound voxelotor, raising concern that this rise in Hb would substantially increase whole-blood viscosity and impose a VOC risk after treatment cessation. Objective: To investigate a potential rebound hyperviscosity effect from GBT021601 cessation in a murine model of SCD by determining the effect on whole-blood viscosity and on RBC deformability under a range of oxygen concentrations using oxygen gradient ektacytometry. Methods: Nine-week-old Townes HbSS mice were fed chows containing 0.2% GBT021601 for 5 weeks, then transitioned to control chow. Whole blood was collected in an EDTA tube. All tests were conducted within 4 hours of collection. Using an Element HT5 hematology analyzer (Heska), complete blood count was obtained from mice while on GBT021601 (day 0) and then on days 1, 3, and 5 after transition to control chow. Blood viscosity was measured with a cone/plate viscometer (AMETEK Brookfield) at 37 °C at shear rates of 45 s-1 and 225 s-1. Hematocrit to viscosity ratio (HVR) was calculated to determine RBC oxygen-carrying capacity. RBC deformability was measured by oxygen gradient ektacytometry (Lorrca, RR Mechatronics). Results: Treatment with GBT021601 significantly increased Hb from 8.0 to 12.9 g/dL (P<0.0001) without increasing HVR. Compared with previous studies with the voxelotor analog GBT1118, Hb dropped off more slowly with GBT021601 treatment; this allowed for the ideal assessment of whole-blood viscosity and HVR when the drug had cleared, and HbS was no longer modified, despite elevated Hb levels. HVR at both 45 s-1 and 225 s-1 showed no significant change with drug clearance. GBT021601 treatment also improved RBC deformability, as indicated by a higher elongation index (EI) under both normoxic (EImax, P<0.001) and hypoxic (EImin, P<0.0001) conditions. Point of sickling (PoS) was also reduced (P<0.0001), indicating that treated RBCs tolerated lower oxygen concentrations before sickling. When the drug was withdrawn, PoS and EImin returned to baseline levels, whereas EImax remained elevated until day 5 off treatment. Hb also remained elevated until day 5 off treatment, despite the return to baseline levels of the EImin, PoS, and p50 (partial oxygen pressure at which Hb is 50% saturated with oxygen), indicating drug clearance. Conclusions: In response to GBT021601, sickle cell mice showed a robust increase in Hb, comparable to that of a HbAS mouse, as well as a marked improvement in PoS and RBC deformability. When normalized to HVR, there was no significant difference in blood viscosity during treatment and after cessation of treatment. This finding suggests that oxygen-carrying capacity is not impaired and provides no indication for rebound hyperviscosity with drug clearance. Moreover, the observation that EImax remained elevated until 5 days after drug cessation suggests that some residual rheological improvement occurred in the RBC population that did not dissipate when the drug was cleared. In vitro addition of GBT021601 (and other related compounds) to blood rapidly increases the EImin and reduces the PoS of RBCs but does not alter the EImax. In contrast, an increase in the EImax of RBCs has been consistently observed with GBT021601 treatment in vivo, including in early clinical studies in patients with SCD. The precise mechanism by which this benefit is obtained requires further study. Funding: Global Blood Therapeutics.

P-Selectin Blockage Potentiates the Inhibiting Effects of Inflammasome Cytokine Neutralization on Leukocyte Recruitment in an In Vivo Model of Acute Sickle Cell Vaso-Occlusion

The vaso-occlusive (VO) processes that characterize sickle cell disease (SCD) pathophysiology result from a complex interplay between altered blood rheology, inflammatory mechanisms, pan-cellular activation and adhesive interactions between red blood cells, leukocytes (WBCs), platelets and the endothelium. Inhibition of the binding of the P-selectin (CD62P) adhesion molecule, expressed on activated endothelium and platelets, in the form of crizanlizumab monoclonal antibody (mAb) therapy, has demonstrated documented success for preventing VO crisis (VOC) in SCD patients (N Engl J Med (2017) 376(5):429-39), leading to marketing authorization by numerous agencies. In turn, blockade of interleukin (IL)-1β activity, in the form of canakinumab administration, was well tolerated in SCD patients and associated with inflammatory marker reduction in a multicenter phase 2a study (Blood (2022)139(17):2642-52), with in vivo studies suggesting a role for the inflammasome-processed cytokines, IL-1β and IL-18, in amplifying vaso-occlusion in SCD mice (Blood Abstract: (2021) 138 (S1):856). As targeting multiple mechanisms may enhance VOC prevention in SCD, we evaluated the combined effects of P-selectin blockage, together with IL-1β and/or IL-18 neutralization, on WBC recruitment during tumor necrosis factor-α (TNF)-induced VO processes in SCD mice. Townes mice (5-mo old) received (i.p.) either saline, anti-murine IL-1β mAb (01BSUR IgG2a; 200 µg/mouse), and/or anti-murine IL-18 (SK113AE-4 IgG1; 500 µg/mouse), and/or anti-murine CD62P (RB40.34 IgG1, BD; 30 µg/mouse), or an IgG1 antibody (iProt105125 or A110-1, BD). Previous dosing intravital microscopy (IM) protocols determined the optimal concentrations of antibodies in TNF-stimulated Townes mice (doses tested in µg/mouse; 100, 200 anti-IL-1β [N=3; 4]; 250, 500 anti-IL-18, [N=3; 4]; 10-60 anti-CD62P [N=4-7 each]; data not shown). VO-like processes were induced in mice at 30 min after anti-CD62P treatment or 21h post anti-IL1β / anti-IL-18 administrations by TNF injection (0.5μg, i.p.). At 3h after TNF, the cremaster muscles of the anesthetized mice were surgically exposed, and the rolling, adhesion and extravasation of WBCs in 6-10 venules each were observed using IM. As expected, anti-CD62P administration significantly inhibited the rolling of leukocytes along venule walls of TNF-induced SCD mice, but did not significantly affect WBC adhesion (Fig. 1A). In contrast, the administration of anti-IL-1β mAb significantly inhibited both WBC rolling and adhesion, when compared to saline (Fig 1B); likewise, IL-18 neutralization significantly reduced WBC rolling and adhesion at the venule walls, when compared to IgG1 (Fig. 1C). As previously demonstrated, the combined administration of anti-IL-1β and anti-IL-18 did not further reduce TNF-induced WBC rolling or adhesion in the microvasculature, compared to anti-IL-18 alone (Fig. 1D). Importantly, the co-administration of anti-CD62P, together with either anti-IL1β or anti-IL-18, further inhibited WBC rolling (Fig. 1B and C) and amplified the effect of IL-18 neutralization on WBC adhesion (Fig. 1C). Finally, CD62P inhibition in association with the neutralization of both IL1β and IL-18 further reduced WBC rolling in a non-significant manner and incurred a further significant decrease in the adhesion of WBCs (Fig. 1D). As regards WBC extravasation from cremaster venules, when evaluating the effect of CD62P inhibition together with either anti-IL1β or anti-IL-18, the additive effect observed was not significant (P>0.05; N=4-5); however, CD62P inhibition further reduced the extravasation of WBC in mice subjected to both IL1β and IL-18 neutralization (reduced by 71.1±8.3%, P=0.002; N=4-5). In conclusion, P-selectin blockage potentiates the inhibiting effects of IL-1β and IL-18 neutralization on TNF-induced WBC adhesion to the microvasculature of a murine model of SCD. This potentiating effect may be mediated by further inhibition of WBC rolling and tethering to the endothelium, but concurrent inhibition of platelet-mediated cellular interactions by P-selectin inhibition should not be ruled out. Given that WBC recruitment to blood vessel walls is associated with the initiation of VO processes, we suggest that the combined use of biological therapies may boost these approaches’ individual beneficial effects on SCD VOC. Financial support: Novartis Pharma AG. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased Susceptibility for Adverse Reactions to Ultrasound Enhancing Agents in Sickle Cell Disease

Pain-related adverse events (AEs) to ultrasound enhancing agents (UEAs) have been reported in patients with sickle cell disease (SCD). The aims of this study were to characterize the scope of these AEs in the SCD population and to investigate potential mechanisms on the basis of pathways involved in SCD vaso-occlusive crisis (VOC) and pain. The prevalence and classification of AEs were analyzed from two clinical trials in which high-dose Definity infusions were used in patients with SCD (n=55) or matched control subjects (n=43) to study muscle or myocardial microvascular perfusion. Because complement (C') activation can trigger VOC in SCD, C' activation and surface adhesion of C' proteins on lipid UEAs were studied invitro. C'-mediated UEA attachment to bone marrow immune cells was assessed using flow cytometry in a murine SCD model (Townes mice). Blood from patients receiving Definity was obtained to measure specific lysophospholipid metabolites of lipids in Definity thought to mediate SCD pain. Moderate or greater AEs, all of which were nociceptive (back or bone pain), occurred in one control subject and nine SCD subjects (2% vs 16%, P=.02). Patients with SCD who had AEs tended to have more severe manifestations of SCD. Three of the subjects with SCD had previously received Definity without complications. In patients with SCD, four AEs were classified as severe in intensity and as serious AEs on the basis of need for medical intervention. AEs were described to be similar to SCD-related pain, but there was no evidence for VOC, hemolysis, hypotension, or hypoxemia. At baseline, markers of C' activation were greater in patients with SCD than control subjects. However, after administration of lipid UEAs, SCD and control subjects were similar with regard to C' activation response, anaphylatoxin production, bone marrow microbubble retention, and production of lysophospholipids. There was a trend toward increased deposition of C3b and C3bi on lipid UEAs exposed to serum from patients with SCD. Patients with SCD are particularly susceptible to nociceptive AEs when given Definity at high doses. The mechanism for these AEs remains unclear but most are not related to the triggering of classic VOC.

Interleukin-1 receptor antagonism leads to improved anaemia in a murine model of sickle cell disease and is associated with reduced ex vivo platelet-mediated erythrocyte sickling.

Sickle cell disease (SCD) is associated with haemolytic anaemia and secondary activation of leucocytes and platelets, which in turn may further exacerbate haemolysis. As cytokine signalling pathways may participate in this cycle, the present study investigated whether pharmacological blockade of the interleukin-1 receptor (IL-1R) would mitigate anaemia in a murine model of SCD. Within 2 weeks of treatment, reduced markers of haemolysis were observed in anakinra-treated mice compared to vehicle-treated mice. After 4 weeks of anakinra treatment, mice showed increased numbers of erythrocytes, haemoglobin, and haematocrit, along with reduced reticulocytes. Blood from anakinra-treated mice was less susceptible to ex vivo erythrocyte sickling and was resistant to exogenous IL-1β-mediated sickling. Supernatant generated from IL-1β-treated platelets was sufficient to promote erythrocyte sickling, an effect not observed with platelet supernatant generated from IL-1R-/- mice. The sickling effect of IL-1β-treated platelet supernatant was inhibited by a transforming growth factor-β (TGF-β) neutralising antibody, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition, and superoxide scavengers, but replicated by recombinant TGF-β. In conclusion, pharmacological IL-1R antagonism leads to improved anaemia in a murine SCD model. IL-1β stimulation of platelets promotes erythrocyte sickling. This effect may be mediated by platelet-derived TGF-β-induced reactive oxygen species generation though erythrocyte NADPH oxidase.

Ftx-6058 Induces Fetal Hemoglobin Production and Ameliorates Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a genetic disorder of the red blood cells caused by a mutation in the HBB gene, which results in red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications. Increasing HbF has the potential to prevent or reduce disease-related pathophysiology, including the risk of recurring events such as hemolysis and VOCs. Individuals with SCD who also have hereditary persistence of fetal hemoglobin (HPFH) and exhibit HbF levels of 25 - 30% are often asymptomatic from their SCD, underscoring the protective effect of increased HbF in SCD. Preclinical results with FTX-6058 demonstrate robust target engagement and corresponding increases in HbF levels up to approximately 40% of total hemoglobin, demonstrating the potential to have a significant impact in people living with SCD.FTX-6058 is an investigational, potent, and selective oral small-molecule inhibitor of Embryonic Ectoderm Development (EED) that has been demonstrated to induce robust fetal hemoglobin (HbF) protein expression in cell and murine models of SCD. Here, we report the in vivo effects of EED modulation, and subsequent polycomb repressive complex 2 (PRC2) inhibition in the Townes mouse model of Sickle Cell Disease. Compared with untreated and hydroxyurea treated animals, FTX-6058 (QD, 5mg/kg) exhibits potent target engagement as evidenced by ~70% reduction in H3K27me3 levels, the key epigenetic mark catalyzed by PRC2. Consistent with the robust target engagement observed with FTX-6058, a 2 - 3 fold increase in F cells and HbF protein were observed after 13 and 21 days of FTX-6058 treatment, which was pharmacologically superior to the fetal hemoglobin induction observed with 100 mg/kg hydroxyurea (~25% relative increase in F cells and HbF protein). A linear correlation was also observed between F cells and HbF protein production with FTX-6058 treatment, further supporting the robust HbF induction observed. Consistent with SCD disease presentation, Townes model mice have high reticulocyte counts as a result of premature RBC destruction and hemolysis. Townes model mice treated for 21 days with FTX-6058 ameliorated hemolysis as evidenced by decreases in reticulocytes and increases in red blood cells and total hemoglobin levels. Furthermore, FTX-6058 demonstrated the ability to impact inflammatory drivers of disease as evidenced by decreases in neutrophils and white blood cells. FTX-6058 did not have effects on other hematopoietic lineages. FTX-6058 also impacted pathophysiologic symptoms (e.g., splenomegaly) associated with SCD, as evidenced by ~25% reduction in spleen weight. While some trends were detected, no statistically significant changes were observed in hematological parameters or pathophysiologic symptoms with hydroxyurea, the current standard of care in SCD. These results observed with hydroxyurea underscore the need for new, novel therapies in SCD and other hemoglobinopathies.Taken together, these studies further support the therapeutic rationale of PRC2 modulation in SCD. The fetal hemoglobin induction and effects on hematological parameters and pathophysiologic symptoms observed with FTX-6058 have the potential to translate to meaningful clinical benefits in SCD and other hemoglobinopathies. DisclosuresMatson: Fulcrum Therapeutics, Inc.: Current Employment. Xie: Fulcrum Therapeutics, Inc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Roth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Stuart: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Bruno: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Efremov: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Thompson: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Silver: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Moxham: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Daily Users of Marijuana with Sickle Cell Disease Report More Severe Pain Crisis Yet Have Similar Rates of Admission to Non-Users

▪Introduction: Pain in sickle cell disease (SCD) starts as episodes of recurrent pain and progresses to chronic pain with age. Pain is typically treated with opioids which are often insufficient. One reason for this may be that opioids likely do not effectively treat neuropathic or inflammatory pain in SCD. Cannabinoids, such as marijuana, alleviate neuropathic pain and reduce inflammation. Our SCD patients report marijuana improves their pain and studies in murine models of SCD show cannabinoids reduce mast cell activation, inflammation, and hyperalgesia. Medical marijuana is now legal in 28 states and 4 of them list SCD as a qualifying condition. However, there are minimal data on the use of marijuana in SCD. Thus, we performed a pilot study of adults with SCD who use daily marijuana. Our hypothesis was that daily marijuana users would have worse pain scores from non-daily users which drives their drug use. Our secondary hypotheses were that daily marijuana users may have decreased levels of opioid use, hospital utilization, quality of life measures (QoL) or inflammation.Methods: We conducted a cross-sectional pilot study of adults living with SCD who do and do not use daily marijuana. Subjects were enrolled at scheduled clinic visits, any subject not at their heathy baseline was excluded. Subjects were asked if they had used marijuana in the past 30 days, reasons for use, and rate of use. Subjects who reported daily use were compared to those who used less often or reported no marijuana use. Our primary outcome was pain as defined by Adult Sickle Cell Quality of Life measurement information system (ASCQ-Me) pain domains. ASCQ-Me has separate domains for pain crisis frequency, pain crisis severity, and 7-day pain interference which allows for measurement of both crisis pain and chronic pain. Our secondary outcomes were differences in hospital utilization, opioid use, QoL measures included in ASCQ-ME: sleep, stiffness, social impact and emotional impact, Patient Reported Outcome Measurement Information System domains for nausea and anxiety, and inflammation as demonstrated by WBC and platelet counts, C reactive protein (CRP), markers of mast cell and neutrophil activation and endothelial damage. Hospital utilization (total annual admissions and emergency room visits) was assessed by medical record review over a one-year period (1/1/2016 to 12/31/2016). Weekly opioid use was estimated by review of dispensed prescriptions using the Connecticut Prescription Monitoring Program converted into average weekly oral morphine equivalents. Flow cytometry was performed for levels of: neutrophils and activated neutrophils, monocytes, non-classical and inflammatory monocytes, circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs). The R statistical software program was used to perform t-tests and Wilcoxon rank-sum tests where normality was not met to compare daily users to non-daily users. Significance was set at p<0.05.Results: We enrolled 29 subjects,16 (55%) of whom reported marijuana use in the past 30 days and of those 6 (21%) reported daily marijuana use. Pain relief was endorsed as a reason for marijuana use in 93% of users and 100% of daily users. There was no difference in mean age (32.0 SD 8.6 vs 32.4 SD 12.6 years p=1.0) or gender (% female: 67 vs 52% p=0.7) between groups. Daily marijuana users had higher pain crisis severity scores (61.7 vs 50.0 p=0.03). There was no significant difference in scores for pain crisis frequency, pain interference, or other QoL measures (Table). Daily users had similar median annual admissions and emergency room visits to non-daily users (1.0 (IQR 0.25-1.0) vs 2.0 (IQR 0 - 5.0) admissions p=0.3; 1.0 (IQR 0.25-1.75) vs 0 (IQR 0-1.5) ED visits p=0.6) and used similar doses of weekly opioids (274.5 [IQR 91.5-3447.9] vs 28.6 [IQR 3.03-135.6] mg weekly po morphine equivalents p=0.5). There were no differences in CRP, CBC, CECs or EPCs, or in levels of all neutrophils and monocytes types.Conclusion: In our pilot study, we found daily marijuana users reported more severe pain crisis, but had similar rates of hospital utilization and similar amounts of opioids dispensed. We posit that patients with more severe pain crisis may use daily marijuana to effectively treat their pain allowing them to have similar hospital and opioid utilization to those with less severe pain. Larger prospective studies should be performed to test these hypotheses. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cardiomyopathy Associated with Worsening Intravascular Hemolysis in Transgenic Knock-in Mouse Model of Sickle Cell Disease

Cardiopulmonary complications are the leading cause of death in sickle cell disease (SCD). Cardiac complications, including left ventricular (LV) dysfunction, are prevalent in both young and adult SCD patients. Our studies in the Townes transgenic murine model indicate mice with homozygous SCD (SS) recapitulate the age-related clinical deterioration seen in humans. This is evidenced by the sudden decline in survival of SS mice during the transition from adolescence to adulthood (between the ages of three to six months); we have previously determined that the severity of intravascular hemolysis increases during this transition period in SS mice (Ghosh et al JCI Insight 2016; 1(4): e81090). Hitherto, the cardiac phenotype of this murine model of SCD, which appears the most versatile, has not been defined. In this study, we imaged the hearts of one, three, and six month old Townes SS mice and littermates with sickle cell trait (AS mice) using high resolution ultrasound (Vevo 2100, VisualSonics). Standard indices of LV systolic function, size, and mass, were derived from measurements made on short axis B-mode images of the heart. Ejection fraction and fractional shortening were similar for AS and SS mice, signifying preservation of systolic function in both groups up to 6 months of age. The LV mass, LV internal dimensions (LVID), systolic and diastolic volumes as well as stroke volume significantly increased with age in the SS but not in the AS. Major differences in the cardiac phenotype in SS compared to AS littermates were observed only at six-months. At six months, the SS mice had larger hearts ( 236.5 ± 20.1 mg, n=7, p=0.005) compared to AS littermates (180.2 ± 9.6 mg, n=6). The LVID in both systole (3.1 ± 0.2 mm versus2.1± 0.3, p=0.03) and diastole (4.7 ± 0.2 mmversus 3.9 ± 0.1 mm, p=0.002) were larger in the SS mice, accompanied by higher LV Mass Index (4.6 ± 0.2, n=7 versus 3.1 ± 0.3, n= 6 , p=0.03) indicating LV hypertrophy in the SS mice. RT-PCR analysis of hypertrophy markers in the LV revealed increased expression of Atrial Natriuretic Peptide (ANP, p=0.001) and β-Myosin Heavy Chain (p=0.03) genes in the SS mice compared to AS mice. In agreement with findings in humans, at 6 months, systolic volume (38.4± 6.6 µ L, n=7,versus 17.2±5.3 µ L, n=6, p=0.003) , diastolic volume (101.3± 8.4 µ L, n=7, versus 62.3 ± 4.6 µ L , n=6, p=0.0002), stroke Index (2.2± 0.2, n=7, versus 1.5±0.2, n=6, p=0.03)and cardiac index (1.0±0.1, n=7, versus 0.7±0.1, n=6, p=0.047)were all higher in the six-month SS mice compared to AS littermates. To evaluate vascular inflammation in the heart, P-selectin was measured by ELISA; Compared to AS littermates, SS mice hearts showed a 2-fold higher expression of P-selectin (p=0.009). The severity of anemia and intravascular hemolysis increased with aging in the SS mice. Our study shows progression of LV enlargement and dilation in the Townes SS mice concomitant with increased severity of intravascular hemolysis with aging. Future studies are aimed at delineating the specific molecules and cognate pathways downstream of intravascular hemolysis that promote cardiomyopathy in SCD. DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties.

Soluble MD-2 and Heme in Sickle Cell Disease Plasma Promote Pro-Inflammatory Signaling in Endothelial Cells.

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.

Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

The Mitochondrion: A Link Between Hemolysis and Platelet Activation

Patients with Sickle Cell Disease (SCD) demonstrate characteristics of chronic hemostatic activation including elevated baseline platelet activation. While it is well established that platelet activation is positively correlated with the magnitude of erythrocytic hemolysis in these patients, the mechanisms linking hemolysis to platelet activation remain unclear. In this study, we investigate the role of the platelet mitochondrion as the molecular link between hemolysis and downstream platelet activation. Using extracellular flux analysis, we demonstrate that platelets isolated from patients with SCD show a distinct alteration in mitochondrial function characterized by a decrease in the activity of the mitochondrial ATP Synthase (electron transport chain Complex V), which leads to enhanced mitochondrial membrane potential and elevated reactive oxygen species (ROS) production by the electron transport chain. Notably, levels of platelet mitochondrial ROS production was significantly correlated with markers of hemolysis (lactate dehydrogenase and plasma free hemoglobin concentration) as well as platelet activation in this cohort. Moreover, the mitochondrial dysfunction observed in platelets from SCD patients was recapitulated by exposing healthy platelets to cell-free hemoglobin ex vivo. Importantly, augmented mitochondrial ROS generation appears to initiate platelet activation, as scavenging of mitochondrial ROS inhibited cell free hemoglobin-induced activation of healthy platelets. Studies in the Berkley (BERK) transgenic murine model of SCD supported ex vivo data demonstrating that hemolysis-induced mitochondrial ROS production stimulates platelet activation. Homozygous BERK mice showed significantly inhibited platelet mitochondrial complex V activity and elevated mitochondrial ROS production, concomitant with enhanced baseline platelet activation compared to hemizygote mice. When subjected to a ferric chloride-induced vascular injury model of thrombosis, BERK homozygous mice showed a significantly shorter time to vascular occlusion due to thrombosis compared to hemizygote mice. Treatment of homozygous BERK mice with the mitochondrial ROS scavenger MitoTEMPO (supplemented in drinking water) significantly increased time to vascular occlusion in the ferric chloride thrombosis model. These ex vivo human platelet data and in vivo studies in murine models demonstrate that hemolysis induces platelet mitochondrial ROS production, which stimulates downstream platelet activation. Ongoing studies are focused on determining the molecular mechanisms by which cell free hemoglobin elicits an inhibition of platelet mitochondrial Complex V and implicate a role for activation of the platelet surface toll like receptor 4 (TLR4). Collectively, these data identify the platelet mitochondrion as an important signaling hub that links hemolysis to platelet activation in SCD patients. The potential of therapeutically modulating platelet mitochondrial ROS production to attenuate hemolysis induced thrombotic activation will be discussed. Disclosures No relevant conflicts of interest to declare.

Sensitization of nociceptors by prostaglandin E2–glycerol contributes to hyperalgesia in mice with sickle cell disease

AbstractPain is a characteristic feature of sickle cell disease (SCD), 1 of the most common inherited diseases. Patients may experience acute painful crises as well as chronic pain. In the Berkley transgenic murine model of SCD, HbSS-BERK mice express only human hemoglobin S. These mice share many features of SCD patients, including persistent inflammation and hyperalgesia. Cyclooxygenase-2 (COX-2) is elevated in skin, dorsal root ganglia (DRG), and spinal cord in HbSS-BERK mice. In addition to arachidonic acid, COX-2 oxidizes the endocannabinoid 2-arachidonoylglycerol (2-AG) to produce prostaglandin E2 (PGE2)–glycerol (PGE2-G); PGE2-G is known to produce hyperalgesia. We tested the hypotheses that PGE2-G is increased in DRGs of HbSS-BERK mice and sensitizes nociceptors (sensory neurons that respond to noxious stimuli), and that blocking its synthesis would decrease hyperalgesia in HbSS-BERK mice. Systemic administration of R-flurbiprofen preferentially reduced production of PGE2-G over that of PGE2 in DRGs, decreased mechanical and thermal hyperalgesia, and decreased sensitization of nociceptors in HbSS-BERK mice. The same dose of R-flurbiprofen had no behavioral effect in HbAA-BERK mice (the transgenic control), but local injection of PGE2-G into the hind paw of HbAA-BERK mice produced sensitization of nociceptors and hyperalgesia. Coadministration of a P2Y6 receptor antagonist blocked the effect of PGE2-G, indicating that this receptor is a mediator of pain in SCD. The ability of R-flurbiprofen to block the synthesis of PGE2-G and to normalize levels of 2-AG suggests that R-flurbiprofen may be beneficial to treat pain in SCD, thereby reducing the use of opioids to relieve pain.

Medical Marijuana for Sickle Cell Disease: Results of Two Years of Certification in an Adult Sickle Cell Center

Background: The hallmark of sickle cell disease (SCD) is pain, which can be both acute and chronic. Pain in patients with SCD leads to hospital admissions and diminishes quality of life. Pain in patients with SCD is primarily treated with opioid medications, which are often insufficient. In 2017 the Committee on the Health Effects of Marijuana of the National Academies of Science, Engineering, and Medicine reported that there is “conclusive or substantial evidence that cannabis or cannabinoids are effective for the treatment of chronic pain in adults”, but the report made no mention of chronic pain in patients with SCD. Currently, 30 states and Washington D.C. have medical marijuana (MM) laws, and 5 states list SCD as a qualifying condition. SCD was added to the list of qualifying conditions in Connecticut in February 2016. Since then we have offered certification to patients in our program who make regular clinic visits and for whom we think MM will be safe. However, not all patients who are certified go on to complete the state regulatory paperwork and obtain MM. We hypothesized that those who had obtained MM would show an improvement in clinical outcomes compared to those patients who had been certified but not obtained MM.Methods: All patients who requested certification were educated on safety risks of MM. We conducted a review of all certified patients using our electronic medical record and the Connecticut Prescription Monitoring Program, which provides dispensing reports for schedule I-IV medications including MM. Our primary outcome was admissions in the 6 months after compared to the 6 months before obtaining MM, or the date certified for those who did not obtain MM. Our secondary outcomes were acute care services utilization (emergency department and outpatient infusion center visits) for the treatment of acute pain, and opioid use. Baseline hospital admissions, acute services utilization, and daily opioid use were defined as use in the 6 months prior to obtaining MM or certification for those who did not obtain MM. Opioid use was calculated as total oral morphine equivalents (OME) dispensed in a 6-month period and expressed as OME per day. Previous marijuana use was defined as 1 or more urine studies positive for cannabinoids before MM certification. Genotype was divided into more clinically severe (HbSS/Hbβ0) or less severe (HbSC/Hbβ+/Persistent HbF). We compared admissions, acute services, and opioid use for those who obtained MM to those who did not using a difference in differences analysis. Baseline admissions, baseline acute services, baseline opioid use, age, gender, genotype, hydroxyurea use, previous marijuana use, and insurance type of those who obtained MM were compared to those who did not using Student's t tests.Results: 52 patients requested certification, and 50 patients were certified. 2 patients were not certified due to concerns about inappropriate use based on their past history. Twenty-eight patients who were certified obtained MM; 22 did not. Baseline hospital admissions, acute services utilization, and daily opioid were similar between the two groups. Patients who obtained MM were more likely to be genotype HbSS/HbSβ0. Age, gender, hydroxyurea use, previous marijuana use, and insurance type were similar between the two groups. (Table 1). Patients obtained MM a median of 109.5 days (IQR 54.8 - 188.8) after certification. Two patients concurrently started L-Glutamine within 6 months of MM certification, one who obtain MM and one who did not. Obtaining MM was associated with a decrease in admission rates in the next 6 months compared to those who did not obtain MM (-1.1 admissions 95% CI -0.1 - -2.0, p=0.03). Obtaining MM was not associated with a significant change in acute services utilization (0.3 visits 95% CI -1.4 - 1.9, p=0.8) or daily opioid use (-0.1 OME 95% CI -19.6 - 19.3, p= 1.0).Conclusion: Though there was no difference in admission rates between the two groups we examined prior to MM certification, and the only clinical difference was that those who obtained MM were more likely to be in the moresevere genotype, those who obtained MM showed a decrease in admissions in the next 6 months compared to those who did not. We suggest that MM allowed patients to improve their pain relief and thereby reduce admission rates. This is consistent with data showing cannabinoid agonists improve pain in murine models of SCD. Larger, randomized and controlled studies of MM for pain in SCD should be conducted. DisclosuresNo relevant conflicts of interest to declare.