New Therapeutic Options for the Treatment of Sickle Cell Disease

Alessandro Matte¹, Francesco Zorzi¹, Filippo Mazzi¹, Enrica Federti¹, Oliviero Olivieri¹ and Lucia De Franceschi¹.

 ¹ Department of Medicine, University of Verona and AOUI Verona, Verona. Italy

Introduction

Sickle cell disease (SCD) is a hemoglobinopathy which affects approximately 100,000 individuals in the United States and almost 20,000-25,000 subjects in Europe, mainly immigrants from endemic areas such as Sub-Saharan Africa to European countries.[1-3] Estimates of the number of affected newborn in 2010 are of approximately 312,302 subjects with 75.5% being born in Africa.[4] The invalidating impact of SCD on patient survival, quality of life and cost for health systems,[2] requires the development of new therapeutic options to treat sickle cell related acute and chronic complications.
SCD is caused by a point mutation in the β-globin gene resulting in the synthesis of pathological hemoglobin S (HbS). HbS displays peculiar biochemical characteristics, polymerizing when deoxygenated with associated reduction in cell ion and water content (cell dehydration), increased red cell density and further acceleration of HbS polymerization (Figure 1).[5-7]

---

Figure 1. Schematic diagram of the mechanisms involved in the pathogenesis of acute sickle cell related vaso-occlusive events. These involve the adherence of sickle red blood cells (RBCs) or reticulocytes and neutrophils to the abnormally activated endothelial cells, with the participation of activated and phosphatidyl- Serine (PS)-rich platelets (PLTs), activation of the coagulation system, and activation of a cytokine storm. PS: Phosphatidyl-Serine; TSP: thrombospondine; vWF: von Willebrand factor; BCAM/LU: Lutheran blood group protein; ICAM-4: Landstein-Weiner (LW) blood group glycoprotein; MPs: microparticles; Mac1: β2 integrins (αMβ2 or CD11b/CD18); ESL-1: neutrophil E- selectin ligand -1; Hb: hemoglobin; ROS: reactive oxygen species; iNKT: invariant natural killer T cells; ET-1: endothelin-1; NO: nitric oxide (modified from De Franceschi L et al. Seminars in Thrombosis, 37: 266; 2011).

---

Pathophysiological studies have shown that dense, dehydrated red cells play a central role in acute and chronic clinical manifestations of SCD, in which intravascular sickling in capillaries and small vessels leads to vaso-occlusion and impaired blood flow with ischemic/reperfusion injury.[5,8-10] In microcirculation, vaso-occlusive events (VOC) result from a complex and still partially known scenario, involving the interactions between different cell types, including dense red cells, reticulocytes, abnormally activated endothelial cells, leukocytes, platelets and plasma factors (Figure 1).[5,9-13] Acute VOCs have been associated with increased expression of pro-adhesion molecules such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) or selectins (Figure 1).[5,9,11,12,14,15] These molecules are important in recruitment and adhesion of both neutrophils and sickle red cells to the abnormally activated vascular endothelial surface.[11,16] In addition, the presence of free Hb and free heme contribute to the local reduction of nitric oxide (NO) bioavailability, establishing an endovascular high pro- oxidant and pro-inflammatory environment. This is associated with modulation of innate immunity and increased iNKT lymphocytes, increase levels of vascular active cytokines such as endothelin 1, combined with the final contribution of platelets (Figure 1).[5,9,14,17-20]

Hydroxyurea is the Gold- Standard Treatment for Sickle Cell Disease

Hydroxyurea or hydroxycarbamide (HU) is the key therapeutic tool for SCD approved by Food and Drug Administration (FDA) and European Medical Agency (EMEA). US and European guidelines highlighted that HU should be available for all SCD patients from pediatric to adult populations.[21,22]
Studies in SCD show a multimodal action of HU, which (i) increases HbF production, resulting in delayed HbS polymerization; (ii) reduces hemolysis and increase NO availability targeting cGMP production; (iii) modulates endothelial activation and reduces neutrophil counts, contributing to the reduction of chronic inflammation (Figure 2).[23-27] Long-term use of HU has been shown to be safe and well-tolerated in large cohorts of children and adults with SCD, reducing mortality and morbidity of both children and adult patients.[21,28-31] Indeed, HU reduces (i) the frequency of VOC and the rate of hospitalization; (ii) the incidence of ACS; (iii) the transfusion requirements; and (iv) the severity of dactilitis in SCD pediatric population.[21,32-36] HU might also be used in combination with transfusion regimen in selected SCD population such as SCD children with progressive cerebrovascular disease in the absence of antigen- matched sibling donor.[37] Furthermore, recent reports propose HU as acceptable alternative to chronic transfusion regimen in SCD patients with history of abnormalities at the transcranial doppler scan (TCD), used to screen for cerebrovascular disease in pediatric patients.[38-40] This requires a close follow-up by TCD scan every 3 months, with the possibility to switch-back to chronic transfusion regimen if abnormal transcranial velocities are again documented.[38-40] Noteworthy, increase reticulocyte count before HU treatment and high leukocyte count after HU have been identified as risk factor for reversion to abnormal TCD velocities in SCD pediatric patients. Thus, again chronic inflammation and vasculopathy seems to be key determinants of severe chronic complications in SCD.[38-40]

---

Figure 2. Schematic diagram of multimodal therapeutic action of hydroxyurea (HU) in sickle cell disease. ROS: reactive oxygen species; Hb: hemoglobin; NO: nitric oxide; HbS: sickle hemoglobin; HbF: fetal hemoglobin.

---

Although HU should be available for all SCD subjects, the major limitation is the poor adherence of adults SCD patients to HU therapy. Different studies have identified multiple factors to be involved in reduced adherence of SCD patients to HU such as (i) chronicity of the treatment; (ii) socio-economic reasons; and (iii) adhesion barriers related to the transition from pediatric to adult care system.[41-44]
The dissemination of the use of HU is particularly important in underdeveloped countries with high incidence of SCD such as in the sub-Saharan African areas.[45] Recently, Opoka et al. reported safety of use for HU at the dosage of ~20 mg/Kg/d in African children from Uganda, a malaria endemic area (NOHARM study, NCT01976416).[46] This study further supports the importance of HU as a front-line medical treatment for SCD patients all over the world. Noteworthy, in geographical context where frequent hematologic monitoring is not available, Toya et al. have recently reported the beneficial effects of low dose HU (10 mg/Kg/d) on SCD acute clinical manifestations in Nigerian patients.[47]

Novel Therapeutic Approaches to Treat Sickle Cell Disease

In the last two decades, the availability of mouse models for SCD has allowed both characterization of the pathogenesis of sickle cell related organ damage(s) and identification of pathophysiology- based new therapeutic options in addition to HU.[5,7,11,12,48-50]
As shown in Table 1, pathophysiology related novel therapies for SCD can be divided into:
•    Agents which reduce/prevent sickle red cell dehydration or red cell sickling or HbF inducers;
•    Agents targeting SCD vasculopathy and sickle cell- endothelial adhesive events;
•    Anti-oxidant agents.

---

Table 1. Targets in SCD and Experimental Treatments.

---

Agents Which Reduce/Prevent Sickle Red Cell Dehydration and Sickling. Different agents targeting sickle red cells have been developed to prevent or limit HbS polymerization or to block the mechanism(s) involved in red cell dehydration.[14,18,19,48,51-55] Targeting the reduction of circulating dense red cells and/or sickled red cells is very important, since these cells are easily trapped in microcirculation and participate to the pathogenesis of acute VOC.
Recent reports indicate GBT440, an oral direct anti-sickling agent, to be beneficial in SCD. GBT440 (or voxelotor) blocks HbS intermolecular contacts, preventing the generation of HbS fibers and red cell sickling.[56-60] GBT440 has been shown (i) to ameliorate in vitro SCD red cell features such as red cell deformability or viscosity and (ii) to improve sickle red cell survival with decrease reticulocyte count.[56-60] Preliminary data on phase I/II double blind placebo study with GBT440 in healthy volunteers and few SCD patients show safety and tolerability of GBT440 associated with an amelioration of hemolytic indices and a reduction in reticulocyte count (#NCT02285088).[55,61,62] Blyden et al. have reported the compassionate use of voxelotor, at the dosage of 900 mg/d up to 1500 mg/d for 24 weeks in a small group of subjects with severe untreatable SCD. Voxelotor beneficially impacts SCD patient well-being with a reduction in number of hospitalization for severe VOC compared to patient’s clinical history.[63] These data further support the on-going phase III clinical trial on voxelotor in a larger population of SCD patients (HOPE; #NCT03036813).

Agents Targeting SCD Vasculopathy and Sickle Cell-Endothelial Adhesive Events. SCD is not only a hemolytic anemia but also a chronic inflammatory disorder characterized by abnormally activated vascular endothelial cells, amplified inflammatory response, and the release of soluble factors, which promote abnormal adhesive interactions between erythrocytes, endothelial cells, and neutrophils.[5,7,10,12,64,65] An increased number of circulating, abnormally activated endothelial cells has been identified in SCD patients during acute VOCs, indicating the presence of chronic vasculopathy, worsened by acute events.[66] Thus, SCD is characterized by a chronic inflammatory vasculopathy that favors the recruitment of leukocytes and the entrapment of dense red cells with the generation of heterotypic aggregates (thrombi) with ischemic/reperfusion local damage.
In this context, the major objectives of therapeutic strategies targeting sickle cell vasculopathy are to reduce or prevent vascular endothelial activation and damage. The end-point of anti-adherence therapy, alternatively, is to interfere with the initialization and/or amplification of adhesive events.
In SCD, agents targeting SCD vasculopathy and sickle cell-endothelial adhesive events (Figure 3) can be divided into:
i.    Molecules targeting hemolysis-induced vasculopathy;
ii.   Agents that modulate the abnormal vascular tone;
iii.  Agents interfering with red cell vascular adhesion events.

---

Figure 3. Schematic diagram of the mechanisms of action of pathophysiology based new therapeutic options for treatment of sickle cell disease and sickle cell vasculopathy. Hp: haptoglobin; Hx: hemopexin; NAC: N-Acetyl-cysteine; Ab: antibody; ROS: reactive oxygen species; iNKT: invariant natural killer T cells; NKTT120: humanized monoclonal antibody specifically depleting iNKT; NO: nitric oxide; ET-1: endothelin- 1; ET-R: endothelin-1 receptor.

---

i. Molecules targeting hemolysis-induced vasculopathy. The chronic hemolytic anemia of SCD is for one-third intravascular and for two-third extravascular, via the reticulo-endothelial systems. Free Hb is present in the peripheral circulation of SCD patients, reacting with plasma nitric oxide (NO) with production of reactive oxygen species (ROS) and generation of MetHb. This is a key step for the release of free heme.[9,67,68]
The physiological systems binding free Hb or free heme are haptoglobin (Hp) and hemopexin (Hx), respectively.
In SCD patients, both Hp and Hx levels are significantly reduced in steady state compared to healthy controls; they further decrease during acute VOCs.[67,69] The highly pro-oxidant environment with the presence of free heme and free Hb promotes inflammation and abnormal vascular activation with increased expression of adhesion vascular molecules such as VCAM-1, ICAM-1 or E-selectin.[67,69] Studies in mouse models for SCD have shown that free heme induces vascular stasis and leukocyte extravasation with the trapping of dense red cells and neutrophils in microcirculation.[70-72]
In human SCD patients, free Hb and free heme increase during acute VOCs with further reduction in Hp and Hx levels (Figure 1).[72,73] Noteworthy, Hp levels correlate with pulmonary hypertension,[67] suggesting that the blockage of free-Hb by Hp might possibly affect SCD related organ damage. In mouse models for SCD, the infusion of Hp has been shown to prevent vascular stasis. Encouraging data from small, in vivo human studies with infused Hp show that Hp protects the kidneys from free Hb-related tubular damage in patients who have undergone cardiopulmonary surgery or endoscopic sclerotherapy.[67] Few case reports are present in the literature on the use of Hp in patients with hemolytic crisis and inherited red cell disorders.[67,74] Thus, Hp might be as a possible new therapeutic tool to be further explored in SCD.
In the complex scenario of the pathogenesis of SCD vasculopathy, Hx, a high affinity heme binding protein, represents another interesting molecule that might be explored as a novel therapeutic option (Figure 1). The supplementation of Hx in mouse models for SCD has been shown to reduce heme induced oxidative stress, vascular endothelial injury, inflammation, and vascular stasis.[9] Recently, a link between increased free heme and complement activation has been reported in cell- and animal-based model for SCD.[75] Hx significantly reduces complement deposition in kidney from humanized SCD mice, highlighting the importance of controlling free heme plasma level as additional tool to limit inflammatory vasculopathy and related severe organ damage in SCD. The importance of optimal levels of Hp and Hx is also supported by a recent report on the use of therapeutic plasma exchange in SCD with severe VOC, resistant to red cell exchange.[76]
Further studies need be carried out to develop and understand the potential clinical use of Hp and/or Hx in management of severe complication related to excess of free heme in SCD patients.
ii. Agents that modulate the abnormal vascular tone. Vascular tone results from the balance between vaso-dilatatory factors such as nitric oxide (NO) and vaso-constrictor factors such as the endothelin-1 (ET-1) system (Figure 1).[18,77-81] In SCD, reduced NO local bioavailability, a consequence of the presence of free Hb, contributes to chronic vaso-constriction and amplifies the expression of vascular adhesion molecules.[77,82,83] In addition, the release of arginase in peripheral circulation by sickle red cells during chronic hemolysis, subtracts arginine from the urea cycle in endothelial cells, and further contributes to NO deficiency.[77,82-85] Plasma NO metabolites are decreased in SCD patients during acute VOCs and decreased exhaled NO has also been reported. Thus, therapeutic strategies to supplement or modulate NO might beneficially interfere with the pathogenesis of acute SCD related clinical manifestations such as VOCs. Initial trials showed some positive, encouraging effects of inhaled NO on acute VOCs.[82,86,87] However, a subsequent multicentric, double-blind, randomized placebo-controlled study in SCD with VOCs using inhaled NO showed no clinically significant effects.[82] New NO donors such as nitrate or NCX1443 need to be further evaluated in humanized animal-based pre- clinical studies.[83,88] Another possible strategy to increase NO production in SCD is the supplementation of L- Arginine. Oral L-Arginine (i) decreases artery pulmonary pressure in SCD; (ii) improves leg ulcers; and (iii) contributes in pain control in SCD.[84,85,89] The co-administration of L-Arginine with HU has been reported to increase levels of nitrate, suggesting L- Arginine as an adjuvant molecule in treatment of SCD.[84,85,89]
Endothelin-1 is a potent vasoconstrictor and bronchoconstrictor, whose plasma and urinary values are increased in SCD subjects in steady state and during acute VOCs.[18,90,91] In a mouse model for SCD, the ET-1 receptors’ blocker, bosentan, prevented hypoxia induced organ damage and affect neutrophil mediated inflammatory response, suggesting the modulation of the ET-1 system as an additional therapeutic option to interfere with the pathogenesis of SCD related clinical manifestation(s).[18,92,93] It is of interest to note that increased ET-1 and high ET-1 levels have been shown to positively correlate with pain rating in children with SCD.[94] This has been recently investigated in humanized mouse model for SCD, showing that endothelin receptor-type A might be involved in inflammatory mediated pain component throughout the modulation of Nav1.8 channel in primary sensing neurons.[95]
iii. Agents interfering with red cell vascular adhesion events. In SCD, anti-adherence therapeutic strategies might represent an interesting, novel therapeutic strategy to prevent the generation of acute VOCs and to lessen SCD related organ damage (Figure 1 and 3). The anti-adherence therapeutic options might be divided into three groups based on their mechanism of action:
a)    Molecules interfering with the physical properties of the red cell-endothelial adhesion process;
b)    Molecules specifically interfering with sickle cell- endothelial adhesive mechanisms;
c)    Molecules modulating inflammatory pathways involved in sickle cell endothelial adhesion;
d)    Molecules affecting platelet function.
a) Molecules interfering with the physical properties of the red cell-endothelial adhesion process. RheothRx (Poloxamer 188), a non-ionic surfactant copolymer was shown to improve microvascular blood flow by lowering viscosity and frictional forces. RheothRx was shown some beneficial effects on pain intensity and duration of hospitalization in a pilot study on SCD patients experiencing moderate to severe vaso- occlusive crisis.[96] RheothRx was tested in a phase III clinical study for treatment of VOCs in SCD. Although P188 has been shown to shorten the duration of pain crisis, its effects on acute events were limited.[97,98] Mast Therapeutics announced in 2016 negative results for a new phase III trial with Vepoloxamer (MST-188), a IV agent tested to assess its effect on reducing the duration of vaso-occlusive crises.[99]
b) Molecules interfering with sickle cell-endothelial adhesive mechanisms. Recent studies in SCD have identified different mechanisms involved in sickle cell-endothelium adhesive events, which may be of therapeutic relevance (Figure 1): (i) the integrin α4β1 receptor of fibronectin and the vascular adhesion molecule-1 (VCAM-1), E-selectin and P-selectin; (ii) the thrombospondin and/or collagen and receptor CD36, present on the surface of endothelial cells, platelets and reticulocyte-rich subpopulations of normal and sickle red cells; (iii) the sulfate glycolipids, which bind thrombospondin, von-Willebrand factor multimer and laminin; (iv) the Lutheran blood group proteins (BCAM/LU), whose expression is increased in red cells from SCD patients; (v) the ICAM-4 (Landstein-Weiner blood group glycoprotein-LW), which binds αVβ3 integrin receptors; and (vi) the exposure of PS detectable in a subpopulation of sickle red cells, which participates both in cell-cell adhesion to activated vascular endothelium surface and in the activation of a coagulation system. Monoclonal antibodies against the adhesion molecules or short synthetic peptides interfering with ICAM-4 or αVβ3 integrin have been shown to reduce adhesion events in SCD mouse models (Figure 1). It is of interest to note that antibodies against adhesion molecules block the heme induced vascular stasis, supporting again the connection between heme, vasculopathy, and adhesion events in SCD.[68,100,101] Among the agents interfering with red cell vascular adhesion events, the blockade of adhesion mechanisms through interference with Selectins seems to be a novel powerful therapeutic option for clinical management of SCD. Selectins are a family of molecules mediating adhesion of blood cells with activated vascular endothelial cells. and play a key role in leukocyte recruitment as well as in sickle red cell adhesion to inflammatory activated vascular endothelium. In addition, studies have shown that P-selectin are increased in plasma of SCD patients.[65,102-106] Different therapeutic strategies have been developed, to block selectins: (i) pan-Selectin antagonist (GMI-1070, rivipansel); (ii) humanized anti-P-Selectin antibody (SelG1, crinalizumab); (iii) P-selectin-aptamer; and (iv) sevuparin.[11,12,15,50,65,104,107-112] Rivipansel is a glycomimetic pan-selectin antagonist, which was tested in phase-I and -II studies in SCD. Rivipansel showed a safe profile, reducing the levels of E-Selectin in SCD patients during acute VOCs.[107,113] In phase II study, rivipansel beneficially affected the number of pain crisis in a small number of SCD subjects (#NCT01119833). However, these data were obtained including some SC patients, which generates some difficulties in their interpretation. An on-going phase II study is focused on SCD children.
Crinalizumab is a humanized P-Selectin antibody, which has been tested in a multinational double-blind placebo-controlled trial (SUSTAIN, #NCT0185361).[15,111] SCD subjects (SS, SC, Sβ⁺ and Sβ⁰ genotype) were treated with crinalizumab either 2.5 or 5 mg/Kg every 4 weeks. Crinalizumab at the dosage of 5 mg/Kg every 4 weeks reduced the number of pain crisis and increased the time between VOCs in SCD independently from possible preceding HU treatment.[15,111,112]
An additional strategy targeting P-Selectins is represented by the use of low molecular weight heparins, such as tinzaparin, which has been shown to block the P-Selectin system and to reduce the duration and the severity of VOCs in few cases of SCD patients.[12,50] Sevuparin is a derivative of low-molecular weight heparin, lacking anticoagulant activity and it has been evaluated in SCD.[109,114] Sevuparin acts on multiple targets: (i) P and L-selectins; (ii) thrombospondin- Fibronectin-Von Willebrand factor; and (iii) sickle- leukocyte-endothelial cells interaction. A phase II multicenter international trial on sevuparin in acute VOCs is ongoing.
c) Molecules modulating inflammatory pathways involved in sickle cell endothelial adhesion. Another set of novel therapeutic option is represented by agents modulating the inflammatory pathways that participate to adhesion events in SCD.
Studies in different models of hypoxia/reoxygenation stress have shown that adenosine is released from cells and interacts with A (1-3) receptors (AR), which are present on endothelial cells, leukocytes and iNKT cells. This promotes the activation of the transcriptional factor NF-kB, which orchestrates the inflammatory response. iNKT are a subgroup of T lymphocytes that affects both innate and adaptive immunity, participating to inflammatory cascade.[115-117] ncreased iNKT circulating cells have been observed in SCD subjects on both steady state and during acute VOCs. Antibodies against iNKT cells (NKTT120) have been developed, based on the key role that adhesion and inflammation are involved in the pathogenesis of severe acute complication of SCD (#NCT01783691).[15,99,115] Field et al. recently reported the failure of regadenoson in reducing iNKT activation and in interfering with the severity of the acute clinical manifestations of SCD patients enrolled in randomized phase II clinical trial (#NCT01788631).[118]
An attempt to target inflammatory vasculopathy and to modulate inflammatory response has been made based on the evidences in other diseases such as in cardiovascular disease looking to dietary manipulation with omega-3 fatty acids (ω-3 PUFAs). Supplementation with omega-3 fatty acids has been reported to (i) beneficially affect red cell membrane lipid composition; (ii) modulate soluble and cellular inflammatory response and coagulation cascade; and (iii) to favor NO production.[119-122] In SCD, the fatty acid profile of sickle erythrocytes is altered compared to healthy controls, with a relative increase in the ratio of ω-6 to ω-3 PUFAs, in agreement with sustained chronic inflammation.[123,124] In humanized mouse model for SCD, PUFA supplementation protects against acute sickle cell-related lung and liver damages during hypoxia/reoxygenation induced VOCs.[14] A phase II multicenter randomized double-blind placebo- controlled study in SCD patients reported that ω-3 fatty acid supplementation reduced pain episode in SCD subjects (SCOT, #NCT02973360).[125-128]
d) Molecules affecting platelet function. The role of platelets in clinical manifestations of SCD on both steady state and acute events has been only partially characterized and much still remains to be investigated.[5,11,50] Early evidence on the beneficial effects of ticlopidine on reducing the rate of pain crisis highlighted the potential role of platelet activation and aggregation during acute events in SCD.[129] However, a multicentric phase 2 study on prasugrel, a third- generation anti-platelet agent, in adult with SCD showed a reduction of platelet activation without change in pain rate.[130] Recently, ticagrelor, a direct anti-platelet agent with some effects on vascular tone and inflammatory response has been evaluated in a dose-finding study on SCD children (HESTIA1, #NCT02214121).[11,131] Ticagrelor was well tolerated without significant drug related adverse events, in particular no hemorrhagic events were reported. Noteworthy, in SCD children ticagrelor induced platelet inhibition similar to that reported in adults with acute coronary disease.[131] A phase III clinical trial with ticagrelor in adults with SCD is on-going (#NCT02482298).[11,131]

Anti-Oxidant Agents and Sickle Cell Disease. SCD is also characterized by a highly pro-oxidant environment due to the elevated production of reactive oxygen species (ROS) generated by increased levels of pathological free heme and iron and a reduction in anti- oxidant systems such as GSH (Figure1).[5,7,12,132,133] N- Acetyl-Cysteine (NAC), an exogenous thiol donor, has been studied both in vitro and in vivo in SCD patients. NAC supplementation (1,200-2,400 mg/day) was shown to reduce the formation of dense red cells and the rate of hemolysis and to increase GSH levels in SCD subjects. However, Sins et al. have recently reported a randomized, placebo-, double-blind trial (#NCT01849016) on NAC in SCD. Although the study shows a failure of NAC in affecting acute clinical manifestations of SCD, the Authors point out that the low adherence of SCD to NAC treatment might be responsible for the reduced biological effect of NAC in SCD. A clinical trial with high dose of NAC during acute VOCs related to SCD is ongoing (#NCT 01800526).[134-136]
L-Glutamine is a likely anti-oxidant agent in SCD. Glutamine is involved in GSH metabolism since it preserves NADPH levels required for GSH recycling, and it is the precursor for nicotinamide adenine dinucleotide (NAD) and arginine.[137-139] A first randomized, double blind, placebo-controlled parallel group trial with L-glutamine supplementation in SCD patients showed reduction in number of hospitalization compared to historic patients data.[138] Recently, a multicenter, randomize, placebo-controlled double- blind phase III clinical trial with L-glutamine (0.3 g/Kg twice a day) involving 230 SS/Sbeta0 patients with > 2 pain crisis showed that L-glutamine supplementation reduced the mean number and length of hospitalization, associated with increased median time to the first crisis.[137] Both studies have several limitations such as (i) the high rate of patient drop-out; (ii) the presence of fatal events due to multiorgan failure in L-glutamine arm; (iii) the lack of effects on hematologic parameters and hemolytic indices; and (iv) the absence of clear data on L-glutamine mechanism of action.[137,140] Since no information are available on log-term use of L- glutamine supplementation as well on the systemic effects of L-glutamine, the sickle cell scientific community should use caution in prescribing L- glutamine supplement for both adult and pediatric SCD patients.[140]  Future studies are required to further define the role of anti-oxidant treatments in the clinical management of SCD subjects.

Curative Options in Sickle Cell Disease

Conclusions

Acknowledgments

Competing Interests and Funding

1. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organization 2008;86:480-487. https://doi.org/10.2471/BLT.06.036673 PMid:18568278 PMCid:PMC2647473
   
2. Murray CJ, Vos T, Lozano R, et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012;380:2197-2223. https://doi.org/10.1016/S0140-6736(12)61689-4  
   
3. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem. Bulletin of the World Health Organization 2001;79:704-712. PMid:11545326 PMCid:PMC2566499
   
4. Piel FB, Patil AP, Howes RE, et al. Global epidemiology of sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. Lancet 2013;381:142-151. https://doi.org/10.1016/S0140-6736(12)61229-X 
   
5. De Franceschi L, Cappellini MD, Olivieri O. Thrombosis and sickle cell disease. Semin Thromb Hemost 2011;37:226-236. https://doi.org/10.1055/s-0031-1273087 PMid:21455857
   
6. Eaton WA, Hofrichter J. Sickle cell hemoglobin polymerization. Advances in protein chemistry 1990;40:63-279. https://doi.org/10.1016/S0065-3233(08)60287-9
   
7. De Franceschi L, Corrocher R. Established and experimental treatments for sickle cell disease. Haematologica 2004;89:348-356. PMid:15020275
   
8. Ballas SK, Smith ED. Red blood cell changes during the evolution of the sickle cell painful crisis. Blood 1992;79:2154-2163. PMid:1562742
   
9. Vinchi F, De Franceschi L, Ghigo A, et al. Hemopexin therapy improves cardiovascular function by preventing heme-induced endothelial toxicity in mouse models of hemolytic diseases. Circulation 2013;127:1317-1329. https://doi.org/10.1161/CIRCULATIONAHA.112.130179 PMid:23446829
   
10. Hebbel RP, Vercellotti G, Nath KA. A systems biology consideration of the vasculopathy of sickle cell anemia: the need for multi-modality chemo-prophylaxsis. Cardiovasc Hematol Disord Drug Targets 2009;9:271-292. https://doi.org/10.2174/1871529X10909040271 PMid:19751187 PMCid:PMC2914570
    
11. Telen MJ. Beyond hydroxyurea: new and old drugs in the pipeline for sickle cell disease. Blood 2016;127:810-819. https://doi.org/10.1182/blood-2015-09-618553 PMid:26758919 PMCid:PMC4760087
    
12. Manwani D, Frenette PS. Vaso-occlusion in sickle cell disease: pathophysiology and novel targeted therapies. Blood 2013;122:3892-3898. https://doi.org/10.1182/blood-2013-05-498311 PMid:24052549 PMCid:PMC3854110
    
13. Hebbel RP. Adhesion of sickle red cells to endothelium: myths and future directions. Transfus Clin Biol 2008;15:14-18. https://doi.org/10.1016/j.tracli.2008.03.011 PMid:18501652
    
14. Kalish BT, Matte A, Andolfo I, et al. Dietary omega-3 fatty acids protect against vasculopathy in a transgenic mouse model of sickle cell disease. Haematologica 2015;100:870-880. https://doi.org/10.3324/haematol.2015.124586 PMid:25934765 PMCid:PMC4486221
    
15. Ataga KI, Kutlar A, Kanter J, et al. Crizanlizumab for the Prevention of Pain Crises in Sickle Cell Disease. N Engl J Med 2017;376:429-439. https://doi.org/10.1056/NEJMoa1611770 PMid:27959701 PMCid:PMC5481200
    
16. Hidalgo A, Chang J, Jang JE, et al. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury. Nature medicine 2009;15:384-391. https://doi.org/10.1038/nm.1939 PMid:19305412 PMCid:PMC2772164
    
17. Dalle Carbonare L, Matte A, Valenti MT, et al. Hypoxia-reperfusion affects osteogenic lineage and promotes sickle cell bone disease. Blood 2015;126:2320-2328. https://doi.org/10.1182/blood-2015-04-641969 PMid:26330244
    
18. Sabaa N, de Franceschi L, Bonnin P, et al. Endothelin receptor antagonism prevents hypoxia-induced mortality and morbidity in a mouse model of sickle-cell disease. J Clin Invest 2008;118:1924-1933. https://doi.org/10.1172/JCI33308 PMid:18382768 PMCid:PMC2276396
    
19. Wieschhaus A, Khan A, Zaidi A, et al. Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology. Biochem J 2012;448:141-152. https://doi.org/10.1042/BJ20121008 PMid:22870887 PMCid:PMC3955119
    
20. Siciliano A, Turrini F, Bertoldi M, et al. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease. Blood Cells Mol Dis 2010;44:233-242. https://doi.org/10.1016/j.bcmd.2010.02.007 PMid:20206558
    
21. Yawn BP, Buchanan GR, Afenyi-Annan AN, et al. Management of sickle cell disease: summary of the 2014 evidence-based report by expert panel members. JAMA 2014;312:1033-1048. https://doi.org/10.1001/jama.2014.10517 PMid:25203083
    
22. Engert A, Balduini C, Brand A, et al. The European Hematology Association Roadmap for European Hematology Research: a consensus document. Haematologica 2016;101:115-208. https://doi.org/10.3324/haematol.2015.136739 PMid:26819058 PMCid:PMC4938336
    
23. Platt OS. Hydroxyurea for the treatment of sickle cell anemia. N Engl J Med 2008;358:1362-1369. https://doi.org/10.1056/NEJMct0708272 PMid:18367739
    
24. Yarbro JW. Mechanism of action of hydroxyurea. Semin Oncol 1992;19:1-10. PMid:1641648
    
25. Charache S. Mechanism of action of hydroxyurea in the management of sickle cell anemia in adults. Semin Hematol 1997;34:15-21. PMid:9317197
    
26. Charache S, Terrin ML, Moore RD, et al. Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia [see comments]. New England Journal of Medicine 1995;332:1317-1322. https://doi.org/10.1056/NEJM199505183322001 PMid:7715639
    
27. Saleh AW, Hillen HF, Duits AJ. Levels of endothelial, neutrophil and platelet-specific factors in sickle cell anemia patients during hydroxyurea therapy. Acta Haematol 1999;102:31-37. https://doi.org/10.1159/000040964 PMid:10473885
    
28. Ware RE, de Montalembert M, Tshilolo L, et al. Sickle cell disease. Lancet 2017;390:311-323. https://doi.org/10.1016/S0140-6736(17)30193-9  
    
29. Rigano P, De Franceschi L, Sainati L, et al. Real-life experience with hydroxyurea in sickle cell disease: A multicenter study in a cohort of patients with heterogeneous descent. Blood Cells Mol Dis 2018;69:82-89. https://doi.org/10.1016/j.bcmd.2017.08.017 PMid:29107441
    
30. Pule GD, Mowla S, Novitzky N, et al. A systematic review of known mechanisms of hydroxyurea-induced fetal hemoglobin for treatment of sickle cell disease. Expert Rev Hematol 2015;8:669-679. https://doi.org/10.1586/17474086.2015.1078235 PMid:26327494 PMCid:PMC4829639
    
31. Jison ML, Munson PJ, Barb JJ, et al. Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease. Blood 2004;104:270-280. https://doi.org/10.1182/blood-2003-08-2760 PMid:15031206 PMCid:PMC5560446
    
32. Stettler N, McKiernan CM, Melin CQ, et al. Proportion of adults with sickle cell anemia and pain crises receiving hydroxyurea. JAMA 2015;313:1671-1672. https://doi.org/10.1001/jama.2015.3075 PMid:25919532
    
33. Wong TE, Brandow AM, Lim W, et al. Update on the use of hydroxyurea therapy in sickle cell disease. Blood 2014;124:3850-3857. https://doi.org/10.1182/blood-2014-08-435768 PMid:25287707 PMCid:PMC4271176 
34. Crosby WH, Dameshek W. The significance of hemoglobinemia and associated hemosideriinuria, with particular references to various types of hemolytic anemia. J Lab Clin Med 1951;38:829. PMid:14889070
    
35. Voskaridou E, Christoulas D, Bilalis A, et al. The effect of prolonged administration of hydroxyurea on morbidity and mortality in adult patients with sickle cell syndromes: results of a 17-year, single-center trial (LaSHS). Blood 2010;115:2354-2363. https://doi.org/10.1182/blood-2009-05-221333 PMid:19903897
    
36. Wang WC, Ware RE, Miller ST, et al. Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG). Lancet 2011;377:1663-1672. https://doi.org/10.1016/S0140-6736(11)60355-3 
    
37. Brousse V, Gandhi S, de Montalembert M, et al. Combined blood transfusion and hydroxycarbamide in children with sickle cell anaemia. Br J Haematol 2013;160:259-261. https://doi.org/10.1111/bjh.12104 PMid:23116405
    
38. Bernaudin F, Verlhac S, Arnaud C, et al. Long-term treatment follow-up of children with sickle cell disease monitored with abnormal transcranial Doppler velocities. Blood 2016;127:1814-1822. https://doi.org/10.1182/blood-2015-10-675231 PMid:26851292
    
39. Ware RE, Davis BR, Schultz WH, et al. Hydroxycarbamide versus chronic transfusion for maintenance of transcranial doppler flow velocities in children with sickle cell anaemia-TCD With Transfusions Changing to Hydroxyurea (TWiTCH): a multicentre, open-label, phase 3, non-inferiority trial. Lancet 2016;387:661-670. https://doi.org/10.1016/S0140-6736(15)01041-7 
    
40. Helton KJ, Adams RJ, Kesler KL, et al. Magnetic resonance imaging/angiography and transcranial Doppler velocities in sickle cell anemia: results from the SWiTCH trial. Blood 2014;124:891-898.
    https://doi.org/10.1182/blood-2013-12-545186 PMid:24914136 PMCid:PMC4126329
    
41. Inoue S, Kodjebacheva G, Scherrer T, et al. Adherence to hydroxyurea medication by children with sickle cell disease (SCD) using an electronic device: a feasibility study. Int J Hematol 2016;104:200-207. https://doi.org/10.1007/s12185-016-2027-x PMid:27225236
    
42. Han J, Bhat S, Gowhari M, et al. Impact of a Clinical Pharmacy Service on the Management of Patients in a Sickle Cell Disease Outpatient Center. Pharmacotherapy 2016;36:1166-1172. https://doi.org/10.1002/phar.1834 PMid:27639254 PMCid:PMC5373798
    
43. Green NS, Manwani D, Qureshi M, et al. Decreased fetal hemoglobin over time among youth with sickle cell disease on hydroxyurea is associated with higher urgent hospital use. Pediatr Blood Cancer 2016;63:2146-2153. https://doi.org/10.1002/pbc.26161 PMid:27573582 PMCid:PMC5072999
    
44. Smaldone A, Findley S, Manwani D, et al. HABIT, a Randomized Feasibility Trial to Increase Hydroxyurea Adherence, Suggests Improved Health-Related Quality of Life in Youths with Sickle Cell Disease. J Pediatr 2018;197:177-185 e172.
    
45. Ansong D, Akoto AO, Ocloo D, et al. Sickle cell disease: management options and challenges in developing countries. Mediterr J Hematol Infect Dis 2013;5:e2013062. https://doi.org/10.4084/mjhid.2013.062 PMid:24363877 PMCid:PMC3867228
    
46. Opoka RO, Ndugwa CM, Latham TS, et al. Novel use Of Hydroxyurea in an African Region with Malaria (NOHARM): a trial for children with sickle cell anemia. Blood 2017;130:2585-2593. https://doi.org/10.1182/blood-2017-06-788935  PMid:29051184
    
47. Tayo BO, Akingbola TS, Saraf SL, et al. Fixed Low-Dose Hydroxyurea for the Treatment of Adults with Sickle Cell Anemia in Nigeria. Am J Hematol 2018.
    
48. De Franceschi L. Pathophisiology of sickle cell disease and new drugs for the treatment. Mediterr J Hematol Infect Dis 2009;1:e2009024.
    
49. Stocker JW, De Franceschi L, McNaughton-Smith GA, et al. ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice. Blood 2003;101:2412-2418. https://doi.org/10.1182/blood-2002-05-1433 PMid:12433690
    
50. De Franceschi L, Saadane N, Trudel M, et al. Treatment with oral clotrimazole blocks Ca(2+)-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease. J Clin Invest 1994;93:1670-1676. https://doi.org/10.1172/JCI117149 PMid:7512989 PMCid:PMC294212
    
51. Telen MJ. Developing new pharmacotherapeutic approaches to treating sickle-cell disease. ISBT Sci Ser 2017;12:239-247. https://doi.org/10.1111/voxs.12305 PMid:28484512 PMCid:PMC5418585
    
52. De Franceschi L, Franco RS, Bertoldi M, et al. Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease. FASEB J 2013;27:750-759. https://doi.org/10.1096/fj.12-217836 PMid:23085996 PMCid:PMC3545531
    
53. McNaughton-Smith GA, Burns JF, Stocker JW, et al. Novel inhibitors of the Gardos channel for the treatment of sickle cell disease. J Med Chem 2008;51:976-982. https://doi.org/10.1021/jm070663s PMid:18232633
    
54. De Franceschi L, Brugnara C, Rouyer-Fessard P, et al. Formation of dense erythrocytes in SAD mice exposed to chronic hypoxia: evaluation of different therapeutic regimens and of a combination of oral clotrimazole and magnesium therapies. Blood 1999;94:4307-4313. PMid:10590075
    
55. Li Q, Henry ER, Hofrichter J, et al. Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease. Proc Natl Acad Sci U S A 2017;114:E689-E696. https://doi.org/10.1073/pnas.1619054114 PMid:28096387 PMCid:PMC5293101
    
56. Dufu K, Oksenberg D. GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro. Hematol Rep 2018;10:7419. https://doi.org/10.4081/hr.2018.7419 PMid:30046411 PMCid:PMC6036981
    
57. Metcalf B, Chuang C, Dufu K, et al. Discovery of GBT440, an Orally Bioavailable R-State Stabilizer of Sickle Cell Hemoglobin. ACS Med Chem Lett 2017;8:321-326. https://doi.org/10.1021/acsmedchemlett.6b00491 PMid:28337324 PMCid:PMC5346980
    
58. Oksenberg D, Dufu K, Patel MP, et al. GBT440 increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease. Br J Haematol 2016;175:141-153. https://doi.org/10.1111/bjh.14214 PMid:27378309
    
59. Dufu K OD, Zhou C, Hutchaleelaha A, Archer DR. GTx011, a potent allosteric modifier of hemoglobin oxygen affinity, prevents RBC sickling in whole blood and prolongs RNC half-life in vivo in a murine model of sickle cell disease. In: Blood, editor. American Society of Hematology; 2014. p a217.  
    
60. Patel M CP, Dufu K, Metcalf B, Sinha U. GTx011, an anti-sickling compound, improves SS blood rheology by reduction of HbS polymerization via allosteric modulation of O2 affinity. In: Blood, editor. American Society of Hematology 2014. p a1370.
    
61. Blyden G, Bridges KR, Bronte L. Case series of patients with severe sickle cell disease treated with voxelotor (GBT440) by compassionate access. Am J Hematol 2018.
    
62. Telfer P, Agodoa I, Fox KM, et al. Impact of voxelotor (GBT440) on unconjugated bilirubin and jaundice in sickle cell disease. Hematol Rep 2018;10:7643. https://doi.org/10.4081/hr.2018.7643 PMid:30046415 PMCid:PMC6036983
    
63. Estepp JH. Voxelotor (GBT440), a first-in-class hemoglobin oxygen-affinity modulator, has promising and reassuring preclinical and clinical data. Am J Hematol 2018;93:326-329. https://doi.org/10.1002/ajh.25042 PMid:29352729
    
64. Kato GJ, Hebbel RP, Steinberg MH, et al. Vasculopathy in sickle cell disease: Biology, pathophysiology, genetics, translational medicine, and new research directions. Am J Hematol 2009;84:618-625. https://doi.org/10.1002/ajh.21475 PMid:19610078 PMCid:PMC3209715
    
65. Kato GJ, Piel FB, Reid CD, et al. Sickle cell disease. Nat Rev Dis Primers 2018;4:18010. https://doi.org/10.1038/nrdp.2018.10 PMid:29542687
    
66. Solovey A, Gui L, Ramakrishnan S, et al. Sickle cell anemia as a possible state of enhanced anti-apoptotic tone: survival effect of vascular endothelial growth factor on circulating and unanchored endothelial cells. Blood 1999;93:3824-3830. PMid:10339489
    
67. Schaer DJ, Buehler PW, Alayash AI, et al. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 2013;121:1276-1284. https://doi.org/10.1182/blood-2012-11-451229 PMid:23264591 PMCid:PMC3578950
    
68. Belcher JD, Chen C, Nguyen J, et al. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 2014;123:377-390. https://doi.org/10.1182/blood-2013-04-495887 PMid:24277079 PMCid:PMC3894494
    
69. Reiter CD, Wang X, Tanus-Santos JE, et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002;8:1383-1389. https://doi.org/10.1038/nm1202-799 PMid:12426562
    
70. Belcher JD, Vineyard JV, Bruzzone CM, et al. Heme oxygenase-1 gene delivery by Sleeping Beauty inhibits vascular stasis in a murine model of sickle cell disease. J Mol Med (Berl) 2010;88:665-675. https://doi.org/10.1007/s00109-010-0613-6 PMid:20306336 PMCid:PMC2877767
    
71. Belcher JD, Beckman JD, Balla G, et al. Heme degradation and vascular injury. Antioxid Redox Signal 2010;12:233-248. https://doi.org/10.1089/ars.2009.2822 PMid:19697995 PMCid:PMC2821146
    
72. Muller-Eberhard U, Javid J, Liem HH, et al. Plasma concentrations of hemopexin, haptoglobin and heme in patients with various hemolytic diseases. Blood 1968;32:811-815. PMid:5687939
    
73. Ballas SK, Marcolina MJ. Hyperhemolysis during the evolution of uncomplicated acute painful episodes in patients with sickle cell anemia. Transfusion 2006;46:105-110. https://doi.org/10.1111/j.1537-2995.2006.00679.x PMid:16398738
    
74. Ohga S, Higashi E, Nomura A, et al. Haptoglobin therapy for acute favism: a Japanese boy with glucose-6-phosphate dehydrogenase Guadalajara. Br J Haematol 1995;89:421-423. https://doi.org/10.1111/j.1365-2141.1995.tb03322.x PMid:7873396
    
75. Merle NS, Grunenwald A, Rajaratnam H, et al. Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles. JCI Insight 2018;3.
    
76. de Franceschi L, Malpeli G, Scarpa A, et al. Protective effects of S-nitrosoalbumin on lung injury induced by hypoxia-reoxygenation in mouse model of sickle cell disease. Am J Physiol Lung Cell Mol Physiol 2006;291:L457-465. https://doi.org/10.1152/ajplung.00462.2005 PMid:16603592
    
77. Louie JE, Anderson CJ, Fayaz MFK, et al. Case series supporting heme detoxification via therapeutic plasma exchange in acute multiorgan failure syndrome resistant to red blood cell exchange in sickle cell disease. Transfusion 2018;58:470-479. https://doi.org/10.1111/trf.14407 PMid:29193101
    
78. Kato GJ, Gladwin MT. Evolution of novel small-molecule therapeutics targeting sickle cell vasculopathy. JAMA 2008;300:2638-2646. https://doi.org/10.1001/jama.2008.598 PMid:19066384 PMCid:PMC2756016
    
79. Belcher JD, Young M, Chen C, et al. MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice. Blood 2013;122:2757-2764. https://doi.org/10.1182/blood-2013-02-486282 PMid:23908468 PMCid:PMC4067504
    
80. de Franceschi L, Baron A, Scarpa A, et al. Inhaled nitric oxide protects transgenic SAD mice from sickle cell disease-specific lung injury induced by hypoxia/reoxygenation. Blood 2003;102:1087-1096. https://doi.org/10.1182/blood-2002-07-2135 PMid:12689931
    
81. De Franceschi L, Platt OS, Malpeli G, et al. Protective effects of phosphodiesterase-4 (PDE-4) inhibition in the early phase of pulmonary arterial hypertension in transgenic sickle cell mice. FASEB J 2008;22:1849-1860. https://doi.org/10.1096/fj.07-098921 PMid:18245171
    
82. Gladwin MT, Kato GJ, Weiner D, et al. Nitric oxide for inhalation in the acute treatment of sickle cell pain crisis: a randomized controlled trial. JAMA 2011;305:893-902. https://doi.org/10.1001/jama.2011.235 PMid:21364138 PMCid:PMC3403835
    
83. Kim-Shapiro DB, Gladwin MT. Nitric oxide pathology and therapeutics in sickle cell disease. Clin Hemorheol Microcirc 2018;68:223-237. https://doi.org/10.3233/CH-189009 PMid:29614634
    
84. Bakshi N, Morris CR. The role of the arginine metabolome in pain: implications for sickle cell disease. Journal of pain research 2016;9:167-175. PMid:27099528 PMCid:PMC4821376
    
85. Morris CR. Alterations of the arginine metabolome in sickle cell disease: a growing rationale for arginine therapy. Hematology/ Oncology Clinics of North America 2014;28:301-321. https://doi.org/10.1016/j.hoc.2013.11.008 PMid:24589268
    
86. Weiner DL, Hibberd PL, Betit P, et al. Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive crisis in pediatric patients with sickle cell disease. JAMA 2003;289:1136-1142. https://doi.org/10.1001/jama.289.9.1136 PMid:12622584
    
87. Head CA, Swerdlow P, McDade WA, et al. Beneficial effects of nitric oxide breathing in adult patients with sickle cell crisis. Am J Hematol 2010;85:800-802. https://doi.org/10.1002/ajh.21832  PMid:20799359
    
88. Abid S, Kebe K, Houssaini A, et al. New Nitric Oxide Donor NCX 1443: Therapeutic Effects on Pulmonary Hypertension in the SAD Mouse Model of Sickle Cell Disease. J Cardiovasc Pharmacol 2018;71:283-292. https://doi.org/10.1097/FJC.0000000000000570 PMid:29438213
    
89. Morris CR, Kato GJ, Poljakovic M, et al. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. JAMA 2005;294:81-90. https://doi.org/10.1001/jama.294.1.81 PMid:15998894 PMCid:PMC2065861
    
90. Tharaux PL, Hagege I, Placier S, et al. Urinary endothelin-1 as a marker of renal damage in sickle cell disease. Nephrol Dial Transplant 2005;20:2408-2413. https://doi.org/10.1093/ndt/gfi111 PMid:16144850
    
91. Hammerman SI, Kourembanas S, Conca TJ, et al. Endothelin-1 production during the acute chest syndrome in sickle cell disease. American journal of respiratory and critical care medicine 1997;156:280-285. https://doi.org/10.1164/ajrccm.156.1.9611085 PMid:9230761
    
92. Koehl B, Nivoit P, El Nemer W, et al. The endothelin B receptor plays a crucial role in the adhesion of neutrophils to the endothelium in sickle cell disease. Haematologica 2017;102:1161-1172. https://doi.org/10.3324/haematol.2016.156869 PMid:28385784 PMCid:PMC5566019
    
93. Taylor C, Kasztan M, Tao B, et al. Combined hydroxyurea and ETA receptor blockade reduces renal injury in the humanized sickle cell mouse. Acta Physiol (Oxf) 2018:e13178. https://doi.org/10.1111/apha.13178 PMid:30144292
    
94. Smith TP, Haymond T, Smith SN, et al. Evidence for the endothelin system as an emerging therapeutic target for the treatment of chronic pain. Journal of Pain Research 2014;7:531-545. https://doi.org/10.2147/JPR.S65923 PMid:25210474 PMCid:PMC4155994
    
95. Lutz BM, Wu S, Gu X, et al. Endothelin type A receptors mediate pain in a mouse model of sickle cell disease. Haematologica 2018;103:1124-1135. https://doi.org/10.3324/haematol.2017.187013 PMid:29545351 PMCid:PMC6029538
    
96. Adams-Graves P, Kedar A, Koshy M, et al. RheothRx (poloxamer 188) injection for the acute painful episode of sickle cell disease: a pilot study. Blood 1997;90:2041-2046. PMid:9292541
    
97. Orringer EP, Casella JF, Ataga KI, et al. Purified poloxamer 188 for treatment of acute vaso-occlusive crisis of sickle cell disease: A randomized controlled trial. JAMA 2001;286:2099-2106. https://doi.org/10.1001/jama.286.17.2099 PMid:11694150
    
98. Koo S, Yang Y, Neu B. Poloxamer 188 reduces normal and phosphatidylserine-exposing erythrocyte adhesion to endothelial cells in dextran solutions. Colloids and surfaces B, Biointerfaces 2013;112:446-451. https://doi.org/10.1016/j.colsurfb.2013.07.035 PMid:24055859
    
99. Archer N, Galacteros F, Brugnara C. 2015 Clinical trials update in sickle cell anemia. Am J Hematol 2015;90:934-950. https://doi.org/10.1002/ajh.24116 PMid:26178236 PMCid:PMC5752136
    
100. Kaul DK, Liu XD, Zhang X, et al. Peptides based on alphaV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation. Am J Physiol Cell Physiol 2006;291:C922-930. https://doi.org/10.1152/ajpcell.00639.2005 PMid:16738001
     
101. Kaul DK, Liu XD, Zhang X, et al. Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants. Am J Physiol Heart Circ Physiol 2006;291:H167-175. https://doi.org/10.1152/ajpheart.01096.2005 PMid:16443674
     
102. Pan J, Xia L, McEver RP. Comparison of promoters for the murine and human P-selectin genes suggests species-specific and conserved mechanisms for transcriptional regulation in endothelial cells. J Biol Chem 1998;273:10058-10067. https://doi.org/10.1074/jbc.273.16.10058 PMid:9545353
     
103. Matsui NM, Borsig L, Rosen SD, et al. P-selectin mediates the adhesion of sickle erythrocytes to the endothelium. Blood 2001;98:1955-1962. https://doi.org/10.1182/blood.V98.6.1955 PMid:11535535
     
104. Kutlar A, Ataga KI, McMahon L, et al. A potent oral P-selectin blocking agent improves microcirculatory blood flow and a marker of endothelial cell injury in patients with sickle cell disease. Am J Hematol 2012;87:536-539. https://doi.org/10.1002/ajh.23147 PMid:22488107
     
105. Turhan A, Weiss LA, Mohandas N, et al. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm. Proc Natl Acad Sci U S A 2002;99:3047-3051. https://doi.org/10.1073/pnas.052522799 PMid:11880644 PMCid:PMC122470
     
106. Blann AD, Mohan JS, Bareford D, et al. Soluble P-selectin and vascular endothelial growth factor in steady state sickle cell disease: relationship to genotype. J Thromb Thrombolysis 2008;25:185-189. https://doi.org/10.1007/s11239-007-0177-7 PMid:18080800
     
107. Wun T, Styles L, DeCastro L, et al. Phase 1 study of the E-selectin inhibitor GMI 1070 in patients with sickle cell anemia. PLoS One 2014;9:e101301. https://doi.org/10.1371/journal.pone.0101301 PMid:24988449 PMCid:PMC4079300
     
108. Chang J, Patton JT, Sarkar A, et al. GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice. Blood 2010;116:1779-1786. https://doi.org/10.1182/blood-2009-12-260513 PMid:20508165 PMCid:PMC2947397
     
109. Telen MJ, Batchvarova M, Shan S, et al. Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion. Br J Haematol 2016;175:935-948. https://doi.org/10.1111/bjh.14303 PMid:27549988
     
110. Gutsaeva DR, Parkerson JB, Yerigenahally SD, et al. Inhibition of cell adhesion by anti-P-selectin aptamer: a new potential therapeutic agent for sickle cell disease. Blood 2011;117:727-735. https://doi.org/10.1182/blood-2010-05-285718 PMid:20926770 PMCid:PMC3031491
     
111. Ataga KI, Kutlar A, Kanter J. Crizanlizumab in Sickle Cell Disease. N Engl J Med 2017;376:1796. https://doi.org/10.1056/NEJMoa1611770 PMCid:PMC5481200
     
112. Slomski A. Crizanlizumab Prevents Sickle Cell Pain Crises. JAMA 2017;317:798. https://doi.org/10.1001/jama.2017.0355  
     
113. Telen MJ, Wun T, McCavit TL, et al. Randomized phase 2 study of GMI-1070 in SCD: reduction in time to resolution of vaso-occlusive events and decreased opioid use. Blood 2015;125:2656-2664. https://doi.org/10.1182/blood-2014-06-583351 PMid:25733584 PMCid:PMC4408290
     
114. White J, Lindgren M, Liu K, et al. Sevuparin blocks sickle blood cell adhesion and sickle-leucocyte rolling on immobilized L-selectin in a dose dependent manner. Br J Haematol 2018.
     
115. Field JJ, Nathan DG. Advances in sickle cell therapies in the hydroxyurea era. Mol Med 2014;20 Suppl 1:S37-42.
     
116. Field JJ, Ataga KI, Majerus E, Eaton CA, Mashal R, Nathan DG. A phase I single ascending dose study of NKTT120 in stable adult sickle cell patients. In: Blood, editor. American Society of Hematology; 2014. p a977.
     
117. Field JJ, Nathan DG, Linden J. The role of adenosine signaling in sickle cell therapeutics. Hematology/Oncology Clinics of North America 2014;28:287-299. https://doi.org/10.1016/j.hoc.2013.11.003  PMid:24589267 PMCid:PMC3997263
     
118. Field JJ, Majerus E, Gordeuk VR, et al. Randomized phase 2 trial of regadenoson for treatment of acute vaso-occlusive crises in sickle cell disease. Blood Adv 2017;1:1645-1649. https://doi.org/10.1182/bloodadvances.2017009613 PMid:29296811 PMCid:PMC5728341
     
119. Massaro M, Scoditti E, Carluccio MA, et al. Basic mechanisms behind the effects of n-3 fatty acids on cardiovascular disease. Prostaglandins Leukot Essent Fatty Acids 2008;79:109-115. https://doi.org/10.1016/j.plefa.2008.09.009 PMid:18951002
     
120. Calder PC. Omega-3 fatty acids and inflammatory processes. Nutrients 2010;2:355-374. https://doi.org/10.3390/nu2030355 PMid:22254027 PMCid:PMC3257651
     
121. Rangel-Huerta OD, Aguilera CM, Mesa MD, et al. Omega-3 long-chain polyunsaturated fatty acids supplementation on inflammatory biomakers: a systematic review of randomised clinical trials. Br J Nutr 2012;107 Suppl 2:S159-170.
     
122. Russo C, Olivieri O, Girelli D, et al. Omega-3 polyunsaturated fatty acid supplements and ambulatory blood pressure monitoring parameters in patients with mild essential hypertension. J Hypertens 1995;13:1823-1826. https://doi.org/10.1097/00004872-199512010-00059 PMid:8903660
     
123. Ren H, Obike I, Okpala I, et al. Steady-state haemoglobin level in sickle cell anaemia increases with an increase in erythrocyte membrane n-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids 2005;72:415-421. https://doi.org/10.1016/j.plefa.2005.03.005 PMid:15876528
     
124. Ren H, Ghebremeskel K, Okpala I, et al. Abnormality of erythrocyte membrane n-3 long chain polyunsaturated fatty acids in sickle cell haemoglobin C (HbSC) disease is not as remarkable as in sickle cell anaemia (HbSS). Prostaglandins Leukot Essent Fatty Acids 2006;74:1-6. https://doi.org/10.1016/j.plefa.2005.10.002 PMid:16314081
     
125. Daak AA, Ghebremeskel K, Hassan Z, et al. Effect of omega-3 (n-3) fatty acid supplementation in patients with sickle cell anemia: randomized, double-blind, placebo-controlled trial. Am J Clin Nutr 2013;97:37-44. https://doi.org/10.3945/ajcn.112.036319 PMid:23193009
     
126. Daak AA, Dampier CD, Fuh B, et al. Double-blind, randomized, multicenter phase 2 study of SC411 in children with sickle cell disease (SCOT trial). Blood Adv 2018;2:1969-1979. https://doi.org/10.1182/bloodadvances.2018021444 PMid:30097463 PMCid:PMC6093734
     
127. Daak A, Rabinowicz A, Ghebremeskel K. Omega-3 fatty acids are a potential therapy for patients with sickle cell disease. Nat Rev Dis Primers 2018;4:15. https://doi.org/10.1038/s41572-018-0012-9 PMid:30093627
     
128. Tomer A, Kasey S, Connor WE, et al. Reduction of pain episodes and prothrombotic activity in sickle cell disease by dietary n-3 fatty acids. Thromb Haemost 2001;85:966-974. https://doi.org/10.1055/s-0037-1615948 PMid:11434703
     
129. Cabannes R, Lonsdorfer J, Castaigne JP, et al. Clinical and biological double-blind-study of ticlopidine in preventive treatment of sickle-cell disease crises. Agents and actions Supplements 1984;15:199-212. PMid:6385647
     
130. Heeney MM, Hoppe CC, Abboud MR, et al. A Multinational Trial of Prasugrel for Sickle Cell Vaso-Occlusive Events. N Engl J Med 2016;374:625-635. https://doi.org/10.1056/NEJMoa1512021 PMid:26644172
     
131. Hsu LL, Sarnaik S, Williams S, et al. A dose-ranging study of ticagrelor in children aged 3-17 years with sickle cell disease: a two-part phase 2 study. Am J Hematol 2018. https://doi.org/10.1002/ajh.25273 
     
132. Reid M, Badaloo A, Forrester T, et al. In vivo rates of erythrocyte glutathione synthesis in adults with sickle cell disease. American journal of physiology Endocrinology and metabolism 2006;291:E73-79. https://doi.org/10.1152/ajpendo.00287.2005  PMid:16434557
     
133. Silva DG, Belini Junior E, de Almeida EA, et al. Oxidative stress in sickle cell disease: an overview of erythrocyte redox metabolism and current antioxidant therapeutic strategies. Free Radic Biol Med 2013;65:1101-1109. https://doi.org/10.1016/j.freeradbiomed.2013.08.181  PMid:24002011
     
134. Joep W.R. Sins XF, Karin Fijnvandraat, Melissa Dominguez, A. W. Rijneveld, Jean-Louis Kerkhoffs, A van Meurs, M. R. De Groot, H Heijboer, Erfan Nur, Brenda M Luken, Sacha S Zeerleder, Marie-Françoise Dresse, Phu-Quoc Le, Philippe Hermans, Anna Vanderfaeillie, Eric Van Den Neste, Fleur Samantha Benghiat, Rachel Kesse-Adu, Andre Delannoy, Andre Efira, Marie-Agnes Azerad, C A de Borgie, Junmei Chen, Jose A. Lopez and Bart J. Biemond. Effects of Oral N -Acetylcysteine on Oxidative Stress in Patients with Sickle Cell Disease. In: Blood, editor. ASH. Atlanta; 2017.
     
135. Sins JWR, Fijnvandraat K, Rijneveld AW, et al. Effect of N-acetylcysteine on pain in daily life in patients with sickle cell disease: a randomised clinical trial. Br J Haematol 2017. PMid:28643376
     
136. Joep W.R. Sins KF, Anita W. Rijneveld, Martine B. Boom, Jean-Louis Kerkhoffs, Alfred H. van Meurs, Marco R De Groot, Harriet Heijboer, Marie-Françoise Dresse, Alina Ferster, Philippe Hermans, Anna Vanderfaeillie, Eric W Van Den Neste, Fleur Samantha Benghiat, Jo Howard, Rachel Kesse-Adu, Andre Delannoy, Andre Efira, Marie-Agnes Azerad, Corianne A.J.M. de Borgie and Bart J. Biemond. N-Acetylcysteine in Patients with Sickle Cell Disease: A Randomized Controlled Trial. In: Blood, editor. ASH: Blood; 2016.
     
137. Niihara Y, Miller ST, Kanter J, et al. A Phase 3 Trial of l-Glutamine in Sickle Cell Disease. N Engl J Med 2018;379:226-235. https://doi.org/10.1056/NEJMoa1715971 PMid:30021096
     
138. Niihara Y KH, Tran L, Razon R, Macan H, Stark C, Wun T, Adams-Graves P. A phase 3 study of L-Glutamine Therapy for sickle cell anemia and sickle b0-thalassemia. In: Blood, editor. American Society of Hematology: Blood; 2014. p a86.
     
139. Niihara Y, Zerez CR, Akiyama DS, et al. Oral L-glutamine therapy for sickle cell anemia: I. Subjective clinical improvement and favorable change in red cell NAD redox potential. Am J Hematol 1998;58:117-121. https://doi.org/10.1002/(SICI)1096-8652(199806)58:2<117::AID-AJH5>3.0.CO;2-V 
     
140. Quinn CT. l-Glutamine for sickle cell anemia: more questions than answers. Blood 2018;132:689-693. https://doi.org/10.1182/blood-2018-03-834440 
     
141. Lagresle-Peyrou C, Lefrere F, Magrin E, et al. Plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion. Haematologica 2018;103:778-786. https://doi.org/10.3324/haematol.2017.184788 PMid:29472357 PMCid:PMC5927997
     
142. Esrick EB, Bauer DE. Genetic therapies for sickle cell disease. Semin Hematol 2018;55:76-86. https://doi.org/10.1053/j.seminhematol.2018.04.014 PMid:29958563
     
143. Leonard A, Tisdale JF. Stem cell transplantation in sickle cell disease: therapeutic potential and challenges faced. Expert Rev Hematol 2018;11:547-565. https://doi.org/10.1080/17474086.2018.1486703 PMid:29883237
     
144. Angelucci E, Matthes-Martin S, Baronciani D, et al. Hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel. Haematologica 2014;99:811-820. https://doi.org/10.3324/haematol.2013.099747 PMid:24790059 PMCid:PMC4008115
     
145. Saraf SL, Oh AL, Patel PR, et al. Haploidentical Peripheral Blood Stem Cell Transplantation Demonstrates Stable Engraftment in Adults with Sickle Cell Disease. Biol Blood Marrow Transplant 2018;24:1759-1765. https://doi.org/10.1016/j.bbmt.2018.03.031 PMid:29656137
     
146. Ribeil JA, Hacein-Bey-Abina S, Payen E, et al. Gene Therapy in a Patient with Sickle Cell Disease. N Engl J Med 2017;376:848-855. https://doi.org/10.1056/NEJMoa1609677 PMid:28249145
     
147. Antoniani C, Meneghini V, Lattanzi A, et al. Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human beta-globin locus. Blood 2018;131:1960-1973. https://doi.org/10.1182/blood-2017-10-811505 PMid:29519807
     
148. Sato M, Saitoh I, Inada E. Efficient CRISPR/Cas9-based gene correction in induced pluripotent stem cells established from fibroblasts of patients with sickle cell disease. Stem Cell Investig 2016;3:78. https://doi.org/10.21037/sci.2016.11.05 PMid:28066780 PMCid:PMC5182212
     
149. Ye L, Wang J, Tan Y, et al. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and beta-thalassemia. Proc Natl Acad Sci U S A 2016;113:10661-10665. https://doi.org/10.1073/pnas.1612075113 PMid:27601644 PMCid:PMC5035856