L-glutamine for sickle cell disease: more than reducing redox.

Oxidative stress is a major contributor to the pathophysiology of sickle cell disease (SCD) including hemolysis and vaso-occlusive crisis (VOC). L-glutamine is a conditionally essential amino acid with important roles, including the synthesis of antioxidants, such as reduced glutathione and the cofactors NAD(H) and NADP(H), as well as nitric oxide. Given the increased levels of oxidative stress and lower (NADH):(NAD + + NADH) ratio in sickle erythrocytes that adversely affects the blood rheology compared to normal red blood cells, L-glutamine was investigated for its therapeutic potential to reduce VOC. While L-glutamine was approved by the United States (US) Food and Drug Administration to treat SCD, its impact on the redox environment in sickle erythrocytes is not fully understood. The mechanism through which L-glutamine reduces VOC in SCD is also not clear. In this paper, we will summarize the results of the Phase 3 study that led to the approval of L-glutamine for treating SCD and discuss its assumed mechanisms of action. We will examine the role of L-glutamine in health and propose how the extra-erythrocytic functions of L-glutamine might contribute to its beneficial effects in SCD. Further research into the role of L-glutamine on extra-erythrocyte functions might help the development of an improved formulation with more efficacy.

Haematological profile of malaria patients with G6PD and PKLR variants (erythrocytic enzymopathies): a cross-sectional study in Thailand.

BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PKLR) deficiencies are common causes of erythrocyte haemolysis in the presence of antimalarial drugs such as primaquine and tafenoquine. The present study aimed to elucidate such an association by thoroughly investigating the haematological indices in malaria patients with G6PD and PKLRR41Q variants. METHODS: Blood samples from 255 malaria patients from Thailand, Myanmar, Laos, and Cambodia were collected to determine haematological profile, G6PD enzyme activity and G6PD deficiency variants. The multivariate analysis was performed to investigate the association between anaemia and G6PD MahidolG487A, the most common mutation in this study. RESULTS: The prevalence of G6PD deficiency was 11.1% (27/244) in males and 9.1% (1/11) in female. The MAFs of the G6PD MahidolG487A and PKLRR41Q variants were 7.1% and 2.6%, respectively. Compared with patients with wildtype G6PD after controlling for haemoglobinopathies, G6PD-deficient patients with hemizygous and homozygous G6PD MahidolG487A exhibited anaemia with low levels of haemoglobin (11.16 ± 2.65 g/dl, p = 0.041). These patients also exhibited high levels of reticulocytes (3.60%). The median value of G6PD activity before treatment (Day 0) was significantly lower than that of after treatment (Day 28) (5.51 ± 2.54 U/g Hb vs. 6.68 ± 2.45 U/g Hb; p < 0.001). Reticulocyte levels on Day 28 were significantly increased compared to that of on Day 0 (2.14 ± 0.92% vs 1.57 ± 1.06%; p < 0.001). PKLRR41Q had no correlation with anaemia in malaria patients. The risk of anaemia inpatients with G6PD MahidolG487A was higher than wildtype patients (OR = 3.48, CI% 1.24-9.75, p = 0.018). Univariate and multivariate analyses confirmed that G6PD MahidolG487A independently associated with anaemia (< 11 g/dl) after adjusted by age, gender, Plasmodium species, parasite density, PKLRR41Q, and haemoglobinopathies (p < 0.001). CONCLUSIONS: This study revealed that malaria patients with G6PD MahidolG487A, but not with PKLRR41Q, had anaemia during infection. As a compensatory response to haemolytic anaemia after malaria infection, these patients generated more reticulocytes. The findings emphasize the effect of host genetic background on haemolytic anaemia and the importance of screening patients for erythrocyte enzymopathies and related mutations prior to anti-malarial therapy.

Characterisation of individual ferritin response in patients receiving chelation therapy.

AIMS: To develop a drug-disease model describing iron overload and its effect on ferritin response in patients affected by transfusion-dependent haemoglobinopathies and investigate the contribution of interindividual differences in demographic and clinical factors on chelation therapy with deferiprone or deferasirox. METHODS: Individual and mean serum ferritin data were retrieved from 13 published studies in patients affected by haemoglobinopathies receiving deferiprone or deferasirox. A nonlinear mixed effects modelling approach was used to characterise iron homeostasis and serum ferritin production taking into account annual blood consumption, baseline demographic and clinical characteristics. The effect of chelation therapy was parameterised as an increase in the iron elimination rate. Internal and external validation procedures were used to assess model performance across different study populations. RESULTS: An indirect response model was identified, including baseline ferritin concentrations and annual blood consumption as covariates. The effect of chelation on iron elimination rate was characterised by a linear function, with different slopes for each drug (0.0109 [90% CI: 0.0079-0.0131] vs. 0.0013 [90% CI: 0.0008-0.0018] L/mg mo). In addition to drug-specific differences in the magnitude of the ferritin response, simulation scenarios indicate that ferritin elimination rates depend on ferritin concentrations at baseline. CONCLUSION: Modelling of serum ferritin following chronic blood transfusion enabled the evaluation of drug-induced changes in iron elimination rate and ferritin production. The use of a semi-mechanistic parameterisation allowed us to disentangle disease-specific factors from drug-specific properties. Despite comparable chelation mechanisms, deferiprone appears to have a significantly larger effect on the iron elimination rate than deferasirox.

Revisiting fetal hemoglobin inducers in beta-hemoglobinopathies: a review of natural products, conventional and combinatorial therapies.

Beta-hemoglobinopathies exhibit a heterogeneous clinical picture with varying degrees of clinical severity. Pertaining to the limited treatment options available, where blood transfusion still remains the commonest mode of treatment, pharmacological induction of fetal hemoglobin (HbF) has been a lucrative therapeutic intervention. Till now more than 70 different HbF inducers have been identified. The practical usage of many pharmacological drugs has been limited due to safety concerns. Natural compounds, like Resveratrol, Ripamycin and Bergaptene, with limited cytotoxicity and high efficacy have started capturing the attention of researchers. In this review, we have summarized pharmacological drugs and bioactive compounds isolated from natural sources that have been shown to increase HbF significantly. It primarily discusses recently identified synthetic and natural compounds, their mechanism of action, and their suitable screening platforms, including high throughput drug screening technology and biosensors. It also delves into the topic of combinatorial therapy and drug repurposing for HbF induction. Overall, we aim to provide insights into where we stand in HbF induction strategies for treating ß-hemoglobinopathies.

Curcuminoids supplementation ameliorates iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfusion-dependent ß-thalassemia/Hb E patients.

Curcuminoids, polyphenol compounds in turmeric, possess several pharmacological properties including antioxidant, iron-chelating, and anti-inflammatory activities. Effects of curcuminoids in thalassemia patients have been explored in a limited number of studies using different doses of curcuminoids. The present study aims to evaluate the effects of 24-week curcuminoids supplementation at the dosage of 500 and 1000 mg/day on iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfused ß-thalassemia/Hb E patients. In general, both curcuminoids dosages significantly lowered the levels of oxidative stress, hypercoagulability, and inflammatory markers in the patients. In contrast, reductions in iron parameter levels were more remarkable in the 1000 mg/day group. Subgroup analysis revealed that a marker of hypercoagulability was significantly decreased only in patients with baseline ferritin ≤ 1000 ng/ml independently of curcuminoids dosage. Moreover, the alleviation of iron loading parameters was more remarkable in patients with baseline ferritin > 1000 ng/ml who receive 1000 mg/day curcuminoids. On the other hand, the responses of oxidative stress markers were higher with 500 mg/day curcuminoids regardless of baseline ferritin levels. Our study suggests that baseline ferritin levels should be considered in the supplementation of curcuminoids and the appropriate curcuminoids dosage might differ according to the required therapeutic effect. Thai Clinical Trials Registry (TCTR): TCTR20200731003; July 31, 2020 "retrospectively registered".

Klf10 Gene, a Secondary Modifier and a Pharmacogenomic Biomarker of Hydroxyurea Treatment Among Patients With Hemoglobinopathies.

BACKGROUND: The klf10 gene could indirectly modify γ-globin chain production and hence the level of fetal hemoglobin (HbF) ameliorating the phenotype of ß-hemoglobinopathies and the response to hydroxycarbamide (hydroxyurea [HU]) therapy. In this study, we aimed to evaluate the frequency of different genotypes for the klf10 gene in ß-thalassemia major (B-TM), ß-thalassemia intermedia (B-TI), and sickle cell disease (SCD) patients by polymerase chain reaction and to assess its relation to disease phenotypes and HU response. METHODS: This cross-sectional study included 75 patients: 50 B-TM, 12 SCD, and 13 B-TI patients (on stable HU dose). The relation of the klf10 gene polymorphism (TIEG, TIEG1, EGRα) (rs3191333: c*0.141C>T) to phenotype was studied through baseline mean corpuscular volume, HbF, and transfusion history, whereas evaluation of response to HU therapy was carried out clinically and laboratory. RESULTS: The frequency of the mutant klf10 genotype (TT) and that of the mutant allele (T) was significantly higher among B-TM patients compared with those with B-TI and SCD patients. Only homozygous SCD patients for the wild-type allele within the klf10 gene had a significantly lower transfusion frequency. The percentage of HU responders and nonresponders between different klf10 polymorphic genotypes among B-TI or SCD patients was comparable. CONCLUSIONS: Although the klf10 gene does not play a standalone role as an HbF modifier, our data support its importance in ameliorating phenotype among ß-hemoglobinopathies.

Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.

HbSD-Punjab: clinical and hematological profile of a rare hemoglobinopathy.

PURPOSE: Compound heterozygous HbSD-Punjab is an uncommon hemoglobinopathy encountered in Indians. Limited literature is available about its clinical course. The aim of this study was to describe the clinical and hematological profile of HbSD-Punjab patients from North India. MATERIALS AND METHODS: HbSD-Punjab patients diagnosed in the hematology clinics between year 2000 and 2010 were reviewed retrospectively. The diagnosis was established using high-performance liquid chromatography, molecular analysis, and family screening. Clinical details, laboratory parameters, and therapy details were recorded from case records. RESULTS: Ten patients were identified. Median age at onset of symptoms was 3.5 years (interquartile range [IQR], 1.9 to 7.2). Clinical presentation included: anemia in 3, painful vaso-occlusive crisis in 2, acute chest syndrome in 2, and 3 were diagnosed incidentally. All had moderate to severe anemia (mean hemoglobin [Hb]: 6.8 ± 1.2 g/dL). Eight required red cell transfusions (median: 3 [IQR, 2 to 8]). On high-performance liquid chromatography, median HbF, HbD, and HbS were 12.1% (IQR, 9 to 18.3), 39.7% (IQR, 35 to 42), and 38.5% (IQR, 29 to 43). Five patients received hydroxyurea (HDU), median dose: 20 mg/kg/d (IQR, 18 to 23) with median duration of 7 months (IQR; 6, 45). Increment in Hb and reduction in painful crisis was observed in response to HDU. CONCLUSIONS: HbSD-Punjab has a heterogeneous clinical presentation. Anemia and sickle crises are quite common. HDU may be considered for those presenting with severe phenotype.

Oxidative stress changes the effectiveness of artemisinin in Plasmodium falciparum.

Malaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in Plasmodium falciparum is a complex phenotype linked to the parasite's tolerance of the activated drug's oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in P. falciparum to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite's adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite's survival responses to oxidative stress. In this study, we investigated these relationships further using an in vitro model to evaluate if oxidative stress and heat-shock conditions could alter the parasite's response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin. IMPORTANCE: Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite's responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients.

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.

Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNA-binding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.

Unique Hemoglobinopathy Pattern Following Treatment with Voxelotor.

OBJECTIVE: Voxelotor, a FDA-approved drug for the treatment of patients with sickle cell disease (SCD), inhibits hemoglobin S (HbS) polymerization and increases total hemoglobin via hemolysis reduction. This drug has shown unique patterns in hemoglobin fractionation, affecting its interpretation. We aimed to evaluate whether these voxelotor-induced changes can be linked to improvement of hemolysis markers in pediatric patients on voxelotor. METHODS: A total of 15 patients (age 12 to 20 years; 40% females) on voxelotor were evaluated to compare changes in the hemoglobin fractionation by capillary electrophoresis, total hemoglobin, reticulocyte percentage (retic%), lactate dehydrogenase (LDH), and bilirubin measurements before and after the recorded date of voxelotor prescription. RESULTS: Hemoglobin fractionation showed changes in the profile of 60% (9/15) of the patients studied. Out of the 9 patients for which voxelotor showed changes in the hemoglobin fractionation, 44% (4/9) had an increase of >1 g/dL in their total hemoglobin after voxelotor treatment was started. Assessment of other hemolysis markers available showed decreased LDH (4/4), retic % (6/8), and bilirubin (3/4). CONCLUSIONS: Unique pattern of hemoglobin fractionation analysis following therapy with voxelotor has potential as a tool for the assessment of response and/or compliance to voxelotor for the treatment of SCD.

Effect of hydroxyurea on erythrocyte apoptosis in hemoglobinopathy patients.

BACKGROUND: Hydroxyurea (HU) therapy improves the clinical severity of patients with hemoglobinopathies. Few studies have documented some mechanisms of HU, but the exact mechanism of action is unknown. Phosphatidylserine on erythrocytes is responsible for apoptosis. In this study, we investigate the expression of phosphatidylserine on the erythrocytes surface of hemoglobinopathies before and after HU treatment. RESEARCH DESIGNS AND METHODS: Blood samples from 45 thalassemia intermedia and 40 SCA and 30 HbE-b-thalassemia patients were analyzed before and after 3 and 6 months of HU treatment. The profile of phosphatidylserine was determined by flow-cytometry using the Annexin V-RBC apoptosis kit. RESULTS: Hydroxyurea proved effective in improving clinical severity of hemoglobinopathies. After treatment with hydroxyurea, the percentage of phosphatidylserine-positive cells was significantly reduced in all 3 patient groups (p < 0.0001). Correlation analysis using different hematological parameters as independent variables and % phosphatidylserine  as dependent variable showed a negative relationship with HbF, RBC, and hemoglobin in all 3 patient groups. CONCLUSION: Hydroxyurea reduces the expression of phosphatidylserine on erythrocytes, contributing to the beneficial effects of this therapy. We suggest that the use of such a biological marker in conjunction with HbF levels may provide valuable insights into the biology and consequences of early RBC apoptosis.

The study investigated the role of hydroxyurea in reducing the externalization of phosphatidylserine on the surface of the erythrocyte membrane of patients with hemoglobinopathies. In patients treated with hydroxyurea for 3 and 6 months, the percentage of phosphatidylserine exposure on the erythrocyte surface was reduced compared with baseline. The decreased percentage of phosphatidylserine correlated negatively with hematologic parameters such as red blood cell (RBC), hemoglobin, and fetal hemoglobin (HbF) in patients at baseline and after HU therapy. Treatment with hydroxyurea decreases the percentage of PS exposure on the surface of RBCs, contributing to the beneficial effects of this therapy. We, therefore, suggest that the use of such a biological marker on the erythrocyte cell surface in conjunction with HbF levels may provide valuable insights into the biology and consequences of early erythrocyte apoptosis.

No difference in myocardial iron concentration and serum ferritin with deferasirox and deferiprone in pediatric patients with hemoglobinopathies: A systematic review and meta-analysis.

OBJECTIVES: Iron overload is a common complication experienced by transfusion-dependent children with hemoglobin disorders. Chelators such as deferasirox (DFX) and deferiprone (DFP) are effective in overcoming this problem. We conducted this systematic review and meta-analysis to evaluate the effectiveness of DFX compared to DFP in treating iron overload amongst pediatric patients with hemoglobin disorders. MATERIAL AND METHODS: PubMed and Cochrane Central were searched from their inception until Dec 21 2021, for randomized clinical trials (RCTs) and observational studies, which assessed the efficacy of DFX compared to DFP in the treatment of inherited hemoglobin disorders. The outcomes of interest included myocardial iron concentration (MRI T2*) at the end of the trial and change in mean serum ferritin (SF) levels at the 6 and 12 months mark. Weighted mean differences (WMDs) with their corresponding 95% confidence intervals (CIs) were calculated for continuous outcomes using random effects model. RESULTS: A total of 5 studies comprising 607 children were included. The results of our analysis revealed no significant difference between DFX and DFP in MRI T2* at the end of treatment (WMD: -0.92; 95% CI [-3.35, 1.52]; p = 0.46; I2 = 0). Moreover, there has been no significant difference noted in SF levels at both 6 months (WMD: 97.31; 95% CI [-236.16, 430.77]; p = 0.57; I2 = 0) and 12 months (WMD: 46.99; 95% CI [-191.42, 285.40]; p = 0.70; I2 = 0) respectively. CONCLUSION: Our analysis shows no significant difference between the efficacy of DFX and DFP in the management of iron overload in children with inherited blood disorders. Future large-scale clinical trials are required to further validate our results.

A comprehensive review of hydroxyurea for ß-haemoglobinopathies: the role revisited during COVID-19 pandemic.

BACKGROUND: Hydroxyurea is one of the earliest drugs that showed promise in the management of haemoglobinopathies that include ß-thalassaemia and sickle cell disease. Despite this, many aspects of hydroxyurea are either unknown or understudied; specifically, its usefulness in ß-thalassaemia major and haemoglobin E ß-thalassaemia is unclear. However, during COVID-19 pandemic, it has become a valuable adjunct to transfusion therapy in patients with ß-haemoglobinopathies. In this review, we aim to explore the available in vitro and in vivo mechanistic data and the clinical utility of hydroxyurea in ß-haemoglobinopathies with a special emphasis on its usefulness during the COVID-19 pandemic. MAIN BODY: Hydroxyurea is an S-phase-specific drug that reversibly inhibits ribonucleoside diphosphate reductase enzyme which catalyses an essential step in the DNA biosynthesis. In human erythroid cells, it induces the expression of γ-globin, a fetal globin gene that is suppressed after birth. Through several molecular pathways described in this review, hydroxyurea exerts many favourable effects on the haemoglobin content, red blood cell indices, ineffective erythropoiesis, and blood rheology in patients with ß-haemoglobinopathies. Currently, it is recommended for sickle cell disease and non-transfusion dependent ß-thalassaemia. A number of clinical trials are ongoing to evaluate its usefulness in transfusion dependent ß-thalassaemia. During the COVID-19 pandemic, it was widely used as an adjunct to transfusion therapy due to limitations in the availability of blood and logistical disturbances. Thus, it has become clear that hydroxyurea could play a remarkable role in reducing transfusion requirements of patients with haemoglobinopathies, especially when donor blood is a limited resource. CONCLUSION: Hydroxyurea is a well-tolerated oral drug which has been in use for many decades. Through its actions of reversible inhibition of ribonucleoside diphosphate reductase enzyme and fetal haemoglobin induction, it exerts many favourable effects on patients with ß-haemoglobinopathies. It is currently approved for the treatment of sickle cell disease and non-transfusion dependent ß-thalassaemia. Also, there are various observations to suggest that hydroxyurea is an important adjunct in the treatment of transfusion dependent ß-thalassaemia which should be confirmed by randomised clinical trials.

Recent advances in lentiviral vectors for gene therapy.

Lentiviral vectors (LVs), derived from human immunodeficiency virus, are powerful tools for modifying the genes of eukaryotic cells such as hematopoietic stem cells and neural cells. With the extensive and in-depth studies on this gene therapy vehicle over the past two decades, LVs have been widely used in both research and clinical trials. For instance, third-generation and self-inactive LVs have been used to introduce a gene with therapeutic potential into the host genome and achieve targeted delivery into specific tissue. When LVs are employed in leukemia, the transduced T cells recognize and kill the tumor B cells; in ß-thalassemia, the transduced CD34+ cells express normal ß-globin; in adenosine deaminase-deficient severe combined immunodeficiency, the autologous CD34+ cells express adenosine deaminase and realize immune reconstitution. Overall, LVs can perform significant roles in the treatment of primary immunodeficiency diseases, hemoglobinopathies, B cell leukemia, and neurodegenerative diseases. In this review, we discuss the recent developments and therapeutic applications of LVs. The safe and efficient LVs show great promise as a tool for human gene therapy.

Cilostazol-mediated reversion of γ-globin silencing is associated with a high level of HbF production: A potential therapeutic candidate for ß-globin disorders.

Reversal of fetal hemoglobin (HbF) silencing is an attractive therapeutic intervention for ß-thalassemia and sickle cell anemia. The current study proposes the therapeutic of repurposing of cilostazol, an FDA-approved antithrombotic agent, as a promising HbF inducer. Preliminary, we report that cilostazol induced erythroid differentiation and hemoglobinization of human erythroleukemia K562 cells. The erythroid differentiation was accompanied by increased expression of γ-globin mRNA transcripts and HbF production. Cilostazol induced erythroid differentiation and HbF production, without significantly affecting proliferation and viability of hemoglobin producing cells at maximum erythroid inducing concentration. Moreover, we investigated the effect of cilostazol on human ß- and γ-globin transgenes in in vivo ß-YAC transgenic mice, harboring human ß-locus along with ß-LCR. A good in vitro correlation was found with substantial up-regulation in fetal globin mRNA; whereas, the ß-globin gene expression was not significantly changed. F-cells, analysis in the peripheral blood of cilostazol-treated mice, revealed a significant increase in the F-cells population as compared with sham control groups. Together, these findings support the potential of cilostazol as an HbF inducer, which can be evaluated further to develop a new HbF inducer.

Cucurbitacin D induces fetal hemoglobin synthesis in K562 cells and human hematopoietic progenitors through activation of p38 pathway and stabilization of the γ-globin mRNA.

The search for novel therapeutic candidates targeting fetal hemoglobin (HbF) activation to reduce the imbalance of globin genes is regarded as a promising approach for the clinical management of sickle cell disease and ß-thalassemia. For the first time, we identified cucurbitacin D (CuD), an oxygenated tetracyclic triterpenoid, as a molecular entity inducing γ-globin gene expression and HbF synthesis in K562 cells and human hematopoietic progenitors from a ß-thalassemia patient. CuD demonstrated a higher potency in HbF induction when compared with hydroxyurea, which was revealed by the evidence that CuD results in a higher fetal cell percentage and greater HbF content in K562 cells, in addition, to being less cytotoxic. Moreover, CuD also promotes higher HbF expression in primary erythroid cells. In the study to elucidate the molecular mechanisms of CuD's action, our data indicated that CuD-stimulated HbF synthesis was mediated by p38 pathway activation. At the post-transcriptional level, CuD treatment led to a significant elongation of the γ-globin mRNA half-life in K562 cells. Taken together, the results suggest that CuD may be a potential therapeutic agent for ß-hemoglobinopathies, including sickle cell anemia and ß-thalassemia.

Partial glutathione reductase deficiency as a cause of diverse clinical manifestations in a family with unstable hemoglobin (Hemoglobin Haná, ß63(E7) His-Asn).

Hemoglobin Haná [ß63(E7) His-Asn] is an unstable hemoglobin variant that was described in a Czech proband and her sister with Heinz body hemolytic anemia. The mother bearing the same mutation was asymptomatic; nevertheless, all three carriers had the same proportion of the mutant globin chains. Assessment of several erythrocyte antioxidant parameters revealed that both symptomatic children, unlike their asymptomatic mother, had significantly decreased glutathione reductase (GR) activity. Their GR activities were restorable in vitro by flavin adenine dinucleotide. The riboflavin supplementation improved their glutathione metabolism and ameliorated their hemolysis. Pre- and post-treatment assessment of the B(2) vitamers indicated suboptimal pre-treatment vitamin B(2) status in both children. This study provides evidence that partial GR deficiency may alter the clinical manifestation of an unstable hemoglobinopathy.

Targeted Protein Degradation as a Promising Tool for Epigenetic Upregulation of Fetal Hemoglobin.

The level of fetal hemoglobin (HbF) is an important disease modifier for ß-thalassemia and sickle cell disease patients. Indeed, genetic tinkering with the HbF repression machinery has demonstrated great potential for disease mitigation. Such genetic treatments are costly and the high incidence of ß-hemoglobinopathies in low-income countries, therefore, calls for the development of affordable, off-the-shelf, oral treatments. The use of PROTAC (PRoteolysis TArgeting Chimeras) technology to influence the epigenetic mechanisms involved in HbF suppression may provide a solution. In this minireview, we briefly explain the HbF repression network highlighting the epigenetic factors that could be targeted for degradation by PROTACs. We hope that this review will inspire clinicians, molecular and chemical biologists to collaborate and contribute to this fascinating field, which should ultimately deliver drugs that reactivate HbF expression with high specificity and low toxicity.

A hypothesis about the role of fetal hemoglobin in COVID-19.

COVID-19 infection is less common in children (with higher fetal hemoglobin levels). In our preliminary study, we also observed a low prevalence and fatality of COVID-19 in countries with high rate of hemoglobinopathy carries. Given these two facts, the hemoglobin structure can play a role in the physiopathology of COVID-19 disease. Several drugs are known to increase fetal hemoglobin in adults. Adding these drugs to COVID-19 clinical trials may improve the patients' outcomes.