An Oral Carbon Monoxide-Releasing Molecule Protects against Acute Hyper-hemolysis in Sickle Cell Disease.

Acute hyper-hemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multi-organ failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyper-hemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: 1) prevented the up-regulation of relevant pro-inflammatory, and pro-adhesion controlled by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and 2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-kB pro-inflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyper-hemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as SCD patients with history of DHTR.

SUCCESSES AND PITFALLS IN ORPHAN DRUG DEVELOPMENT FOR SICKLE CELL DISEASE.

Sickle cell disease (SCD) is a hereditary red cell disorder with large global burden problem. In the United States (US) and Europe, medicines may qualify for orphan designation (OD), a regulatory status that provides incentives to boost development. We evaluated the development of new therapies for SCD using data for OD granted in the US and Europe over the last two decades (2000-2021). We analyzed their characteristics, pathophysiological targets, trends, and OD sponsors. We then investigated the approval outcomes, including the phase success rate and reasons for discontinuation across different variables. We identified 57 OD for SCD: 43 (75.4%) small molecules, 32 (56.1%) for oral administration, and 36 (63.1%) for chronic use to prevent SCD complications. At the end of the study (2021) development of 34/57 ODs was completed. Four OD were approved with a success rate of 11.8%. Products targeting upstream causative events of SCD pathophysiology had a 1.8 higher success rate compared to products targeting disease consequences. Large companies showed a fourfold higher success rate compared to small-medium enterprises. Failures in clinical development were mainly seen in Phase 3 for a lack of efficacy on vaso-occlusive crisis as the primary study endpoint, likely related to variable definitions and heterogeneity of pain scoring and treatment. Both advances in SCD knowledge and regulatory incentives paved the way for new therapies for SCD. Our finding of high failure rates in late-stage clinical development signals the need for better early-stage predictive models, also in the context of meaningful clinical endpoints.

Iron restriction in sickle cell disease: When less is more.

Primum non nocere! Can iron deficiency, an abnormality that causes anemia, benefit people with sickle cell disease (SCD) who already have an anemia? The published literature we review appears to answer this question in the affirmative: basic science considerations, animal model experiments, and noncontrolled clinical observations all suggest a therapeutic potential of iron restriction in SCD. This is because SCD's clinical manifestations are ultimately attributable to the polymerization of hemoglobin S (HbS), a process strongly influenced by intracellular HbS concentration. Even small decrements in HbS concentration greatly reduce polymerization, and iron deficiency lowers erythrocyte hemoglobin concentration. Thus, iron deficiency could improve SCD by changing its clinical features to those of a more benign anemia (i.e., a condition with fewer or no vaso-occlusive events). We propose that well-designed clinical studies be implemented to definitively determine whether iron restriction is a safe and effective option in SCD. These investigations are particularly timely now that pharmacologic agents are being developed, which may directly reduce red cell hemoglobin concentrations without the need for phlebotomies to deplete total body iron.

Safety and efficacy of ketorolac continuous infusion for multimodal analgesia of vaso-occlusive crisis in patients with sickle cell disease.

Pain is an hallmark of sickle-cell-related acute clinical manifestations as part of acute vaso-occlusive crisis (VOC). In SCD pain has different origins such as vascular or neuropathic pain, which requires multimodal analgesia. This is based on the administration of drugs with different pharmacological mechanisms of action, maximizing analgesia and minimizing their adverse events and the risk of drug-addition in patients experiencing acute-recurrent pain events as in SCD. Ketorolac is a potent non-narcotic analgesic, being relatively safe and effective during pain-management in children and adults. Up to now, there is a lack of safety information on continuous infusion ketorolac as used to control acute pain in patients with SCD, and the benefits/risks ratio needs to be investigated. Here, we report for the first time the safety profile of ketorolac in the special population of patients with SCD. We confirmed that ketorolac in combination with tramadol, an opioid like molecule, is effective in pain control of adult patients with SCD experiencing acute severe VOCs defined by pain visual analog scale. Our study shows that short term (72 h) continuous infusion of ketorolac plus tramadol is not associated with adverse events such as liver or kidney acute disfunction or abnormalities in coagulation parameters during patients' hospitalization and within 30 days after patients discharge. This is extremely important for patients with SCD, who should have access to multimodal therapy to control recurrent acute pain crisis in order to limit central sensitization a fearsome issue of undertreated recurrent acute pain and of chronic pain.

Nephrological Complications in Hemoglobinopathies: SITE Good Practice.

BACKGROUND: Hemoglobinopathies, among which thalassemic syndromes (transfusion-dependent and non-transfusion dependent thalassemias) and sickle cell disease (SCD), are the most widespread monogenic diseases worldwide. Hemoglobinopathies are endemic and spread-out all-over Italy, as result of internal and external migration flows. Nowadays, the increase therapeutic options associated to the general aging of patients with hemoglobinopathies related to the improvement in clinical management, contribute to the abnormalities in kidney function going from blood and urine test alterations to chronic kidney disease and end stage renal disease. METHODS: Here, we carried out a revision of the literature as panel of recognized experts in hemoglobinopathies with the consultancy and the revision of two nephrologists on kidney alteration and kidney disease in patients with TDT, NTDT and SCD. This is part of the action of the Italian society for the study of thalassemia and hemoglobinopties (SITE). The purpose of this "good practice (GP)" is to provide recommendations for follow-up and therapy for the management of kidney alterations in patients with TDT, NTDT and SCD. The literature review covers the period 1.1.2016 to 31.12.2022. In consideration of the rarity of these diseases, the analysis was extended from 5 to 7 years. Moreover, in the absence of relevant scientific papers in the identified time frame, we referred to pivotal or population studies, when available. Finally, in the absence of evidence-based data from prospective and randomized trials, the authors had to refer to expert opinion (expert consensus) for many topics. RESULTS: We generated question and answer boxes to offer a friendly consultation, using color code strategy and focused answers. CONCLUSIONS: The present GP will help in improving the clinical management, and the quality of care of patients with hemoglobinopathies.

Epeleuton, a novel synthetic ω-3 fatty acid, reduces the hypoxia/reperfusion stress in a mouse model of sickle cell disease.

Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to SCD disease progression. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel orally administered second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory response and improves red cell features, resulting in reduced hemolysis and sickling compared with vehicle treated SCD mice. In addition, epeleuton prevents the hypoxia/reoxygenation induced activation of NF-kB with downregulation of NLRP3 inflammasome in lung, kidney, and liver. This was associated with down-regulation of vascular activation markers in epeleuton treated SCD mice when compared to vehicle treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD.

Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2-/- mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2-/- mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

Low-dose anti-IL 5 treatment in idiopathic hypereosinophilic syndrome: towards a precision medicine approach for remission maintenance.

Mepolizumab at the dose of 300 mg/4 weeks has been recently approved as an add-on therapy for patients with uncontrolled hypereosinophilic syndrome (HES) without any identifiable non-hematologic secondary cause. According to the available real-life evidence mepolizumab 300 mg and 100 mg, licensed for severe eosinophilic asthma, are comparable in terms of drug efficacy. However, the clinical rationale for selecting one dose or the other has not been explored. We investigated the efficacy and safety of mepolizumab 100 mg in idiopathic HES (I-HES) patients as a steroid sparing strategy for disease remission maintenance by assessing clinical conditions, blood eosinophil count (BEC) and adverse events at baseline and at 3-6-12 months follow-up. Overall, 11 patients were enrolled (females 4-36%) with a median age of 62 years (IQR 55.0-72.0). At 3-month visit both prednisone daily dose and BEC significantly decreased from baseline, whilst a substantial improvement of Brief fatigue inventory score (BFI) was not recorded before the 6 months assessment. More than 70% of patients completely stopped prednisone at 12-months follow-up, without any flare in terms of BEC and BFI. No adverse event was registered. Although larger studies are needed, our report firstly describes that in a well-defined population, diagnosed with I-HES and in disease remission, low dose mepolizumab is a safe and effective steroid-sparing option for remission maintenance. It suggests that a personalized treatment dose might be explored according to the disease classification and activity at the time of biologic treatment start.

Management of the Sickle Cell Trait: An Opinion by Expert Panel Members.

The number of individuals with the sickle cell trait exceeds 300 million worldwide, making sickle cell disease one of the most common monogenetic diseases globally. Because of the high frequency of sickle cell disease, reproductive counseling is of crucial importance. In addition, unlike other carrier states, Sickle Cell Trait (SCT) seems to be a risk factor for several clinical complications, such as extreme exertional injury, chronic kidney disease, and complications during pregnancy and surgery. This expert panel believes that increasing knowledge about these clinical manifestations and their prevention and management can be a useful tool for all healthcare providers involved in this issue.

Hidden Comorbidities in Asthma: A Perspective for a Personalized Approach.

Bronchial asthma is the most frequent inflammatory non-communicable condition affecting the airways worldwide. It is commonly associated with concomitant conditions, which substantially contribute to its burden, whether they involve the lung or other districts. The present review aims at providing an overview of the recent acquisitions in terms of asthma concomitant systemic conditions, besides the commonly known respiratory comorbidities. The most recent research has highlighted a number of pathobiological interactions between asthma and other organs in the view of a shared immunological background underling different diseases. A bi-univocal relationship between asthma and common conditions, including cardiovascular, metabolic or neurodegenerative diseases, as well as rare disorders such as sickle cell disease, α1-Antitrypsin deficiency and immunologic conditions with hyper-eosinophilia, should be considered and explored, in terms of diagnostic work-up and long-term assessment of asthma patients. The relevance of that acquisition is of utmost importance in the management of asthma patients and paves the way to a new approach in the light of a personalized medicine perspective, besides targeted therapies.

Hip Labral Morphological Changes in Patients with Femoroacetabular Impingement Speed Up the Onset of Early Osteoarthritis.

Over the last decade, evidence has mounted for a prominent etiologic role of femoroacetabular impingement (FAI) in the development of early hip osteoarthritis (OA). The aim of this study was to compare the ultrastructure and tissue composition of the hip labrum in healthy and pathological conditions, as FAI and OA, to provide understanding of structural changes which might be helpful in the future to design targeted therapies and improve treatment indications. We analyzed labral tissue samples from five healthy multi-organ donors (MCDs) (median age, 38 years), five FAI patients (median age, 37 years) and five late-stage OA patients undergoing total hip replacement (median age, 56 years). We evaluated morpho-functional by histology and transmission electron microscopy. Extracellular matrix (ECM) structure changes were similar in specimens from FAI compared to those from patients with OA (more severe in the latter) showing disorganization of collagen fibers and increased proteoglycan content. In FAI and in OA nuclei the chromatin was condensed, organelle degenerated and cytoplasm vacuolized. Areas of calcification were mainly observed in FAI and OA labrum, as well as apoptotic-like features. We showed that labral tissue of patients with FAI had similar pathological alterations of tissue obtained from OA patients, suggesting that FAI patients might have high susceptibility to develop OA.

In Humanized Sickle Cell Mice, Imatinib Protects Against Sickle Cell-Related Injury.

Drug repurposing is a valuable strategy for rare diseases. Sickle cell disease (SCD) is a rare hereditary hemolytic anemia accompanied by acute and chronic painful episodes, most often in the context of vaso-occlusive crisis (VOC). Although progress in the knowledge of pathophysiology of SCD have allowed the development of new therapeutic options, a large fraction of patients still exhibits unmet therapeutic needs, with persistence of VOCs and chronic disease progression. Here, we show that imatinib, an oral tyrosine kinase inhibitor developed for the treatment of chronic myelogenous leukemia, acts as multimodal therapy targeting signal transduction pathways involved in the pathogenesis of both anemia and inflammatory vasculopathy of humanized murine model for SCD. In addition, imatinib inhibits the platelet-derived growth factor-B-dependent pathway, interfering with the profibrotic response to hypoxia/reperfusion injury, used to mimic acute VOCs. Our data indicate that imatinib might be considered as possible new therapeutic tool for chronic treatment of SCD.

Erythrocyte pyruvate kinase activation in red cell disorders.

PURPOSE OF REVIEW: In red cells, pyruvate kinase is a key enzyme in the final step of glycolytic degradative process, which generates a constant energy supply via ATP production. This commentary discusses recent findings on pyruvate kinase activators as new therapeutic option in hereditary red cell disorders such as thalassemic syndromes or sickle cell disease (SCD). RECENT FINDINGS: Mitapivat and etavopivat are two oral pyruvate kinase activators. Studies in a mouse model for ß thalassemia have shown beneficial effects of mitapivat on both red cell survival and ineffective erythropoiesis, with an amelioration of iron homeostasis. This was confirmed in a proof-of-concept study in patients with nontransfusion-dependent thalassemias. Both mitapivat and etavopivat have been evaluated in mouse models for SCD, showing an increased 2-3DPG/ATP ratio and a reduction in haemolysis as well as in sickling. These data were confirmed in proof-of-concept clinical studies with both molecules carried in patients with SCD. SUMMARY: Preclinical and clinical evidence indicate that pyruvate kinase activators represent new therapeutic option in hemoglobinopathies or SCD. Other red cell disorders such as hereditary spherocytosis or hereditary anaemias characterized by defective erythropoiesis might represent additional areas to investigate the therapeutic impact of pyruvate kinase activators.

Duality of Nrf2 in iron-overload cardiomyopathy.

Cardiomyopathy deeply affects quality of life and mortality of patients with b-thalassemia or with transfusion-dependent myelodysplastic syndromes. Recently, a link between Nrf2 activity and iron metabolism has been reported in liver ironoverload murine models. Here, we studied C57B6 mice as healthy control and nuclear erythroid factor-2 knockout (Nrf2-/-) male mice aged 4 and 12 months. Eleven-month-old wild-type and Nrf2-/- mice were fed with either standard diet or a diet containing 2.5% carbonyl-iron (iron overload [IO]) for 4 weeks. We show that Nrf2-/- mice develop an age-dependent cardiomyopathy, characterized by severe oxidation, degradation of SERCA2A and iron accumulation. This was associated with local hepcidin expression and increased serum non-transferrin-bound iron, which promotes maladaptive cardiac remodeling and interstitial fibrosis related to overactivation of the TGF-b pathway. When mice were exposed to IO diet, the absence of Nrf2 was paradoxically protective against further heart iron accumulation. Indeed, the combination of prolonged oxidation and the burst induced by IO diet resulted in activation of the unfolded protein response (UPR) system, which in turn promotes hepcidin expression independently from heart iron accumulation. In the heart of Hbbth3/+ mice, a model of b-thalassemia intermedia, despite the activation of Nrf2 pathway, we found severe protein oxidation, activation of UPR system and cardiac fibrosis independently from heart iron content. We describe the dual role of Nrf2 when aging is combined with IO and its novel interrelation with UPR system to ensure cell survival. We open a new perspective for early and intense treatment of cardiomyopathy in patients with b-thalassemia before the appearance of heart iron accumulation.

Treatment with recombinant ADAMTS13, alleviates hypoxia/reoxygenation-induced pathologies in a mouse model of human sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is an inherited red blood cell disorder with a causative substitution in the beta-globin gene that encodes beta-globin in hemoglobin. Furthermore, the ensuing vasculopathy in the microvasculature involves heightened endothelial cell adhesion, inflammation, and coagulopathy, all of which contribute to vaso-occlusive crisis (VOC) and the sequelae of SCD. In particular, dysregulation of the von Willebrand factor (VWF) and a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in human SCD pathology. OBJECTIVES: To investigate the beneficial potential of treatment with recombinant ADAMTS13 (rADAMTS13) to alleviate VOC. METHODS: Pharmacologic treatment with rADAMTS13 in vitro or in vivo was performed in a humanized mouse model of SCD that was exposed to hypoxia/reoxygenation stress as a model of VOC. Then, pharmacokinetic, pharmacodynamic, and behavioral analyses were performed. RESULTS: Administration of rADAMTS13 to SCD mice dose-dependently increased plasma ADAMTS13 activity, reduced VWF activity/antigen ratios, and reduced baseline hemolysis (free hemoglobin and total bilirubin) within 24 hours. rADAMTS13 was administered in SCD mice, followed by hypoxia/reoxygenation stress, and reduced VWF activity/antigen ratios in parallel to significantly (p < .01) improved recovery during the reoxygenation phase. Consistent with the results in SCD mice, we demonstrate in a human in vitro system that treatment with rADAMTS13 counteracts the inhibitory activity of hemoglobin on the VWF/ADAMTS13-axis. CONCLUSION: Collectively, our data provide evidence that relative ADAMTS13 insufficiency in SCD mice is corrected by pharmacologic treatment with rADAMTS13 and provides an effective disease-modifying approach in a human SCD mouse model.