Targeted modification of furan-2-carboxaldehydes into Michael acceptor analogs yielded long-acting hemoglobin modulators with dual antisickling activities.

Sickle cell disease (SCD) is the most common genetic disorder, affecting millions of people worldwide. Aromatic aldehydes, which increase the oxygen affinity of human hemoglobin to prevent polymerization of sickle hemoglobin and inhibit red blood cell (RBC) sickling, have been the subject of keen interest for the development of effective treatment against SCD. However, the aldehyde functional group metabolic instability has severly hampered their development, except for voxelotor, which was approved in 2019 for SCD treatment. To improve the metabolic stability of aromatic aldehydes, we designed and synthesized novel molecules by incorporating Michael acceptor reactive centers into the previously clinically studied aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF). Eight such derivatives, referred to as MMA compounds were synthesized and studied for their functional and biological activities. Unlike 5-HMF, which forms Schiff-base interaction with αVal1 nitrogen of hemoglobin, the MMA compounds covalently interacted with ßCys93, as evidenced by reverse-phase HPLC and disulfide exchange reaction, explaining their RBC sickling inhibitory activities, which at 2 mM and 5 mM, range from 0% to 21% and 9% to 64%, respectively. Additionally, the MMA compounds showed a second mechanism of sickling inhibition (12%-41% and 13%-62% at 2 mM and 5 mM, respectively) by directly destabilizing the sickle hemoglobin polymer. In vitro studies demonstrated sustained pharmacologic activities of the compounds compared to 5-HMF. These findings hold promise for advancing SCD therapeutics.

Profiling the Plasmodium falciparum Erythrocyte Membrane Protein 1-Specific Immununoglobulin G Response Among Ghanaian Children With Hemoglobin S and C.

Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are important targets for protective immunity. Abnormal display of PfEMP1 on the surfaces of infected erythrocytes (IEs) and reduced cytoadhesion have been demonstrated in hemoglobin (Hb) AS and HbAC, inherited blood disorders associated with protection against severe P. falciparum malaria. We found that Ghanaian children with HbAS had lower levels of immunoglobulin G against several PfEMP1 variants and that this reactivity increased more slowly with age than in their HbAA counterparts. Moreover, children with HbAS have lower total parasite biomass than those with HbAA at comparable peripheral parasitemias, suggesting impaired cytoadhesion of HbAS IEs in vivo and likely explaining the slower acquisition of PfEMP1-specific immunoglobulin G in this group. In contrast, the function of acquired antibodies was comparable among Hb groups and appears to be intact and sufficient to control parasitemia via opsonization and phagocytosis of IEs.

Doping control approach: Identification of equine in vitro metabolites of voxelotor (GBT440), a hemoglobin S polymerization inhibitor.

RATIONALE: Sickle cell disease, a debilitating genetic disorder affecting numerous newborns globally, has historically received limited attention in pharmaceutical research. However, recent years have witnessed a notable shift, with the Food and Drug Administration approving three innovative disease-modifying medications. Voxelotor, also known as GBT440, is a promising compound that effectively prevents sickling, providing a safe approach to alleviate chronic hemolytic anemia in sickle cell disease. It is a novel, orally bioavailable small molecule that inhibits hemoglobin S polymerization by enhancing oxygen affinity to hemoglobin. The investigation demonstrated that voxelotor led to an unintended elevation of hemoglobin levels in healthy individuals by increasing serum erythropoietin levels. METHODS: Voxelotor and its metabolites in an in vitro setting utilizing equine liver microsomes were discussed. Plausible structures of the identified metabolites were inferred through the application of liquid chromatography in conjunction with high-resolution mass spectrometry. RESULTS: Under the experimental conditions, a total of 31 metabolites were detected, including 16 phase I metabolites, two phase II metabolites, and 13 conjugates of phase I metabolites. The principal phase I metabolites were generated through processes such as hydroxylation, reduction, and dissociation. The presence of glucuronide and sulfate conjugates of the parent drug were also observed, along with hydroxylated, reduced, and dissociated analogs. CONCLUSIONS: The data acquired will accelerate the identification of voxelotor and related compounds, aiding in the detection of their illicit use in competitive sports. It is crucial to emphasize that the metabolites detailed in this manuscript were identified through in vitro experiments and their detection in an in vivo study may not be guaranteed.

Pyruvate kinase activators: targeting red cell metabolism in sickle cell disease.

Hemoglobin S (HbS) polymerization, red blood cell (RBC) sickling, chronic anemia, and vaso-occlusion are core to sickle cell disease (SCD) pathophysiology. Pyruvate kinase (PK) activators are a novel class of drugs that target RBC metabolism by reducing the buildup of the glycolytic intermediate 2,3-diphosphoglycerate (2,3-DPG) and increasing production of adenosine triphosphate (ATP). Lower 2,3-DPG level is associated with an increase in oxygen affinity and reduction in HbS polymerization, while increased RBC ATP may improve RBC membrane integrity and survival. There are currently 3 PK activators in clinical development for SCD: mitapivat (AG-348), etavopivat (FT-4202), and the second-generation molecule AG-946. Preclinical and clinical data from these 3 molecules demonstrate the ability of PK activators to lower 2,3-DPG levels and increase ATP levels in animal models and patients with SCD, as well as influence a number of potential pathways in SCD, including hemoglobin oxygen affinity, RBC sickling, RBC deformability, RBC hydration, inflammation, oxidative stress, hypercoagulability, and adhesion. Furthermore, early-phase clinical trials of mitapivat and etavopivat have demonstrated the safety and tolerability of PK activators in patients with SCD, and phase 2/3 trials for both drugs are ongoing. Additional considerations for this novel therapeutic approach include the balance between increasing hemoglobin oxygen affinity and tissue oxygen delivery, the cost and accessibility of these drugs, and the potential of multimodal therapy with existing and novel therapies targeting different disease mechanisms in SCD.

Vascular pathophysiology of sickle cell disease.

Sickle cell disease (SCD) is an hereditary disorder characterized by the production of an abnormal hemoglobin called hemoglobin S (HbS). HbS may polymerize in deoxygenated conditions, which leads to red blood cell (RBC) sickling. Sickled RBCs are more rigid and fragile, and prone to lysis. SCD patients exhibit various acute and/or chronic complications, which may affect several organs. The clinical presentation of SCD is highly variable from one patient to another and cannot be only explained by RBC sickling. Increased blood viscosity, caused by the presence of RBCs with abnormal deformability and aggregation, may increase vascular resistance and increase the risk of acute and chronic vascular complications. Chronic hemolysis results in decreased nitric oxide (NO) bioavailability which may compromise vasodilation and participate to the development of chronic vasculopathy. Furthermore, chronic hemolysis is responsible for increased inflammation and oxidative stress, which affect the vascular system and may promote the adhesion of circulating cells to endothelial cells. Extracellular vesicles and especially RBC microparticles (massively released in the context of SCD) are also at the origin of vascular damages and increased white blood cells adhesion to the endothelium, which may trigger vaso-occlusive crisis and other vascular-related complications. This review highlights the fact that SCD should not only be considered as a hematological disorder but also as a vascular disease.

[Impact of voxelotor on hemoglobin electrophoretic and chromatographic profiles]. / Conséquences du traitement par voxelotor sur les profils électrophorétiques et chromatographiques de l'hémoglobine.

Voxelotor (GBT440, OXBRYTA®) appeared recently as one of the possible treatments for sickle cell disease. This molecule, by binding the alpha globin of hemoglobin, causes hyperaffinity of the latter for oxygen and reduces its polymerization properties. Several therapeutic trials have been able to show its effectiveness on certain aspects of sickle cell disease; thus, the french HAS (High Authority of Health) college issued an early access authorization and, since 2021, this treatment can be offered to patients under a temporary authorization for use. Consequently, the laboratories that carry out the biological monitoring of sickle cell patients will be confronted with new profiles characteristic of the presence of hemoglobin combined with GBT440. This work presents a collection of images obtained by different techniques: HPLC, capillary electrophoresis, isoelectrofocusing, alkaline gel and acid agar gel electrophoresis in transfused or non-transfused sickle cell disease patients. The ability to observe the presence of GBT440 by these analyzes could be useful in order to characterize the therapeutic follow-up of patients.

Voxelotor does not inhibit sickle hemoglobin fiber formation upon complete deoxygenation.

The drug voxelotor (commercially known as Oxbryta) has been approved by the US Food and Drug Administration for the treatment of sickle cell disease. It is known to reduce disease-causing sickling by inhibiting the transformation of the non-polymerizing, high-oxygen-affinity R quaternary structure of sickle hemoglobin into its polymerizing, low-affinity T quaternary structure. It has not been established whether the binding of the drug has anti-sickling effects beyond restricting the change of quaternary structure. By using a laser photolysis method that employs microscope optics, we have determined that fully deoxygenated sickle hemoglobin will assume the T structure. We show that the nucleation rates essential to generate the sickle fibers are not significantly affected by voxelotor. The method employed here should be useful for determining the mechanism of sickling inhibition for proposed drugs.

An integrated therapeutic approach to sickle cell disease management beyond infancy.

Hydroxyurea, the first approved drug for sickle cell disease, decreases sickle hemoglobin polymerization by inducing fetal hemoglobin. Its effects in young children are excellent; responses in adults are variable and not curative. The goal of pharmacotherapy should not be disease "moderation" but reducing morbidity and mortality by diminishing both hemolytic anemia and vaso-occlusive events. This is best done by preventing sickle hemoglobin polymerization; if anti-polymerization treatment is insufficient, agents disrupting pathophysiologic pathways "downstream" of the sickle hemoglobin polymer should be added. We recommend that all patients should be started first on maximal doses of hydroxyurea. When the clinical and hematologic response to hydroxyurea is insufficient, as it is almost always in adults, we favor adding voxelotor, a hemoglobin-oxygen affinity-shifting agent that, likely in a pancellular distribution, decreases sickle hemoglobin polymerization. The P-selectin inhibitor crizanlizumab reduces sickle cell-endothelial interactions and can be used in patients with continued vaso-occlusive events. There is no physiologic reason that all three drugs could not be combined when the response to monotherapy or dual-drug therapy is poor. Drug therapy must be considered in the context of possibly "curative" cellular therapeutics and if needed, exchange transfusion programs.

A critical role for altered red cell cation permeability in pathogenesis of sickle cell disease and other haemolytic anaemias.

The aetiology of sickle cell disease is well known, but pathogenesis is complicated and details remain uncertain. A thorough understanding may suggest novel ways for designing more effective therapies. One area of importance, covered here in Nader et al., is the altered cation permeability of sickle cells and how the co-ordinated operation of a number of membrane transport proteins contributes to disease progression, all driven by the initial event of HbS polymerisation. There are echoes here of the cation leaks of hereditary stomatocytosis. Nader et al. propose a central role for PIEZO1, a novel mechanosensitive channel found in red cells, which may be aberrantly activated in sickle cells following HbS polymerisation and which may have potential as a novel target for future chemotherapies. Commentary on: Nader et al. Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner. Br J Haematol 2023;202:657-668.

Sickle Cell Hemoglobin Genotypes Affect Malaria Parasite Growth and Correlate with Exosomal miR-451a and let-7i-5p Levels.

Malaria affects a significant portion of the global population, with 247 million cases in 2021, primarily in Africa. However, certain hemoglobinopathies, such as sickle cell trait (SCT), have been linked to lower mortality rates in malaria patients. Hemoglobin (Hb) mutations, including HbS and HbC, can cause sickle cell disease (SCD) when both alleles are inherited (HbSS and HbSC). In SCT, one allele is inherited and paired with a normal allele (HbAS, HbAC). The high prevalence of these alleles in Africa may be attributed to their protective effect against malaria. Biomarkers are crucial for SCD and malaria diagnosis and prognosis. Studies indicate that miRNAs, specifically miR-451a and let-7i-5p, are differentially expressed in HbSS and HbAS compared to controls. Our research examined the levels of exosomal miR-451a and let-7i-5p in red blood cells (RBCs) and infected red blood cells (iRBCs) from multiple sickle Hb genotypes and their impact on parasite growth. We assessed exosomal miR-451a and let-7i-5p levels in vitro in RBC and iRBC supernatants. Exosomal miRNAs exhibited distinct expression patterns in iRBCs from individuals with different sickle Hb genotypes. Additionally, we discovered a correlation between let-7i-5p levels and trophozoite count. Exosomal miR-451a and let-7i-5p could modulate SCD and malaria severity and serve as potential biomarkers for malaria vaccines and therapies.

Design, Synthesis, and Evaluation of Allosteric Effectors for Hemoglobin.

Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in hemoglobin (Hb), the protein in the red blood cell (RBC) responsible for the transport of oxygen (O2) throughout the body. The mutation leads to the expression of sickle cell hemoglobin (HbS). Both Hb and HbS exist in equilibrium between oxygenated and deoxygenated forms; however, deoxygenated HbS can polymerize to form long fibers which distort the shape of RBCs into the characteristic sickled shape. The misshapen RBCs can obstruct blood vessels and capillaries, resulting in a vaso-occlusive crisis. Vaso-occulsion deprives tissues and organs of O2 and can cause intense pain which often results in hospitalization. Chronic organ damage is a major cause of reduced life expectancy for SCD patients.Allosteric effectors are molecules which regulate protein function. HbS allosteric effectors can be used to decrease polymerization by stabilizing the oxygenated form of HbS, which leads to an increase in O2 uptake and a decrease in the sickling of RBCs. Allosteric effectors that have been evaluated for the treatment of SCD include vanillin, 5-hydroxymethyl furfural (5-HMF), and voxelotor, which was approved by the U.S. Food and Drug Administration (FDA) for the treatment of SCD in 2019. 5-HMF did not progress to phase III clinical trials since it suffered from rapid metabolic degradation. However, several derivatives of 5-HMF and vanillin have been synthesized and evaluated as potential candidates for SCD treatment. Derivatives of these compounds have shown promise, but their shortcomings, such as high levels of oxidative metabolism, have prevented them from progressing into marketable drugs. Our efforts have produced multiple 5-HMF derivatives which have been evaluated for their potential to treat SCD. Each derivative was evaluated for its ability to increase O2 affinity (i.e., P50, the partial pressure at which hemoglobin is 50% saturated with O2). The synthesized aryl ether derivatives were evaluated, and results suggest that compounds with multiple aromatic aldehydes may have enhanced biological properties. One such derivative, compound 5, which features two furan aldehyde rings, exhibited increased O2 affinity (P50 = 8.82 ± 1.87 mmHg) over that of unmodified Hb (P50 = 13.67 ± 0.22 mmHg). Future studies include obtaining crystal structures of the 5-HMF derivatives complexed with HbS to confirm the protein-allosteric effector interactions.

In vivo evaluation of the effect of sickle cell hemoglobin S, C and therapeutic transfusion on erythrocyte metabolism and cardiorenal dysfunction.

Despite a wealth of exploratory plasma metabolomics studies in sickle cell disease (SCD), no study to date has evaluate a large and well phenotyped cohort to compare the primary erythrocyte metabolome of hemoglobin SS, SC and transfused AA red blood cells (RBCs) in vivo. The current study evaluates the RBC metabolome of 587 subjects with sickle cell sickle cell disease (SCD) from the WALK-PHaSST clinical cohort. The set includes hemoglobin SS, hemoglobin SC SCD patients, with variable levels of HbA related to RBC transfusion events. Here we explore the modulating effects of genotype, age, sex, severity of hemolysis, and transfusion therapy on sickle RBC metabolism. Results show that RBCs from patients with Hb SS genotypes-compared to AA RBCs from recent transfusion events or SC RBCs-are characterized by significant alterations of RBC acylcarnitines, pyruvate, sphingosine 1-phosphate, creatinine, kynurenine and urate metabolism. Surprisingly, the RBC metabolism of SC RBCs is dramatically different from SS, with all glycolytic intermediates significantly elevated in SS RBCs, with the exception of pyruvate. This result suggests a metabolic blockade at the ATP-generating phosphoenolpyruvate to pyruvate step of glycolysis, which is catalyzed by redox-sensitive pyruvate kinase. Metabolomics, clinical and hematological data were collated in a novel online portal. In conclusion, we identified metabolic signatures of HbS RBCs that correlate with the degree of steady state hemolytic anemia, cardiovascular and renal dysfunction and mortality.

Membrane bending and sphingomyelinase-associated, sulfatide-dependent hypoxic adhesion of sickle mature erythrocytes.

Abnormal erythrocyte adhesion owing to polymerization of sickle hemoglobin is central to the pathophysiology of sickle cell disease (SCD). Mature erythrocytes constitute >80% of all erythrocytes in SCD; however, the relative contributions of erythrocytes to acute and chronic vasculopathy in SCD are not well understood. Here, we showed that bending stress exerted on the erythrocyte plasma membrane by polymerization of sickle hemoglobin under hypoxia, enhances sulfatide-mediated abnormal mature erythrocyte adhesion. We hypothesized that sphingomyelinase (SMase) activity, which is upregulated by accumulated bending energy, leads to elevated membrane sulfatide availability, and thus, hypoxic mature erythrocyte adhesion. We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856 ± 481 vs 78 ± 23, mean ± SEM), whereas sickle reticulocyte (early erythrocyte) adhesion, high to begin with, does not change (1281 ± 299 vs 1258 ± 328, mean ± SEM). We showed that greater mean accumulated bending energy of adhered mature erythrocytes was associated with higher acid SMase activity and increased mature erythrocyte adhesion (P = .022, for acid SMase activity and P = .002 for the increase in mature erythrocyte adhesion with hypoxia, N = 5). In addition, hypoxia results in sulfatide exposure of the erythrocyte membrane, and an increase in SMase, whereas anti-sulfatide inhibits enhanced adhesion of erythrocytes. These results suggest that the lipid components of the plasma membrane contribute to SCD complications. Therefore, sulfatide and the components of its upregulation pathway, particularly SMase, should be further explored as potential therapeutic targets for inhibiting sickle erythrocyte adhesion.

Reconsidering sickle cell trait testing of red blood cell units allocated to children with sickle cell disease.

BACKGROUND: Sickle cell trait (SCT) testing of red blood cell (RBC) units is sometimes performed to identify and divert units containing hemoglobin S (HbS). Recipients strategically guarded against this exposure include fetuses, neonates, and children with sickle cell disease (SCD). The clinical necessity of this practice is unclear. STUDY DESIGN AND METHODS: A one-year audit (2018) was performed at a pediatric tertiary care hospital that tests for SCT in RBC units prescribed to children with SCD and neonates. The impact of incorporating varying numbers of SCT RBC units in a single-unit top-up, partial-manual red cell exchange, and automated erythrocytapheresis was modeled in four typical-parameter age scenarios (2, 5, 10, and 18 years) sharing a high baseline HbS. Additionally, a survey assessing SCT testing practices was administered to Canadian pediatric hospital transfusion laboratories serving hemoglobinopathy programs. RESULTS: Of 2268 donor RBC units tested, one was positive for SCT (0.04% [95% CI: 0.01%-0.24%]), at a cost of $19,384.56 CAD. The impact of SCT unit incorporation on lost HbS reduction was modest (Δ1%-3% [automated erythrocytapheresis] and Δ4%-15% [top-up/partial manual exchange]). The survey (with all 13 sites responding) showed variable SCT testing practice; four (31%) do not test, four (31%) test for children with SCD, and six (46%) test for neonates. CONCLUSION: RBC SCT testing may be more costly than beneficial or necessary in children with SCD. As of 2019, our transfusion service has ceased SCT testing for this population. Further research in the fetal/neonatal populations is needed to overturn this entrenched practice.

Pathophysiological characterization of the Townes mouse model for sickle cell disease.

A deeper pathophysiologic understanding of available mouse models of sickle cell disease (SCD), such as the Townes model, will help improve preclinical studies. We evaluated groups of Townes mice expressing either normal adult human hemoglobin (HbA), sickle cell trait (HbAS), or SCD (HbS), comparing younger versus older adults, and females versus males. We obtained hematologic parameters in steady-state and hypoxic conditions and evaluated metabolic markers and cytokines from serum. Kidney function was evaluated by measuring the urine protein/creatinine ratio and urine osmolality. In vivo studies included von Frey assay, non-invasive plethysmography, and echocardiography. Histopathological evaluations were performed in lung, liver, spleen, and kidney tissues. HbS mice displayed elevated hemolysis markers and white blood cell counts, with some increases more pronounced in older adults. After extended in vivo hypoxia, hemoglobin, platelet counts, and white blood cell counts decreased significantly in HbS mice, whereas they remained stable in HbA mice. Cytokine analyses showed increased TNF-alpha in HbS mice. Kidney function assays revealed worsened kidney function in HbS mice. The von Frey assay showed a lower threshold to response in the HbS mice than controls, with more noticeable differences in males. Echocardiography in HbS mice suggested left ventricular hypertrophy and dilatation. Plethysmography suggested obstructive lung disease and inflammatory changes in HbS mice. Histopathological studies showed vascular congestion, increased iron deposition, and disruption of normal tissue architecture in HbS mice. These data correlate with clinical manifestations in SCD patients and highlight analyses and groups to be included in preclinical therapeutic studies.

Hypercoagulability in Sickle Cell Disease: A Thrombo-Inflammatory Mechanism.

Sickle cell disease (SCD) is a group of inherited disorders characterized by the presence of abnormal hemoglobin S. Patients with SCD suffer from frequent episodes of anemia, chronic hemolysis, pain crisis, and vaso-occlusion. Additionally, SCD is associated with diverse and serious clinical complications, including thrombosis, which can lead to organ failure, increased morbidity, and eventually, mortality. SCD is known to be a hypercoagulable condition, and the cause of hypercoagulability is multifactorial, with the molecular basis of hemoglobin S being the main driver. The presence of hemoglobin S induces sickling of the RBCs and their subsequent hemolysis, as well as oxidative stress. Both of these processes can alter the hemostatic system, through the activation of platelets, coagulation system, and fibrinolysis, as well as depletion of coagulation inhibitors. These changes can also induce the formation of microvesicles and expression of tissue factor, leading to activation of WBCs, endothelial cell damage, and inflammatory response. Understanding the various factors that drive hypercoagulability as a thrombo-inflammatory mechanism in SCD can help provide explanations for the pathogenesis and other complications of the disease.

Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is caused by a single-point mutation, and the ensuing deoxygenation-induced polymerization of sickle hemoglobin (HbS), and reduction in bioavailability of vascular nitric oxide (NO), contribute to the pathogenesis of the disease. In a proof-of-concept study, we successfully incorporated nitrate ester groups onto two previously studied potent antisickling aromatic aldehydes, TD7 and VZHE039, to form TD7-NO and VZHE039-NO hybrids, respectively. These compounds are stable in buffer but demonstrated the expected release of NO in whole blood in vitro and in mice. The more promising VZHE039-NO retained the functional and antisickling activities of the parent VZHE039 molecule. Moreover, VZHE039-NO, unlike VZHE039, significantly attenuated RBC adhesion to laminin, suggesting this compound has potential in vivo RBC anti-adhesion properties relevant to vaso-occlusive events. Crystallographic studies show that, as with VZHE039, VZHE039-NO also binds to liganded Hb to make similar protein interactions. The knowledge gained during these investigations provides a unique opportunity to generate a superior candidate drug in SCD with enhanced benefits.

Sickle cell disease in the new era: advances in drug treatment.

Sickle cell disease is an inherited blood disorder afflicting an estimated 100,000 individuals in the United States and over 20 million people worldwide. The disease is heralded as the first molecular disease. However, despite its genetic simplicity, the pathophysiologic processes leading to its clinical sequelae are complex, heterogeneous and interrelated, making drug development to treat the disease challenging. For over two decades only one drug, hydroxyurea, had been used as disease-modifying therapy. New pharmacologic agents are rapidly evolving with three new drugs, with different mechanisms of action, approved by the United States Food and Drug Administration in recent years (L-glutamine, crizanlizumab and voxelotor). Several therapeutic approaches targeting different pathways in the disease pathophysiology are being investigated. These include inhibition of hemoglobin S polymerization such as by fetal hemoglobin induction or by increasing hemoglobin oxygen affinity, as well as intervention of downstream pathways including inhibiting cellular adhesion, reducing inflammation and oxidant stress, modulating platelet activation and coagulation abnormalities, and targeting nitric oxide signaling. This review will provide an overview of these therapeutic strategies, discuss the four currently approved drugs in detail, and summarize ongoing clinical trials of new drugs or drug indications for the treatment of sickle cell disease in different phases of development excluding those related to cellular therapies.

Simultaneous quantification of blood rheology and oxygen saturation to evaluate affinity-modifying therapies in sickle cell disease.

Sickle cell blood demonstrates oxygen-dependent flow behavior as a result of HbS polymerization during hypoxia, and these rheological changes provide a biophysical metric that can be used to quantify the pathological behavior of the blood. Relating these rheological changes directly to hemoglobin oxygen saturation would improve our understanding of SCD pathogenesis and the potential effects of therapeutic drugs. Towards this end, we have developed a microfluidic platform capable of spectrophotometric quantification of Hb-O2 saturation and simultaneous evaluation of the accompanying rheological changes in SCD blood flow. We demonstrated the ability to measure changes in Hb-O2 affinity and a restoration of oxygen-independent blood flow behavior after incubation with voxelotor, an oxygen affinity modifying drug approved for use in SCD. We also identified regimes in Hb-O2 saturation where the effects of HbS polymerization begin to take effect in contributing to pathological flow behavior, independent of voxelotor treatment. In contrast, incubation with voxelotor recovered oxygen-dependent blood flow over a range of PO2, providing a framework for understanding voxelotor's therapeutic effect in lowering the PO2 at which the requisite Hb-O2 saturation is reached to observe pathological blood flow. These results help explain the mechanistic effects of voxelotor and show the potential of this platform to identify affinity-modifying compounds and evaluate their therapeutic effect on blood flow.

ATR1 Angiotensin II Receptor Reduces Hemoglobin S Polymerization, Phosphatidylserine Exposure, and Increases Deformability of Sickle Cell Disease Erythrocytes.

Angiotensin II (Ang II) regulates blood volume and stimulates erythropoiesis through AT1 (ATR1) and AT2 (ATR2) receptors, found in multiple tissues, including erythrocytes. Sickle cell disease (SCD) patients present altered Ang II levels. Hemoglobin S polymerization, deformability and phosphatidylserine translocation are important features of mature erythrocytes, therefore, our hypothesis is Ang II affects these parameters and, if it does, what would be the influence of AT1R and AT2R on these effects. A polymerization assay (PA), deformability, and annexin V binding were performed in SCD erythrocytes samples adding Ang II, ATR1 antagonist (losartan or eprosartan), and ATR2 antagonist (PD123319). Through the PA test, we observed a dose-dependent polymerization inhibition effect when comparing Ang II to control. Losartan did not affect the level or the rate of Ang II inhibition, while PD123319 showed an increased level of protection against polymerization, and eprosartan brought levels back to control. Ang II was able to reduce the translocation of phosphatidylserine from the inner to the outer leaflet, a marker of eryptosis, in the presence of PD123319. Also, ATR1 showed a positive effect increasing deformability. Our data shows that ATR1 is important for maintenance of erythrocyte physiological function in SCD and for prolonging its life.